African Journal of
Microbiology Research
Volume 9 Number 16, 22 April, 2015
ISSN 1996-0808
ABOUT AJMR
The African Journal of Microbiology Research (AJMR) (ISSN 1996-0808) is published Weekly (one volume per
year) by Academic Journals.
African Journal of Microbiology Research (AJMR) provides rapid publication (weekly) of articles in all areas of
Microbiology such as: Environmental Microbiology, Clinical Microbiology, Immunology, Virology, Bacteriology,
Phycology, Mycology and Parasitology, Protozoology, Microbial Ecology, Probiotics and Prebiotics, Molecular
Microbiology, Biotechnology, Food Microbiology, Industrial Microbiology, Cell Physiology, Environmental
Biotechnology, Genetics, Enzymology, Molecular and Cellular Biology, Plant Pathology, Entomology, Biomedical
Sciences, Botany and Plant Sciences, Soil and Environmental Sciences, Zoology, Endocrinology, Toxicology. The
Journal welcomes the submission of manuscripts that meet the general criteria of significance and scientific
excellence. Papers will be published shortly after acceptance. All articles are peer-reviewed.
Submission of Manuscript
Please read the Instructions for Authors before submitting your manuscript. The manuscript files should be given
the last name of the first author
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With questions or concerns, please contact the Editorial Office at ajmr@academicjournals.org.
Editors
Prof. Dr. Stefan Schmidt,
Applied and Environmental Microbiology
School of Biochemistry, Genetics and Microbiology
University of KwaZulu-Natal
Private Bag X01
Scottsville, Pietermaritzburg 3209
South Africa.
Dr. Thaddeus Ezeji
Assistant Professor
Fermentation and Biotechnology Unit
Department of Animal Sciences
The Ohio State University
1680 Madison Avenue
USA.
Prof. Fukai Bao
Department of Microbiology and Immunology
Kunming Medical University
Kunming 650031,
China
Associate Editors
Dr. Jianfeng Wu
Dept. of Environmental Health Sciences,
School of Public Health,
University of Michigan
USA
Dr. Ahmet Yilmaz Coban
OMU Medical School,
Department of Medical Microbiology,
Samsun,
Turkey
Dr. Seyed Davar Siadat
Pasteur Institute of Iran,
Pasteur Square, Pasteur Avenue,
Tehran,
Iran.
Dr. J. Stefan Rokem
The Hebrew University of Jerusalem
Department of Microbiology and Molecular Genetics,
P.O.B. 12272, IL-91120 Jerusalem,
Israel
Prof. Long-Liu Lin
National Chiayi University
300 Syuefu Road,
Chiayi,
Taiwan
N. John Tonukari, Ph.D
Department of Biochemistry
Delta State University
PMB 1
Abraka, Nigeria
Dr. Mamadou Gueye
MIRCEN/ Laboratoire commun de microbiologie
IRD-ISRA-UCAD, BP 1386,
DAKAR, Senegal.
Dr. Caroline Mary Knox
Department of Biochemistry, Microbiology and
Biotechnology
Rhodes University
Grahamstown 6140
South Africa.
Dr. Hesham Elsayed Mostafa
Genetic Engineering and Biotechnology Research
Institute (GEBRI)
Mubarak City For Scientific Research,
Research Area, New Borg El-Arab City,
Post Code 21934, Alexandria, Egypt.
Dr. Wael Abbas El-Naggar
Head of Microbiology Department,
Faculty of Pharmacy,
Mansoura University,
Mansoura 35516, Egypt.
Dr. Abdel Nasser A. El-Moghazy
Microbiology, Molecular Biology, Genetics Engineering
and Biotechnology
Dept of Microbiology and Immunology
Faculty of Pharmacy
Al-Azhar University
Nasr city,
Cairo, Egypt
Editorial Board
Dr. Barakat S.M. Mahmoud
Food Safety/Microbiology
Experimental Seafood Processing Laboratory
Costal Research and Extension Center
Mississippi State University
3411 Frederic Street
Pascagoula, MS 39567
USA
Prof. Mohamed Mahrous Amer
Poultry Disease (Viral Diseases of poultry)
Faculty of Veterinary Medicine,
Department of Poultry Diseases
Cairo university
Giza, Egypt
Dr. Xiaohui Zhou
Molecular Microbiology, Industrial Microbiology,
Environmental Microbiology, Pathogenesis, Antibiotic
resistance, Microbial Ecology
Washington State University
Bustad Hall 402 Department of Veterinary
Microbiology and Pathology, Pullman,
USA
Dr. R. Balaji Raja
Department of Biotechnology,
School of Bioengineering,
SRM University,
Chennai
India
Dr. Aly E Abo-Amer
Division of Microbiology, Botany Department, Faculty
of Science, Sohag University.
Egypt.
Dr. Haoyu Mao
Department of Molecular Genetics and Microbiology
College of Medicine
University of Florida
Florida, Gainesville
USA.
Dr. Rachna Chandra
Environmental Impact Assessment Division
Environmental Sciences
Sálim Ali Center for Ornithology and Natural History
(SACON),
Anaikatty (PO), Coimbatore-641108, India
Dr. Yongxu Sun
Department of Medicinal Chemistry and
Biomacromolecules
Qiqihar Medical University, Qiqihar 161006
Heilongjiang Province
P.R. China
Dr. Ramesh Chand Kasana
Institute of Himalayan Bioresource Technology
Palampur, Distt. Kangra (HP),
India
Dr. S. Meena Kumari
Department of Biosciences
Faculty of Science
University of Mauritius
Reduit
Dr. T. Ramesh
Assistant Professor
Marine Microbiology
CAS in Marine Biology
Faculty of Marine Sciences
Annamalai University
Parangipettai - 608 502
Cuddalore Dist. Tamilnadu,
India
Dr. Pagano Marcela Claudia
Post doctoral fellowship at Department of Biology,
Federal University of Ceará - UFC,
Brazil.
Dr. EL-Sayed E. Habib
Associate Professor,
Dept. of Microbiology,
Faculty of Pharmacy,
Mansoura University,
Egypt.
Dr. Pongsak Rattanachaikunsopon
Department of Biological Science,
Faculty of Science,
Ubon Ratchathani University,
Warin Chamrap, Ubon Ratchathani 34190,
Thailand
Dr. Gokul Shankar Sabesan
Microbiology Unit, Faculty of Medicine,
AIMST University
Jalan Bedong, Semeling 08100,
Kedah,
Malaysia
Dr. Kwang Young Song
Department of Biological Engineering,
School of Biological and Chemical Engineering,
Yanbian Universityof Science and Technology,
Yanji,
China.
Dr. Kamel Belhamel
Faculty of Technology,
University of Bejaia
Algeria
Dr. Sladjana Jevremovic
Institute for Biological Research
Sinisa Stankovic,
Belgrade,
Serbia
Dr. Tamer Edirne
Dept. of Family Medicine, Univ. of Pamukkale
Turkey
Dr. R. Balaji Raja M.Tech (Ph.D)
Assistant Professor,
Department of Biotechnology,
School of Bioengineering,
SRM University,
Chennai.
India
Dr. Minglei Wang
University of Illinois at Urbana-Champaign,USA
Dr. Mohd Fuat ABD Razak
Institute for Medical Research
Malaysia
Dr. Davide Pacifico
Istituto di Virologia Vegetale – CNR
Italy
Prof. Dr. Akrum Hamdy
Faculty of Agriculture, Minia University, Egypt
Egypt
Dr. Ntobeko A. B. Ntusi
Cardiac Clinic, Department of Medicine,
University of Cape Town and
Department of Cardiovascular Medicine,
University of Oxford
South Africa and
United Kingdom
Prof. N. S. Alzoreky
Food Science & Nutrition Department,
College of Agricultural Sciences & Food,
King Faisal University,
Saudi Arabia
Dr. Chen Ding
College of Material Science and Engineering,
Hunan University,
China
Dr Svetlana Nikolić
Faculty of Technology and Metallurgy,
University of Belgrade,
Serbia
Dr. Sivakumar Swaminathan
Department of Agronomy,
College of Agriculture and Life Sciences,
Iowa State University,
Ames, Iowa 50011
USA
Dr. Alfredo J. Anceno
School of Environment, Resources and Development
(SERD),
Asian Institute of Technology,
Thailand
Dr. Iqbal Ahmad
Aligarh Muslim University,
Aligrah
India
Dr. Josephine Nketsia-Tabiri
Ghana Atomic Energy Commission
Ghana
Dr. Juliane Elisa Welke
UFRGS – Universidade Federal do Rio
Grande do Sul
Brazil
Dr. Mohammad Nazrul Islam
NIMR; IPH-Bangalore & NIUM
Bangladesh
Dr. Okonko, Iheanyi Omezuruike
Department of Virology,
Faculty of Basic Medical Sciences,
College of Medicine,
University of Ibadan,
University College Hospital,
Ibadan,
Nigeria
Dr. Giuliana Noratto
Texas A&M University
USA
Dr. Phanikanth Venkata Turlapati
Washington State University
USA
Dr. Khaleel I. Z. Jawasreh
National Centre for Agricultural Research and
Extension, NCARE
Jordan
Dr. Babak Mostafazadeh, MD
Shaheed Beheshty University of Medical Sciences
Iran
Dr. S. Meena Kumari
Department of Biosciences
Faculty of Science
University of Mauritius
Reduit
Mauritius
Dr. S. Anju
Department of Biotechnology,
SRM University, Chennai-603203
India
Dr. Mustafa Maroufpor
Iran
Prof. Dong Zhichun
Professor, Department of Animal Sciences and
Veterinary Medicine,
Yunnan Agriculture University,
China
Dr. Mehdi Azami
Parasitology & Mycology Dept,
Baghaeei Lab.,
Shams Abadi St.
Isfahan
Iran
Dr. Anderson de Souza Sant’Ana
University of São Paulo.
Brazil.
Dr. Juliane Elisa Welke
UFRGS – Universidade Federal do Rio Grande do Sul
Brazil
Dr. Paul Shapshak
USF Health,
Depts. Medicine (Div. Infect. Disease & Internat Med)
and Psychiatry & Beh Med.
USA
Dr. Jorge Reinheimer
Universidad Nacional del Litoral (Santa Fe)
Argentina
Dr. Qin Liu
East China University of Science
and Technology
China
Dr. Xiao-Qing Hu
State Key Lab of Food Science and Technology
Jiangnan University
P. R. China
Prof. Branislava Kocic
Specaialist of Microbiology and Parasitology
University of Nis, School of Medicine Institute
for Public Health Nis, Bul. Z. Djindjica 50, 18000 Nis
Serbia
Dr. Rafel Socias
CITA de Aragón,
Spain
Prof. Kamal I. Mohamed
State University of New York at Oswego
USA
Prof. Isidro A. T. Savillo
ISCOF
Philippines
Dr. Adriano Cruz
Faculty of Food Engineering-FEA
University of Campinas (UNICAMP)
Brazil
Dr. How-Yee Lai
Taylor’s University College
Malaysia
Dr. Mike Agenbag (Michael Hermanus Albertus)
Manager Municipal Health Services,
Joe Gqabi District Municipality
South Africa
Dr. D. V. L. Sarada
Department of Biotechnology,
SRM University, Chennai-603203
India.
Dr. Samuel K Ameyaw
Civista Medical Center
United States of America
Prof. Huaizhi Wang
Institute of Hepatopancreatobiliary
Surgery of PLA Southwest Hospital,
Third Military Medical University
Chongqing400038
P. R. China
Prof. Bakhiet AO
College of Veterinary Medicine, Sudan
University of Science and Technology
Sudan
Dr. Saba F. Hussain
Community, Orthodontics and Peadiatric Dentistry
Department
Faculty of Dentistry
Universiti Teknologi MARA
40450 Shah Alam, Selangor
Malaysia
Prof. Dr. Zohair I.F.Rahemo
State Key Lab of Food Science and Technology
Jiangnan University
P. R. China
Dr. Afework Kassu
University of Gondar
Ethiopia
Dr. Nidheesh Dadheech
MS. University of Baroda, Vadodara, Gujarat, India.
India
Dr. Omitoyin Siyanbola
Bowen University,
Iwo
Nigeria
Dr. Franco Mutinelli
Istituto Zooprofilattico Sperimentale delle Venezie
Italy
Dr. Chanpen Chanchao
Department of Biology,
Faculty of Science,
Chulalongkorn University
Thailand
Dr. Tsuyoshi Kasama
Division of Rheumatology,
Showa University
Japan
Dr. Kuender D. Yang, MD.
Chang Gung Memorial Hospital
Taiwan
Dr. Liane Raluca Stan
University Politehnica of Bucharest,
Department of Organic Chemistry “C.Nenitzescu”
Romania
Dr. Muhamed Osman
Senior Lecturer of Pathology & Consultant
Immunopathologist
Department of Pathology,
Faculty of Medicine,
Universiti Teknologi MARA,
40450 Shah Alam, Selangor
Malaysia
Dr. Mohammad Feizabadi
Tehran University of medical Sciences
Iran
Prof. Ahmed H Mitwalli
State Key Lab of Food Science and Technology
Jiangnan University
P. R. China
Dr. Mazyar Yazdani
Department of Biology,
University of Oslo,
Blindern,
Oslo,
Norway
Dr. Ms. Jemimah Gesare Onsare
Ministry of Higher, Education
Science and Technology
Kenya
Dr. Babak Khalili Hadad
Department of Biological Sciences,
Roudehen Branch,
Islamic Azad University,
Roudehen
Iran
Dr. Ehsan Sari
Department of Plan Pathology,
Iranian Research Institute of Plant Protection,
Tehran,
Iran.
Dr. Adibe Maxwell Ogochukwu
Department of Clinical Pharmacy and Pharmacy
Management,
University of Nigeria,
Nsukka.
Nigeria
Dr. William M. Shafer
Emory University School of Medicine
USA
Dr. Michelle Bull
CSIRO Food and Nutritional Sciences
Australia
Prof. Dr. Márcio Garcia Ribeiro (DVM, PhD)
School of Veterinary Medicine and Animal ScienceUNESP,
Dept. Veterinary Hygiene and Public Health,
State of Sao Paulo
Brazil
Prof. Dr. Sheila Nathan
National University of Malaysia (UKM)
Malaysia
Prof. Ebiamadon Andi Brisibe
University of Calabar,
Calabar,
Nigeria
Dr. Snjezana Zidovec Lepej
University Hospital for Infectious Diseases
Zagreb,
Croatia
Dr. Julie Wang
Burnet Institute
Australia
Dr. Dilshad Ahmad
King Saud University
Saudi Arabia
Dr. Jean-Marc Chobert
INRA- BIA, FIPL
France
Dr. Adriano Gomes da Cruz
University of Campinas (UNICAMP)
Brazil
Dr. Zhilong Yang, PhD
Laboratory of Viral Diseases
National Institute of Allergy and Infectious Diseases,
National Institutes of Health
Dr. Hsin-Mei Ku
Agronomy Dept. NCHU 250 Kuo
Kuang Rd, Taichung,
Taiwan
Dr. Dele Raheem
University of Helsinki
Finland
Dr. Fereshteh Naderi
Physical chemist,
Islamic Azad University,
Shahre Ghods Branch
Iran
Dr. Li Sun
PLA Centre for the treatment of infectious diseases,
Tangdu Hospital,
Fourth Military Medical University
China
Dr. Biljana Miljkovic-Selimovic
School of Medicine,
University in Nis,
Serbia; Referent laboratory for Campylobacter and
Helicobacter,
Center for Microbiology,
Institute for Public Health, Nis
Serbia
Dr. Pradeep Parihar
Lovely Professional University, Phagwara, Punjab.
India
Dr. Xinan Jiao
Yangzhou University
China
Dr. Kanzaki, L I B
Laboratory of Bioprospection. University of Brasilia
Brazil
Dr. Endang Sri Lestari, MD.
Department of Clinical Microbiology,
Medical Faculty,
Diponegoro University/Dr. Kariadi Teaching Hospital,
Semarang
Indonesia
Prof. Philippe Dorchies
Laboratory of Bioprospection. University of Brasilia
Brazil
Dr. Hojin Shin
Pusan National University Hospital
South Korea
Dr. Yi Wang
Center for Vector Biology, 180 Jones Avenue
Rutgers University, New Brunswick, NJ 08901-8536
USA
Dr. Heping Zhang
The Key Laboratory of Dairy Biotechnology and
Engineering,
Ministry of Education,
Inner Mongolia Agricultural University.
China
Dr. William H Roldán
Department of Medical Microbiology,
Faculty of Medicine,
Peru
Dr. C. Ganesh Kumar
Indian Institute of Chemical Technology,
Hyderabad
India
Dr. Farid Che Ghazali
Universiti Sains Malaysia (USM)
Malaysia
Dr. Samira Bouhdid
Abdelmalek Essaadi University,
Tetouan,
Morocco
Dr. Zainab Z. Ismail
Department of Environmental Engineering, University
of Baghdad.
Iraq
Prof. Natasha Potgieter
University of Venda
South Africa
Dr. Ary Fernandes Junior
Universidade Estadual Paulista (UNESP)
Brasil
Dr. Alemzadeh
Sharif University
Iran
Dr. Papaevangelou Vassiliki
Athens University Medical School
Greece
Dr. Sonia Arriaga
Instituto Potosino de Investigación Científicay
Tecnológica/División de Ciencias Ambientales
Mexico
Dr. Fangyou Yu
The first Affiliated Hospital of Wenzhou Medical
College
China
Dr. Armando Gonzalez-Sanchez
Universidad Autonoma Metropolitana Cuajimalpa
Mexico
Dr. Galba Maria de Campos Takaki
Catholic University of Pernambuco
Brazil
Dr. Kwabena Ofori-Kwakye
Department of Pharmaceutics,
Kwame Nkrumah University of Science & Technology,
KUMASI
Ghana
Dr. Hans-Jürg Monstein
Clinical Microbiology, Molecular Biology Laboratory,
University Hospital, Faculty of Health Sciences, S-581
85 Linköping
Sweden
Prof. Dr. Liesel Brenda Gende
Arthropods Laboratory, School of Natural and Exact
Sciences, National University of Mar del Plata
Buenos Aires,
Argentina.
Dr. Ajith, T. A
Associate Professor Biochemistry, Amala Institute of
Medical Sciences, Amala Nagar, Thrissur, Kerala-680
555
India
Dr. Adeshina Gbonjubola
Ahmadu Bello University,
Zaria.
Nigeria
Dr. Feng-Chia Hsieh
Biopesticides Division, Taiwan Agricultural Chemicals
and Toxic Substances Research Institute, Council of
Agriculture
Taiwan
Prof. Dr. Stylianos Chatzipanagiotou
University of Athens – Medical School
Greec
Dr. Dongqing BAI
Department of Fishery Science,
Tianjin Agricultural College,
Tianjin 300384
P. R. China
Dr. Dingqiang Lu
Nanjing University of Technology
P.R. China
Dr. L. B. Sukla
Scientist –G & Head, Biominerals Department,
IMMT, Bhubaneswar
India
Dr. Hakan Parlakpinar
MD. Inonu University, Medical Faculty, Department
of Pharmacology, Malatya
Turkey
Dr Pak-Lam Yu
Massey University
New Zealand
Dr Percy Chimwamurombe
University of Namibia
Namibia
Dr. Euclésio Simionatto
State University of Mato Grosso do Sul-UEMS
Brazil
Prof. Dra. Suzan Pantaroto de Vasconcellos
Universidade Federal de São Paulo
Rua Prof. Artur Riedel, 275 Jd. Eldorado, Diadema, SP
CEP 09972-270
Brasil
Dr. Maria Leonor Ribeiro Casimiro Lopes Assad
Universidade Federal de São Carlos - Centro de
Ciências Agrárias - CCA/UFSCar
Departamento de Recursos Naturais e Proteção
Ambiental
Rodovia Anhanguera, km 174 - SP-330
Araras - São Paulo
Brasil
Dr. Pierangeli G. Vital
Institute of Biology, College of Science, University of
the Philippines
Philippines
Prof. Roland Ndip
University of Fort Hare, Alice
South Africa
Dr. Shawn Carraher
University of Fort Hare, Alice
South Africa
Dr. José Eduardo Marques Pessanha
Observatório de Saúde Urbana de Belo
Horizonte/Faculdade de Medicina da Universidade
Federal de Minas Gerais
Brasil
Dr. Yuanshu Qian
Department of Pharmacology, Shantou University
Medical College
China
Dr. Helen Treichel
URI-Campus de Erechim
Brazil
Dr. Xiao-Qing Hu
State Key Lab of Food Science and Technology
Jiangnan University
P. R. China
Dr. Olli H. Tuovinen
Ohio State University, Columbus, Ohio
USA
Prof. Stoyan Groudev
University of Mining and Geology “Saint Ivan Rilski”
Sofia
Bulgaria
Dr. G. Thirumurugan
Research lab, GIET School of Pharmacy, NH-5,
Chaitanya nagar, Rajahmundry-533294.
India
Dr. Charu Gomber
Thapar University
India
Dr. Jan Kuever
Bremen Institute for Materials Testing,
Department of Microbiology,
Paul-Feller-Str. 1, 28199 Bremen
Germany
Dr. Nicola S. Flanagan
Universidad Javeriana, Cali
Colombia
Dr. André Luiz C. M. de A. Santiago
Universidade Federal Rural de Pernambuco
Brazil
Dr. Dhruva Kumar Jha
Microbial Ecology Laboratory,
Department of Botany,
Gauhati University,
Guwahati 781 014, Assam
India
Dr. N Saleem Basha
M. Pharm (Pharmaceutical Biotechnology)
Eritrea (North East Africa)
Prof. Dr. João Lúcio de Azevedo
Dept. Genetics-University of São Paulo-Faculty of
Agriculture- Piracicaba, 13400-970
Brasil
Dr. Julia Inés Fariña
PROIMI-CONICET
Argentina
Dr. Yutaka Ito
Kyoto University
Japan
Dr. Cheruiyot K. Ronald
Biomedical Laboratory Technologist
Kenya
Prof. Dr. Ata Akcil
S. D. University
Turkey
Dr. Adhar Manna
The University of South Dakota
USA
Dr. Cícero Flávio Soares Aragão
Federal University of Rio Grande do Norte
Brazil
Dr. Gunnar Dahlen
Institute of odontology, Sahlgrenska Academy at
University of Gothenburg
Sweden
Dr. Pankaj Kumar Mishra
Vivekananda Institute of Hill Agriculture, (I.C.A.R.),
ALMORA-263601, Uttarakhand
India
Dr. Benjamas W. Thanomsub
Srinakharinwirot University
Thailand
Dr. Maria José Borrego
National Institute of Health – Department of Infectious
Diseases
Portugal
Dr. Catherine Carrillo
Health Canada, Bureau of Microbial Hazards
Canada
Dr. Marcotty Tanguy
Institute of Tropical Medicine
Belgium
Dr. Han-Bo Zhang
Laboratory of Conservation and Utilization for Bioresources
Key Laboratory for Microbial Resources of the
Ministry of Education,
Yunnan University, Kunming 650091.
School of Life Science,
Yunnan University, Kunming,
Yunnan Province 650091.
China
Dr. Ali Mohammed Somily
King Saud University
Saudi Arabia
Dr. Nicole Wolter
National Institute for Communicable Diseases and
University of the Witwatersrand,
Johannesburg
South Africa
Dr. Marco Antonio Nogueira
Universidade Estadual de Londrina
CCB/Depto. De microbiologia
Laboratório de Microbiologia Ambiental
Caixa Postal 6001
86051-980 Londrina.
Brazil
Dr. Bruno Pavoni
Department of Environmental Sciences University of
Venice
Italy
Dr. Shih-Chieh Lee
Da-Yeh University
Taiwan
Dr. Satoru Shimizu
Horonobe Research Institute for the Subsurface
Environment,
Northern Advancement Center for Science &
Technology
Japan
Dr. Tang Ming
College of Forestry, Northwest A&F University,
Yangling
China
Dr. Olga Gortzi
Department of Food Technology, T.E.I. of Larissa
Greece
Dr. Mark Tarnopolsky
Mcmaster University
Canada
Dr. Sami A. Zabin
Al Baha University
Saudi Arabia
Dr. Julia W. Pridgeon
Aquatic Animal Health Research Unit, USDA, ARS
USA
Dr. Lim Yau Yan
Monash University Sunway Campus
Malaysia
Prof. Rosemeire C. L. R. Pietro
Faculdade de Ciências Farmacêuticas de Araraquara,
Univ Estadual Paulista, UNESP
Brazil
Dr. Nazime Mercan Dogan
PAU Faculty of Arts and Science, Denizli
Turkey
Dr Ian Edwin Cock
Biomolecular and Physical Sciences
Griffith University
Australia
Prof. N K Dubey
Banaras Hindu University
India
Dr. S. Hemalatha
Department of Pharmaceutics, Institute of
Technology,
Banaras Hindu University, Varanasi. 221005
India
Dr. J. Santos Garcia A.
Universidad A. de Nuevo Leon
Mexico India
Dr. Somboon Tanasupawat
Department of Biochemistry and Microbiology,
Faculty of Pharmaceutical Sciences,
Chulalongkorn University,
Bangkok 10330
Thailand
Dr. Vivekananda Mandal
Post Graduate Department of Botany,
Darjeeling Government College,
Darjeeling – 734101.
India
Dr. Shihua Wang
College of Life Sciences,
Fujian Agriculture and Forestry University
China
Dr. Victor Manuel Fernandes Galhano
CITAB-Centre for Research and Technology of AgroEnvironment and Biological Sciences, Integrative
Biology and Quality Research Group,
University of Trás-os-Montes and Alto Douro,
Apartado 1013, 5001-801 Vila Real
Portugal
Dr. Maria Cristina Maldonado
Instituto de Biotecnologia. Universidad Nacional de
Tucuman
Argentina
Dr. Alex Soltermann
Institute for Surgical Pathology,
University Hospital Zürich
Switzerland
Dr. Dagmara Sirova
Department of Ecosystem Biology, Faculty Of Science,
University of South Bohemia,
Branisovska 37, Ceske Budejovice, 37001
Czech Republic
Dr. Mick Bosilevac
US Meat Animal Research Center
USA
Dr. Nora Lía Padola
Imunoquímica y Biotecnología- Fac Cs Vet-UNCPBA
Argentina
Dr. Maria Madalena Vieira-Pinto
Universidade de Trás-os-Montes e Alto Douro
Portugal
Dr. Stefano Morandi
CNR-Istituto di Scienze delle Produzioni Alimentari
(ISPA), Sez. Milano
Italy
Dr Line Thorsen
Copenhagen University, Faculty of Life Sciences
Denmark
Dr. Ana Lucia Falavigna-Guilherme
Universidade Estadual de Maringá
Brazil
Dr. Baoqiang Liao
Dept. of Chem. Eng., Lakehead University, 955 Oliver
Road, Thunder Bay, Ontario
Canada
Dr. Ouyang Jinping
Patho-Physiology department,
Faculty of Medicine of Wuhan University
China
Dr. John Sorensen
University of Manitoba
Canada
Dr. Andrew Williams
University of Oxford
United Kingdom
Dr. E. O Igbinosa
Department of Microbiology,
Ambrose Alli University,
Ekpoma, Edo State,
Nigeria.
Dr. Chi-Chiang Yang
Chung Shan Medical University
Taiwan, R.O.C.
Dr. Hodaka Suzuki
National Institute of Health Sciences
Japan
Dr. Quanming Zou
Department of Clinical Microbiology and Immunology,
College of Medical Laboratory,
Third Military Medical University
China
Prof. Ashok Kumar
School of Biotechnology,
Banaras Hindu University, Varanasi
India
Dr. Guanghua Wang
Northeast Institute of Geography and Agroecology,
Chinese Academy of Sciences
China
Dr. Chung-Ming Chen
Department of Pediatrics, Taipei Medical University
Hospital, Taipei
Taiwan
Dr. Renata Vadkertiova
Institute of Chemistry, Slovak Academy of Science
Slovakia
Dr. Jennifer Furin
Harvard Medical School
USA
Dr. Julia W. Pridgeon
Aquatic Animal Health Research Unit, USDA, ARS
USA
Dr Alireza Seidavi
Islamic Azad University, Rasht Branch
Iran
Dr. Thore Rohwerder
Helmholtz Centre for Environmental Research UFZ
Germany
Dr. Daniela Billi
University of Rome Tor Vergat
Italy
Dr. Ivana Karabegovic
Faculty of Technology, Leskovac, University of Nis
Serbia
Dr. Flaviana Andrade Faria
IBILCE/UNESP
Brazil
Prof. Margareth Linde Athayde
Federal University of Santa Maria
Brazil
Dr. Guadalupe Virginia Nevarez Moorillon
Universidad Autonoma de Chihuahua
Mexico
Dr. Tatiana de Sousa Fiuza
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African Journal of Microbiology Research
International Journal of Medicine and Medical Sciences
Table of Content: Volume 9 Number 16, 22 April, 2015
ARTICLES
Control of psychrophilic microbiota in shrimp (Litopenaeus vannamei) by
Lactobacillus reuteri
Helen Silvestre da Silva, Marília Miotto, Karin de Medeiros and Cleide Rosana
Werneck Vieira
Antibacterial activity of Andrographis paniculata Nees against selective human
pathogens
V. Baby Shalini and J. Sriman Narayanan
Evaluation of rice landraces against rice root-knot nematode, Meloidogyne
graminicola
H. Ravindra, Mukesh Sehgal, H. B. Narasimhamurthy, H. S. Imran Khan and
S. A. Shruthi
Performance evaluation of laboratory professionals on tuberculosis microscopy at
Hawassa Town, Southern Ethiopia
Mengistu Hailemariam, Abebe Minuta, Gezahegn Bewoket, Tadesse Alehegn,
Yismake Worku and Moges Desta
Biosynthesis of indole-3-acetic acid by plant growth promoting rhizobacteria,
Klebsiella pneumonia, Bacillus amyloliquefaciens and Bacillus subtilis
Vijendra Kumar Mishra and Ashok Kumar
Assessment of phenol biodegradation capacity of indigenous bacteria isolated
from sewage treatment plant
N. R. Das and N. Kumar
Resistance of heavy metals on some pathogenic bacterial species
Aditi Singh, Maitreyi Mishra, Parul Tripathi and Shweta Sachan
Vol. 9(16), pp. 1118-1121, 22 April, 2015
DOI: 10.5897/AJMR2014.7167
Article Number: 6893CE752653
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Control of psychrophilic microbiota in shrimp
(Litopenaeus vannamei) by Lactobacillus reuteri
Helen Silvestre da Silva*, Marília Miotto, Karin de Medeiros and
Cleide Rosana Werneck Vieira
Department of Food Science and Technology, Federal University of Santa Catarina (UFSC), Av. Admar Gonzaga, 1346,
Itacorubi, 88034-001 Florianopolis SC, Brazil.
Received 2 October, 2014; Accepted 13 April, 2015
As an alternative to traditional methods for controlling microorganisms in food, bioprotection using
lactic acid bacteria is promising. Lactobacillus reuteri exhibits an ability to produce reuterin, an
antimicrobial substance presenting effects against Gram-positive and Gram-negative bacteria, as well
as molds, yeasts and protozoa. In the present study, the activity of L. reuteri culture and extract over
psychrophilic microbiota of Litopenaeus vannamei shrimp kept under refrigeration was evaluated.
Sterile extract of L. reuteri was able to reduce the counts of psychrophiles in 2 log cycles, while L.
reuteri culture kept the initial count during the whole storage period. L. reuteri was efficient in
controlling psychrophiles in shrimp suggesting its use as a food bioprotector.
Key words: Lactobacillus reuteri, reuterin, antimicrobial activity.
INTRODUCTION
Cultivation of marine shrimps is a new practice in Brazil.
It is fast growing because of technical and economic
factors in an appropriate price environment and demand
for national and international markets besides internal
stimulus to the development of this activity. This sector
has a strong development potential and became one of
the most important agropecuary activity for Brazilian
economy, mainly in the Northeastern region (BRDE,
2004).
Processing techniques in the national carciniculture,
generally, are not specialized. The operation starts after
finishing the shrimp collection (when shrimps are
removed from fattening tanks) and before commercialization. The product is processed in order to be proper
for buyer demand and, according to specialists, include a
simple procedure without aggregating much value
(BRDE, 2004).
Because of fish products are very perishable they must
be properly stored in order to keep sensorial quality and
shelf life. The decreasing in freshness of fish products
depends on several factors such as collection,
slaughtering and processing conditions.
Food industry always searches for alternative techniques for replacing the traditional methods of microorganisms control in food such as acidification, freezing,
drying, salting or using of chemical agents. Bioconservation can increase shelf life and food safety by using a
natural or controlled microbiota, mainly lactic acid
bacteria (LAB) (Hugas et al., 1995).
Reuterin is produced by Lactobacillus reuteri, a
heterofermentative species inhibiting the GI tract of
humans and animals. It is formed during the anaerobic
*Corresponding author. E-mail: helen_silves@yahoo.com.br. Tel: 55 47 88414749.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
da Silva et al.
growth of L. reuteri by the action of glycerol dehydratase
which catalyzes the conversion of glycerol into reuterin.
Reuterin has been chemically identified to be 3-hydroxy
propanol (β-hydroxyl propionaldehyde) a highly soluble,
pH- neutral compound which is in equilibrium with its
hydrated monomeric and cyclic dimeric forms. Reuterin
exhibits a broad spectrum of antimicrobial activity against
certain Gram-positive and Gramnegative bacteria, yeast,
fungi and protozoa. Spoilage organisms sensitive to
reuterin include species of Salmonella, Shigella,
Clostridium, Staphylocaccus, Listeria, Candida and
Trypanosoma (Mayur and Madhukar, 2014). Reuterin is a
hydrosoluble, active in a wide pH range and resistant to
proteolytic and lipolytic anzymes (El-ziney and Debevere,
1998). The mode of action is not fully understood, but
recent studies have demonstrated that the aldehyde
group of 3-HPA is mainly responsible for the antimicrobial
activity. Reuterin reacts with sulfhydryl groups of proteins
and small molecules inducing oxidative stress responses
(Montiel et al., 2014). The aim of present study was to
evaluate the control of psychrophilic microbiota from
Litopenaeus vannamei shrimp kept under refrigeration
conditions by Lactobacillus reuteri and its sterile extract.
MATERIALS AND METHODS
Microrganisms and growth conditions
L. reuteri ATCC 1428 was kept in MRS Broth with 20% glycerol at 20ºC. For the microorganism reactivation, 1 mL of stock culture was
transferred to 10 mL MRS broth and incubated at 37ºC for 24 h.
The strain had its antimicrobial activity characterized in a previous
study (Silva et al., 2010).
Production of L. reuteri cell free supernatant
Production of the cell free supernatant was carried out according to
Cleusix et al. (2008) with some modifications. One milliliter of the
reactivated culture was transferred to a new tube containing 10 mL
of MRS broth and incubated at 37ºC for 6 h. After incubation, the
whole volume was added to a flask containing 50 mL of MRS broth
and incubated at 37ºC for 12 h. Later, the 61 mL of culture was
added to a new flask containing 500 mL of MRS broth and
incubated at 37ºC for 24 h. Cells were, then, collected by
centrifugation (1500 x g, 10 min, 20ºC), washed in potassium
phosphate buffer (0.1 M, pH 7.0), resuspended in 300 mL of a
sterile aqueous solution containing glycerol (200 mm) and
incubated at 37°C for 3 h under anaerobic conditions
(Anaerogen®). Cells were collected by centrifugation (8000 x g, 10
min) and 150 mL of the supernatant were sterilized by membrane
filtration (0.22 µm, Millipore®, Merck, USA).
Evaluation of reuterin production
A fresh culture of reuterin-producing L. reuteri ATCC 1428 was
inoculated at 1% in 1 L of MRS broth and incubated anaerobically
at 37°C overnight. After growth, cells were harvested by
centrifugation (4500 × g, 5 min) and gently washed in sterile
aqueous solution of glycerol (100 mM). In order to produce reuterin
from glycerol, the obtained cell biomass was resuspended into 250
ml sterile aqueous solution of glycerol (100 mM), and resting cells
1119
were incubated under anaerobic conditions at 37°C for 3 h. After
centrifugation (6600 × g, 5 min), the resulting supernatant was
collected, filter-sterilized (0.22 μm) and maintained at −40°C for
subsequent experiments. The concentration of reuterin in the
supernatant was determined by a colorimetric method as described
by Lüthi-Peng et al. (2002). Acrolein (Fluka; Sigma-Aldrich Quimica
SA, Madrid, Spain) was used for obtaining the standard curve,
since 3-hydroxypropionaldehyde dehydrates in equimolar concentrations to acrolein. Standards were made diluting acrolein in
distilled water. Supernatants containing reuterin were diluted with
distilled water if necessary before the colorimetric reaction. All
determinations were carried out in triplicate.
Sample of shrimp
Samples of 1000 g of fresh and deshelled pacific white shrimp were
bought in the Public Market of Florianopolis, Brazil. Samples were
placed in isothermal boxes containing ice and took to the Food
Microbiology Laboratory (Florianopolis, Brazil) for analysis.
Treatment of samples with L. reuteri
Each sample was divided in portions of 300 g and each portion was
labeled according to the treatment: Treatment 1: portion added with
0.1 mL/g of L. reuteri culture; Treatment 2: portion added with 0.1
mL/g of L. reuteri cell free supernatant; Control: portion added with
0.1 mL/g of sterile distilled water. Each sample was analyzed in
relation to psychrotrophic count and further L. reuteri count for T1
samples: right after treatment (T0), 3 h after treatment (T3 h), 6 h
after treatment (T6 h), 24 h after treatment (T24 h) and 48 h after
treatment (T48 h). Samples were kept under refrigeration (±7ºC)
until analysis. All analyses were carried out in triplicate.
Microbiological determinations
Representative shrimp samples (25 g) were homogenized with 250
mL of sterile 0.1% (w/v) peptone solution in a Stomacher 400 (A. J.
Seward Ltd, London, UK). Decimal dilutions of shrimp
homogenates were prepared in sterile 0.1% (w/v) peptone solution.
L. reuteri counts were determined on duplicate plates of Rogosa
agar (Difco) and incubated at 37°C for 48 h under anaerobic
conditions, and psychrotrophic counts on Plate Count agar (Oxoid)
for five days at 7°C. Psychrotrophic counts in the T1 samples were
expressed as the difference between the total count of
psychrotrophic and Lactobacillus reuteri counts.
Statistical analysis
Results of psychrotrophic counts were submitted to analysis of
variance (ANOVA) and averages of treatments were analyzed by
Tukey’s test in order to verify differences among the treatments.
Confidence level was of 95%. Tests were performed using
STATISTICA 9.0 software (StaSoft).
RESULTS AND DISCUSSION
Standard count carried out at zero time showed that
samples present an initial contamination of about 106
CFU/g. According to Vanderzant et al. (1971), shrimp
collected in tropical waters contains counts ranging from
106 to 107 CFU/g. This result was confirmed by
1120
Afr. J. Microbiol. Res.
Figure 1. Count of psychrophilic microrganisms (Log 10) in shrimp
samples treated with L. reuteri culture (treatment 1) and L. reuteri
extract (treatment 2) for 48 h at 7°C.
Jeyasekaran et al. (2006) which evaluated the increasing
of the microbiota of pacific white shrimp stored under
different refrigeration and modified atmosphere conditions.
Moura et al. (2003), evaluating the microbiological quality
of pink shrimp commercialized in São Paulo, found
4
7
counts ranging from 1.1 x 10 to 3.0 x 10 CFU/g.
International
Commission
on
Microbiological
Specification for Food (ICMSF) (1998) established a limit
of 107 CFU/g of aerobic standard plate counts for frozen
raw crustaceae, with no limit for psychrotrophic
microorga-nisms. Brazilian legislation (Kirschink and
Viegas, 2004; ANVISA, 2010) did not present limits for
mesophilic aerobic and psychrotrophic plate counts in
fish products. However, it is well known that elevated
populations can reduce the shelf life of fish products
(Ruch, 1974).
Counts of psychrophile in control treatment ranged
from 6 to 7 log cycles during storage of shrimp at ±8ºC. In
can be noticed that refrigeration temperature was efficient
in controlling the microbial growth during 48 h, once
averages during the whole storage period were not
statistically different (p > 0.05).
According to Jeyasekaran et al. (2006), storage under
refrigeration in temperatures lower than 6ºC is sufficient
for controlling microbial growth for 72 h. After this period,
psychrotrophic bacteria grow fast, leading to the spoilage
of the product. In shrimps, such psychrotrophic microbita
includes Pseudomonas sp. and Aeromonas sp.
Analyzing the effect of L. reuteri culture (Treatment 1)
over psychrotrophiles in shrimps, it was observed an
increasing on counts of these microorganisms after 3 h of
treatment. L. reuteri culture added to the sample could
influence this count. A slight decrease in psychrophile
counts was observed after 6 h, although results did not
present statistical differences (p>0.05) when comparing
the test sample and the control. After 48 h, microbial
counts were similar to those found for control. It can be
explained by a probable inability of L. reuteri in growing
and producing antimicrobial substances in temperatures
under 10°C. In this way, the effect observed at 6 and 24 h
can be due to a small amount of an antimicrobial
substance produced during the growth, once L. reuteri
can produce small quantities of reuterin at anaerobic
conditions (Ruch, 1975; Tomé, 2006). The effect of
treatment 1 compared to the control during 48 h is
presented in the Figure 1.
Reuterin concentration in cell free supernatant from L.
reuteri, obtained after incubation in 100 mM glycerol, was
approximately 65 mM, as determined by the method
described by Lüthi-Peng et al. (2002). The activity of L.
reuteri cell free supernatant over shrimp microbiota
(Treatment 2) presented singular positive results (Figure
1), with statistical analysis demonstrating differences
between the averages of treatment and control for T6 (p
= 0.002), T24 (p = 0.0002) and T48 (p = 0.0009). This
result shows the efficiency of the L. reuteri cell free
supernatant in reducing and keeping the counts of
psychrophiles low and suggesting an extension in
product shelf life.
Several authors suggest that species of Lactobacillus
genera are the principal antagonists of pathogenic and
spoiling microorganisms in fish products (Lyhs, 2001;
Suárez, 2008). Suárez et al. (2008) evaluated the effect
of the bacteriocin produced by Lactobacillus plantarum
over mesophilic and psychrotrophic bacterial populations,
coliforms at 35°C and thermotolerant coliforms present in
Piaractus brachypomus fillets.
Authors verified that the initial population of
psychrotrophic cycles regarding to control. Souza et al.
(2006) evaluated the reduction of total mesophiles during
fermentation of bonito-da-barriga-listrada using L. sakei
8
as starter culture. Authors reported a decreasing from 10
4
CFU/g to 10 CFU/g for total mesophiles.
The inhibitory effect of reuterin produced by L. reuteri
da Silva et al.
has been investigated in several in vitro studies. Also,
studies applying this antimicrobial substance in food have
been reported. El-ziney and Debevere (1998) reported
the inhibitory effect of reuterin against Listeria
monocytogenes and Escherichia coli O157:H7 in milk
and cottage cheese artificially contaminated.
Arques et al. (2004) reported that reuterin added to milk
at 8 UA/mL exhibited a bacteriostatic effect on L.
monocytogenes in treated milk at 37°C with no regrowth
of the pathogen, fact already observed in a previous
study. L. monocytogenes was completely inactivated
within five days at ±7°C in milk with reuterin at 150
UA/mL (5). According to these authors, the inactivation
rate depends on the concentration of reuterin.
Arques et al. (2007) reported that the presence of
reuterin in milk leads to a decreasing in S. enteric and Y.
enterocolitica counts after 12 days while C. jejuni and A.
hydrophila were completely inactivated after 76 and 12
days, respectively. In this work, authors suggest that
reuterin is very effective when applied to foodstuffs stored
under refrigeration.
According to Spinler et al. (2008), reuterin is produced
during fermentation of glycerol. In this way, it can be
suggested that the inhibitory action of the L. reuteri
extract produced through glycerol fermentation (treatment
2) is due to the presence of reuterin.
The use of reuterin in food can be more explored
considering its efficiency and the safety for human health,
once L. reuteri is an autochthon bacterium from gastrointestinal tract and known a safe for ingestion as probiotic
(Spinler et al. (2008).
Conclusions
L. reuteri exhibited the ability to reduce psychrotrophic
microbiota in pacific white shrimp stored under
refrigeration and can be used as a bioprotector in food.
ACKNOWLEDGEMENT
The National Counsel of Technological and Scientific
Development (CNPq) for financial support during this
assignment.
Conflict of interest
The authors did not declare any conflict of interest.
REFERENCES
Arques JL Fernandez J, Gaya P, Nunez M, Rodriguez E, Medina M
(2004). Antimicrobial activity of reuterin in combination with nisin
against food-borne pathogens. Int. J. Food Microbiol. 95(2):225-229.
Arques JL, Nunez M, Rodriguez E, Medina M (2007). Antimicrobial
Activity of Nisin, Reuterin, and the Lactoperoxidase System on
Listeria monocytogenes and Staphylococcus aureus in Cuajada, a
Semisolid Dairy Product Manufactured in Spain. J. Dairy Sci. 91:7075.
1121
Cleusix V, Lacroix C, Vollenweider S, Le Blay G (2008). Glycerol
induces reuterin production and decreases Escherichia coli
population in na in vitro model of colonic fermentation with
immobilized human feces. FEMS Microbiol. Ecol. 63:56-64.
El-Ziney MG, Debevere JM (1998). The effect of reuterin on Listeria
monocytogenes and Escherichia coli O157:H7 in milk and cottage
cheese. J. Food. Protect. 61:1275-1280.
Hugas M, Garriga M, Aymerich MT, Monfort JM (1995). Inhibition of
Listeria in dry fermented sausages by the bacteriocinogenic
Lactobacillus sakei CTC494. J. Appl. Microbiol. 79(3):322-330.
ICMSF (1998). Pescados y productos derivados. In: Microorganismos
de los alimentos. Ecología microbiana de los productos alimentares.
Zaragoza: Acribia, 121-66.
Jeyasekaran G, Ganesan P, Jeya SR, Maheswari K, Sukumar D
(2006). Quantitative and qualitative studies on the bacteriological
quality of Indian white shrimp (Penaeus indicus) stored in dry ice. Int.
J. Food Microbiol. 23:526-533.
Kirschink PG, Viegas EMM (2004). Changes in quality of freshwater
prawn Macrobrachium rosembergii during storage in ice. Ciencia
Tecnol. Alime. 24(3):p. 407-412.
Lüthi-Peng Q, Schärer S, Puhan Z (2002). Production and stability of 3hydroxypropionaldehyde in Lactobacillus reuteri. Appl. Microbiol. Biot.
60p.
Lyhs U, Lahtinen J, Fredriksson-Ahomaa M, Hyytia-Trees E, Elfing K,
Korkeala H (2001). Microbiological quality and shelf-life of vacuumpackaged gravad rainbow trout stored at 3 and 8°C. Int. J. Food
Microbiol. 70:221-230.
Mayur S, Dinesh RM, Madhukar S (2014). Role of Lactic Acid Bacteria
as Probiotics in Health and Disease. Prensa Med. Argent. 100:4.
Montiel R, Martín-Cabrejas I, Langa S, El Aouad, N, Arqués JL, Reyes
F, Medina M (2014). Antimicrobial activity of reuterin produced by
Lactobacillus reuteri on Listeria monocytogenes in cold-smoked
salmon, Food Microbiol. p. 44.
Moura AFP, Mayer MD, Landgraf M, Tenuta AF (2003). Qualidade
química e microbiológica de camarão-rosa comercializado em São
Paulo. Braz. J. Pharm. Sci. 39:203-208.
Regional Development Bank of the Extreme South-BRDE (2004).
Agency of Florianopolis. Management Planning. Shrimp farming in
Santa Catarina: overview, reproduction and larval rearing.
Florianópolis: BRDE. 101 p.
Ruch FE, Lengeler J, Lin ECC (1975). Independent constitutive
expression of the aerobic and anaerobic pathways of glycerol
catabolism in Klebsiella aerogenes. J. Bacteriol. 124:348-352.
Ruch FE, Lengeler J, Lin ECC (1974). Regulation of glycerol catabolism
in Klebsiella aerogenes. J. Bacteriol. 119:50-56.
Silva HS, Ramos RJ, Miotto M, Cirolini A, Vieira CRW (2010). Atividade
antimicrobiana de Lactobacillus reuteri contra bactérias de interesse
alimentar. Rev. Inst. Adolfo Lutz, [S.l.], 69(4): 584-587. ISSN 19833814.
Souza J, Rodrigues LGG, Gonzalez PNM, Tortato R, Carbonea N,
Espírito Santo MLP (2006). Atividade antimicrobiana do Lactobacillus
sakei na fermentação do bonito-de-barriga-listrada (Euthynnus
pelamis). VETOR. 16:25-36.
Spinler JK, Taweechotipatr M, Rognerud CIL, Ching NO, Tumwasorn S,
Versalovic J (2008). Human-derived probiotic Lactobacillus reuteri
demonstrate antimicrobial activities targeting diverse enteric bacterial
pathogens. Anaerobe 14:166-171.
Suárez HM, Francisco A, Beirão LH (2008). Influencia de bacteriocinas
producidas por Lactobacillus plantarum LPBM10 sobre la vida útil de
filetes del híbrido de cachama Piaractus brachypomus x Colossoma
macropomum empacado al vacío. VITAE-COLUMBIA 15: 32-40.
Tomé E, Teixeira P, Gibbs PA (2006). Anti-listerial inhibitory lactic acid
bacteria isolated from commercial cold smoked salmon. Int. J. Food
Microbiol. 23:399-405.
Vanderzant C, Cobb BF, Thompson CA, Parker JC (1971). Microbial
flora, chemical characteristics and shelf life of four species of pond
reared shrimp. J. Milk Food Technol. 36:443-449.
Vol. 9(16), pp. 1122-1127, 22 April, 2015
DOI: 10.5897/AJMR2015.7515
Article Number: 7EAC54352655
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Antibacterial activity of Andrographis paniculata Nees
against selective human pathogens
V. Baby Shalini* and J. Sriman Narayanan
Department of Microbiology, Faculty of Science, Annamalai University, Annamalainagar- 608002, Cuddalore District,
Tamil Nadu, India.
Received 6 January, 2015; Accepted 13 April, 2015
The present study was conducted to investigate the antibacterial effect of Andrographis paniculata
against selective human pathogens. The plant parts of A. paniculata such as leaf, stem and root were
studied for its antibacterial activity. Different solvents were used to extract the active components from
the plant parts. The antibacterial activity was studied against selective human pathogens viz.,
Staphylococcus sp., Escherichia coli, Salmonella sp. and Pseudomonas sp. Among the different
solvents, methanol extract showed greater antibacterial activity against E. coli (32.8 mm) followed by
Salmonella Typhi (24.7 mm), Pseudomonas sp. (24.2 mm) and Staphylococcus sp. (18.4 mm). So the
future investigation was carried out for leaf extract using methanol as solvent to find out the minimum
inhibitory concentration for selective human pathogens. The results reveal that 75 µl was optimum for
all the test cultures. It shows more activity in E. coli (32.3 mm) followed by S. Typhi (28.1 mm),
Staphylococcus sp. (14.1 mm) and Pseudomonas sp. (13.4 mm).
Key words: Andrographis paniculata, Antibacterial, Andrographolide, minimum inhibitory concentration.
INTRODUCTION
Medicinal plants have grown enormously from the use of
herbal products as natural cosmetics and as selfmedication by the general public scientific for their
beneficial effects (Sharma and Joshi, 2011). In olden
ages, antibiotics were produced mostly from the leaves
and roots of medicinal plants. The extracts of many plant
species have become popular in recent years and
attempts have taken to characterize their bioactive
principles which gained scope for various pharmaceutical
food processing and medical application. Andrographis
paniculata is an herbaceous plant in the family
Acenthaceae, native to India and Srilanka. In North-
Eastern India the plant is known as Maha-tita literally
“King of bitters”, known as various vermicular names
(Abhishek et al., 2010). The Tamil has been using
Nilavembu-as it is called in Tamil – for centuries.
In Siddha medicine A. paniculata used widely to treat
fever like ckikunguny, swine –flu, typhoid, snake bite and
common cold etc. (Dhiman et al., 2012). It is an annual
herb. The leaves are used traditionally in Asian traditional
medicine and particularly in Ayurveda for treatment of
various diseases and illness. The plant is cultivated in
many areas, as well. It grows well in a sunny location.
The seeds are sown during May and June.
*Corresponding author. E-mail: microshal@yahoo.com. Tel: 9025958726.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
Shalini and Narayanan
The seedlings are transplanted at a distance of 30 × 60
cm. The seeds are small and remain dormant for 5-6
months. If possible seedlings should be raised in shade
to protect them from heat (Seema et al., 2002). It is
distributed in tropical Asian countries, often in isolated
patches. It can be found in a variety of habitats, such as
plains, hillsides, coastlines, roadsides, farms, and
wastelands (Prajapati et al., 2003). Approximately, 28
species of are known and indigenous to Asia. The
species also found in Hong Kong, Thailand, Brunei,
Singapore, and other parts of Asia, where it may or may
not be native. It is widely cultivated in southern and South
Eastern Asia to treat infections and some diseases. A.
paniculata grows erect to a height of 30 - 110 cm in
moist, shady places. The slender stem is dark green. The
lance-shaped leaves have hairless blades measuring 8
cm long by 2.5 wide. The small flowers are borne in
spreading racemes. The fruit is a capsule around 2 cm
long and a few millimeters wide. It contains many yellowbrown seeds.
A. paniculata is also used for other medical purpose;
for example, digestive problems, blood cleanser, fever,
sore throat (Sharma and Joshi, 2011). A. paniculata is
used to cure fever and cold (Koul and Kapil, 1994). It is
one of the best anti-malarial agent compared to the
commercial product of quinine (Parvataneni et al., 2010).
The herb has shown an ability to reduce inflammation
(heat) and fight viral infections and is used as a principal
ingredient in traditional Chinese medicinal formulas for
lung support from colds (Sheeja et al., 2006). A.
paniculata is a blood purifier, so it is used to cure turbid
liver, jaundice, dermatological diseases, dyspepsia,
febrifuge and anhelhemic. A. paniculata acts to dispel
heat and remove toxin. Andrographaloid was found to be
more potent and a standard hepato protective agent
(Visen et al., 1993). The whole plant of A. paniculata is
used extensively as an anti-inflammatory and antipyretic
drug for the treatment of laryngitis, diarrhea. The juice of
fresh leaves generally contains andrographolide. It is
used as a domestic remedy in the treatment of colic pain,
loss of appetite, irregular stools and diarrhea (Mishra et
al., 2007). Since ancient times, A. paniculata has been
known in traditional Asian medicine as an immune
system booster, to treat infections in the gastrointestinal
tract and upper respiratory tract, harps, sore throat and a
variety
of
other
chronic
infectious
diseases
(Wangboonskul et al., 2006). Nowadays microbes are
resistance to various antibiotics. The resistant of
microbes is due to indiscriminate utilization of commercial
antimicrobial medicines supported by many scientists
investigation for modern antimicrobial substances from
several medicinal plants (Alagesaboopathi and Kalaiselvi,
2012). Most of the researchers concentrate on screening
and minimum inhibitory concentration (MIC) of extracts of
plants rather than identifying compounds with activity. In
this attempt, we isolated active constituents of the plant
1123
and screened for antimicrobial activity which can be used
further in research to develop antimicrobial compounds
with the isolated compounds or their synthetic analogues
reported by Mohamed et al. (2013). The present
investigation was carried out to study the antibacterial
potential of extract of A. paniculata against selective
human pathogens.
MATERIALS AND METHODS
Collection of plant material
The whole plant of A. paniculata was collected from the trial plots at
campus of Faculty of Agriculture, and verified by Dr. Manivannan
Professor and Head, Department of Horticulture Annamalai
University, Annamalai Nagar, Chidambaram, Tamil Nadu.
Fresh and healthy leaf, stem, and root were used to extract
bioactive fractions of A. paniculata. The parts of plants which were
used for the extract were washed with water to remove soil and
dust particles. Then they were dried under shaded place. Dried
materials were blended to form a fine powder and store in airtight
bottles (Mneenusarma and Sumanjoshi, 2011).
Test organisms
The human pathogens viz., Staphylococcus sp., E. coli, S. Typhi
and Pseudomonas sp., were collected from Jayasekaran hospital,
Nagarcoil, Tamil Nadu, India. The test culture was maintained in
Nutrient agar slant at 4C for further studies.
Preparation of plant extract
The shade dried coarse powder of the leaf stem and root of A.
paniculata were extracted using 250 ml solvent of ethanol, acetone,
methanol and water with the help of soxhlet apparatus. The extracts
were auto-calved to determine the stability of the crude extracts at
the temperature of 121C for 15 min and then the extracts were
stored at 4C for further use (Daniyan and Mohammed, 2008).
Assay of antibacterial activity using the agar well diffusion
method
An agar-well diffusion method was employed for determination of
antibacterial activities. The freeze- dried extract samples of spices
and herbs were dissolved in phosphate buffered saline (PSB, pH
7.0 to 7.2) to the final concentration of 100 mg/ml and sterilized by
filtration through 0.22 µm sterilized Millipore express filter. All
bacteria were suspended in sterile water and diluted to 10 -6
CFU/ml. The suspension (100 µl) was spread onto the surface of
NA medium. Agar wells (4.6 mm in diameter) were cut from the
agar with a sterile borer and 60 µl extract solutions were delivered
into them. The inoculated plates were incubated at 37C for 24 h.
Antibacterial activity was evaluated by measuring the diameter of
inhibition zone of the tested bacteria.
RESULT
The antibacterial activity of different plant parts of A.
paniculata viz., root, stem and leaf were investigated
1124
Afr. J. Microbiol. Res.
Table 1. Antibacterial activity of Andrographis paniculata active against selective human pathogens.
Zone of inhibition (mm) of human pathogens
Staphylococcus sp.
E. Coli Salmonella Typhi Pseudomonas sp.
11.3
16.7
15.2
11.2
12.7
17.3
18.7
14.3
13.2
22.8
18.7
13.8
10.3
14.7
11.7
11.1
Plant parts of Andrographis
paniculata
Solvent used
Root
Ethanol
Acetone
Methanol
Water
Stem
Ethanol
Acetone
Methanol
Water
8.7
13.4
16.6
10.2
11.2
17.6
29.2
10.3
23.3
19.4
18.8
13.3
9.1
9.2
19.6
7.5
Leaf
Ethanol
Acetone
Methanol
Water
9.2
14.9
18.4
8.9
19.4
25.2
32.8
11.9
25.2
28.6
24.7
11.9
18.9
21.5
24.2
16.1
using agar well diffusion method against some of the
selected human pathogens such as Staphylococcus sp.,
E. coli, S. Typhi, and Pseudomonas sp. The extracts
were prepared using various solvents such as ethanol,
acetone, methanol and water. All the examined extract
showed antibacterial activity against human pathogens
(Table 1). Figure 1 depicts the antibacterial activity of leaf
extract of Andrographis paniculata against E. coli.
The antibacterial activity of A. paniculata root extract of
ethanol showed maximum zone of inhibition (16.7 mm)
for E. coli, the minimum zone of inhibition (11.2 mm) for
Pseudomonas sp. Then the activity of root extract of
acetone showed maximum zone of inhibition (18.7 mm)
for Salmonella typhi, the minimum zone of inhibition (12.7
mm) for Staphylococcus sp. The activity of root extract of
methanol showed maximum zone of inhibition (22.8 mm)
for E. coli and the minimum zone of inhibition (13.2 mm)
for Staphylococcus sp. Then, the solvent of water
showed maximum zone of inhibition (14.7mm) for E. coli
and the minimum zone of inhibition (10.3 mm) for
Staphylococcus sp.
The activity of stem extract of ethanol showed
maximum zone of inhibition (23.3 mm) for S. Typhi, and
minimum zone of inhibition (8.7 mm) for Staphylococcus
sp. Then the activity of stem extract by acetone showed
maximum zone of inhibition (19.4 mm) for S. Typhi and
the minimum zone of inhibition (9.2 mm) for
Pseudomonas sp. The activity of stem extract of
methanol showed maximum zone of inhibition (29.2 mm)
for E. coli and the minimum zone of inhibition (16.6 mm)
for Staphylococcus sp., the antibacterial activity of stem
extract of water showed maximum zone of inhibition
(13.3 mm) for S. Typhi, and the minimum zone of
inhibition (7.5 mm) for Pseudomonas sp.
The activity of leaf extracts of ethanol showed
maximum zone of inhibition (25.2 mm) for Salmonella
typhi and the minimum zone of inhibition (9.2 mm) for
Staphylococcus sp. Then the activity of leaf extracts of
acetone showed maximum zone of inhibition (28.6 mm)
for S. Typhi and the minimum zone of inhibition (14.9
mm) for Staphylococcus sp. The activity of leaf extracts
of methanol showed maximum zone of inhibition (32.8
mm) for E. coli and the minimum zone of inhibition (18.4
mm) for Staphylococcus sp. The antibacterial activity of
leaf extracts of water showed maximum zone of inhibition
(11.9 mm) for Salmonella typhi and E. coli the minimum
zone of inhibition (8.9 mm) for Staphylococcus sp.
The result reveals that methanol based leaf extract was
more effective when compared with other extracts and for
all solvent used, methanol was best. So, further,
minimum inhibitory concentration was studied for
methanol extract of leaves. The different concentrations
viz., 0, 25, 50, 75 and 100 µl were taken. Then, the well
diffusion method was followed for all the pathogens
(Table 2).The result reveals that 75 µl was optimum for all
the test cultures and it was found to have more activity for
E. coli (32.3 mm) followed by S. Typhi, (28.1 mm),
Staphylococcus sp., (13.1 mm) and Pseudomonas sp.,
(14.1 mm). Figure 2 depicts the minimum inhibitory
concentration of methanolic leaf extract of A. paniculata
against E. coli.
DISCUSSION
Infectious diseases are a major cause of morbidity and
mortality worldwide. Currently, the ongoing battle against
bacteria prevails certainty of evolving, resistance. On the
Shalini and Narayanan
1125
Ethanol
Acetone
Control
Water
Methanol
Figure 1. Antibacterial activity of Andrographis paniculata active against
selective human pathogens of E.coli.
Table 2. Minimal Inhibitory Concentration of Andrographis paniculata.
Methanol (µl)
0
25
50
75
100
Zone of inhibition of the human pathogen
Staphylococcus sp. E. coli Salmonella sp. Pseudomonas sp.
10.0
25.3
23.7
11.3
11.1
26.0
25.3
12.3
12.0
26.8
25.6
13.0
14.1
32.3
28.1
13.4
13.3
32.5
28.8
14.5
other hand, advancement in medical field results in more
patients being in critical and immune suppressed states,
thus creating a perpetual need for new antibiotics. As a
result, it is the right time to discover new antibiotics
(Mahesh and Satish, 2008). A. paniculata has a several
water soluble lactone andrographoloidic properties.
Medicinal plants are more important in field of
pharmaceutical industries for new drug preparation (Sule
et al., 2010).
Maximum zone of inhibition was recorded with 75 µl
methanol extract against S. aureus, in accordance with the
previous studies reporting that 75 µl methanol is better
than other solvent for antibacterial activity (Pushpendra
Kumar Mishra et al., 2013). Therefore, only the 75 µl of
methanol extract of A. paniculata leaves were used for
further experiments. The present study mainly focused on
assaying the efficiency of different plant parts viz., root,
stem and leaf of Andrograpish paniculata using various
solvent extraction procedures against the selected
human pathogens.
In the preliminary screening of antibacterial activity of
methanol leaf extract of Andrographis paniculata
exhibited maximum activity when compared with other
plant parts and also from different solvent extracts
(Monoharan and Monoharan, 2013). The maximum
activity was observed for the pathogens E. coli followed
by Salmonella typhi, Staphylococcus sp. and
Pseudomonas sp.
The methanolic extracts of Andrographis paniculata at
the highest concentration showed the strongest bacterial
1126
Afr. J. Microbiol. Res.
50 μl
75 μl
0 μl
25 μl
100 μl
Figure 2. Minimal Inhibitory Concentration of Andrographis paniculata.
inhibitory activity of other extracts. This similar
observation reported by many researchers (Negi et al.,
2005; Parekh and Chanda, 2010; Al-Bayati, 2008;
Kaushik and Goyal, 2011).
The methanol leaf extract was studied for minimal
inhibitory concentration concept for various concentrations viz., 0, 25, 50, 75 and 100 µl. Among the different
concentrations, 75 µl showed maximum activity for all the
pathogenic organisms and recorded highest for E. coli
(32.3 mm) and it was on par with 100 µl concentrations.
From the result, it was revealed that plant extracts of A.
paniculata showed strongest antimicrobial activity for a
wide range of pathogens and also more reliable than
commercially available antibiotics.
Conclusion
A. paniculata has been used in Ayurveda, Unani and
Siddha systems of medicine from ancient times. It has
wide spectrum of pharmacological activities either in the
form of powder, extracts or in its isolated compounds with
minimum side effects; several products fortified with
extract or isolated compounds have been launched in
national and international markets for various diseases.
In this context, the present study was carried out to find
out the antibacterial potential of various solvent based
extract of A. paniculata against selective human pathogens. The antibacterial activity of A. paniculata may be
due to the presence of active principle called
andrographoloid. In future, the improvements of active
principle andrographoloid content in A. paniculata using
plant growth promoting rhizobacteria (PGPR) are studied.
The enriched A. paniculata plant extracts are further
studied for their antibacterial properties against selective
human pathogens.
Conflict of interest
The authors did not declare any conflict of interest.
ACKNOWLEDGEMENTS
The authors are very much grateful to the authorities of
Annamalai University for providing the facilities. We also
thank Dr. K. Manivannan, Professor and Head,
Department of Horticulture for confirming the plant
sample.
REFERENCES
Abhishek NSK, Tewari, Alok Lehri (2010). Biological activities of
Kalmegh (Andrographis paniculata Nees) and its active principles –A
review. Indian J. Nat. Products Resour. 1(2):125-135.
Al-Bayati FA (2008). Synergistic antibacterial activity between Thymus
vulgaris and Pimpinella anisum essential oils and methanol extracts.
J. Ethnopharmacol. 116:403-406.
Shalini and Narayanan
Alagesaboopathi C, Kalaiselvi N (2012). Antimicrobial activities of the
root, stem and leaf extracts of Argemone mexicana L. Int. J.
Biosci.2(5):61-68.
Daniyan SY, Muhammad HB (2008). Evaluation of the antimicrobial
activities and phytochemical properties of extracts of Tamaridus
indica against some diseases causing bacteria. Afr. J. Biotechnol.
7(14):2451-2453.
Dhiman A, Goyal J, Sharma KA, Dhiman S (2012). A Review on
Medicinal Prospectives of Andrographis paniculata Nees. JPSI. pp.
1-4.
Kaushik P, Goyal P (2011). “Evaluation of various crude extracts of
Zingiber officinale rhizome for potential antibacterial acitivity: a study
in vitro. Advance Microbiol. 1:7-12.
Koul IB, Kapil A (1994). Effect of diterpenes from Andrographis
paniculata on anti-oxidant defence system and lipid peroxidation. Ind.
J. Pharmacol. 26:296-300.
Mahesh B, Sathis S (2008). Antimicrobial activity of some important
medicinal plants against plant and human pathogens. World J. Agric.
Sci. 4:839-843.
Mishra SK, Sangwan NS, Sangwan RS (2007). Andrographis paniculata
(Kalmegh): A Rev. Pharmacog. Rev. 1(2):283-298.
Monoharan S, Monoharan E (2013). Medicinal and pharmacological
properties of Andrographis paniculata Int. J. Bio. Mol. Biomed. 3(2):112.
Negi PS, Chauhan AS, Sadia GA, Rohinishree YS, Ramteke RS (2005).
Antioxidant and antibacterial activities of various seabuckthorn
(Hippophaerhamnoides L.) seed extracts. Food Chemistry 92:119124.
Parekh J, Chanda S (2010). Antibacterial and phytochemical studies on
twelve species of Indian medicinal plants. Afr. J. Biomed. Res.
10:175-181.
Parvataneni R, Koduru RL (2010). Antimicrobial activity of the
chloroform extracts of the root and the stem Andrographis paniculata
Nees. Int. Res. J. Microbiol. 1(2):37-39.
Prajapati ND, Purohit SS, Sharma AK, Kumar TA (2003). Handbook of
medicinal plants, A complete source book, Agrobios. Jodhpur. India.
pp. 45-46.
Pushpendra KM, Rahul KS, Anamika G, Adya C, Rahul P, Shree PT,
Tribhuban Mohan M (2013). Antibacterial activity of Andrographis
paniculata (Burn.f.) Wall ex Nees leaves against clinical pathogens.
JPR. pp. 459-462.
1127
Seema N, Mohod NB, Wankhade SG, Paturde JT (2002). Effect of
plantanig and harvesting dates on yield and quality of kalmegh
(Andrographis paniculata). JMAPS. 25:981-983.
Sharma M, Joshi S (2011). Comparison of anti-oxidant of Andrographis
paniculata and Tinosporacordifolia leaves. J. Curr. Chem. Pharm. Sc.
1(1):1-8.
Sheeja K, Shihab PK, Kuttan G (2006). Antioxidant and antiinflammatory activities of the plant Andrographis paniculata Nees.
Immunopharmacol. Immunotoxicol. 28:129-140.
Sule A, Ahmed QU, Samah OA, Omar MN (2010). Screening for
Antibacterial Activity of Andrographis paniculata Used in Malaysian
Folkloric Medicine: “A Possible Alternative for the Treatment of Skin
Infections”. Ethnobotanical Leaflets 14:445-56.
Visen PK, Shukla B, Patnaik GK, Dhawan BN (1993). Andrographolide
protects rat hepatocytes against paracetamol induced damage. J.
Ethanopharmacol. 40 (2):131-136.
Wangboonskul J, Daodee S, Jarukamjorn K (2006). Study of
Andrographis paniculata tabulates in healthy thaimale volunteers.
Thai Pharm Health. Sci. J. 1(3):209-218.
Vol. 9(16), pp. 1128-1131, 22 April, 2015
DOI: 10.5897/AJMR2014.7257
Article Number: 59A896652657
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Evaluation of rice landraces against rice root-knot
nematode, Meloidogyne graminicola
H. Ravindra1*, Mukesh Sehgal2, H. B. Narasimhamurthy1, H. S. Imran Khan1 and S. A. Shruthi1
1
Zonal Agricultural and Horticultural Research station, University of Agricultural and Horticultural sciences, Shimoga,
577225, Karnataka, India.
2
Natinal Centre for Integrated Pest Management, LBS Building Pusa Campus, New Delhi, -110012, India.
Received 7 July, 2014; Accepted 13 April, 2015
Of late, rice root-knot nematode Meloidogyne graminicola has become a serious menace in all type of
rice situations in India. A field study was under taken during kharif-2013 to evaluate 135 landraces (local
cultivars) collected and maintained at Organic Farming Research centre of ZAHRS, Navile, Shimoga
against Meloidogyne graminicola. The result reveals that the varieties show varying degrees of
responses. Out of 135 cultivars, 32 cultivars were found to be highly resistant, while, 45 varieties read
were resistant. However, 40 varieties were evaluated to be moderately resistant and nine varieties
susceptible. The remaining nine cultivars were learnt to be highly susceptible.
Key word: Rice, root-knot nematode.
INTRODUCTION
Rice is an important cereal crop of India and is the
second most staple food crop of the world next to wheat
and staple food for two thirds of world’s population
(Abodolereza and Racionzer, 2009). More than 90% of
the world’s rice area is in Asia, which is the home for
more than half of the world’s poor, and more than half of
the world’s rice cultivators (Rao et al., 2010).
Meloidogyne graminicola is known to infect and cause
serious damage to cereals, especially rice, in many
countries (Port and Matias, 1995; Padgham et al., 2004).
In India, M. graminicola has been found in Assam,
Andhra Pradesh, Karnataka, West Bengal, Orissa,
Kerala, Tripura and Madhya Pradesh (Prasad et al.,
1987). It is not only a serious problem in nurseries and
upland rice but also found to be widespread in the
deepwater and irrigated rice in many states of India
(Prasad et al., 1985; MacGowan, 1989; Jairajpuri and
Baqri, 1991). Yield loss up to 50% might be incurred due
to severe infestation of M. graminicola in upland, rainfed
and direct seeded rice (Lorenzana et al., 1998). The use
of resistant cultivars is a low cost and sustainable option
for the control of nematodes in the long term which does
not impose unwanted changes in traditional agronomic
practices (Amoussou et al., 2004).
Rice root-knot nematode appeared in devastating form
in parts of major rice growing areas of Shimoga during
2001, which was a first report from Karnataka and
subsequently, reported from Mandya district of the state
(Krishnappa et al., 2001). Severe outbreak of M.
graminicola is also observed in Shimoga, Karnataka
*Corresponding author. E-mail: ravindranema@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
Ravindra et al.
1129
Table 1. Root-Knot Index 0 to 5 scales for Meloidogyne spp.
Grade
0
1
2
3
4
5
Description
No galls
1-2 galls / root system
3-10 galls / root system
11- 30 galls / root system
31-100 galls / root system
>100 galls / root system
Reaction
Immune
Resistant
Moderately resistant
Moderately susceptible
Susceptible
Highly susceptible
Table 2. Reaction of Rice landraces against Meloidogyne graminicola.
Reaction
Highly
resistant
Resistant
Moderately
resistant
Susceptible
Highly
susceptible
Root-knot
Index
Varieties
Nazar bad, Bheemasaale, Nazarbaik, Kichadi samba, Kichadi samba, kattaru, B.P.T, Mouruda,
Bagashaparimalaakki, Kempukaalu, Malgudisanna, Delhibogabhattha, Sannakki, Padma rekha, Chitiga,
Sharavathikempu, Andrabasumati, Orrisabhattha, Kasubai, Bangarakaddi, KH-4, Meese bhattha,
Kemputadi, Kavadhari, Jeerige samba, Tunuru, Dappavalya, Rasakadari, Nagabhatha, Baiganmanja, Bud
bhattha, Siddasanna.
Chinnaponni, Bangarasanna, Jasmine, H.M.T,
Siggikaimai, Sannavallya, Kalalajeera, Aravatellu,
Karijeddu,
vijayanandeda,
Raichursanna,
Bangarugundu,
Yelatakkigidda,
hasudi,
Marudi,
Aanekombinabhattha,
Sambhamasana,
Sannamuttige, Karigajavilai,
Bilidaddibudda,
Kagga,
kuduvekalaje, Hole saala, Chippiga, H.M.T, Dappabhattha, Kempudaddegidda, Gandhasaale, KA-1,
Eppattu, Manja kai mai, Sasti, Kari basumati, kallunundigar, Malabar, bilijeddu, Neragulabhattha, Bangla
rice, Madaikar, Togarsi, maranellu, Champakali, Red jasmine,Doddiga, Netibhattha, Baredabinasaale.
Yedikani, Kundapulan, Kempujeddu, Kiruvagi Jaya, Biriya black, Mukkanna, Bagyajyothi, Selamsanna,
Valaiponni, Puttabhattha, Kagisale, Rajbhoga, Sughandhi, Kerekalumuttiga, guddapairunellu, Gangadalai,
SampigeGilisaale, Ulkad rice, Honnasu, Akkalu, Madras sanna, Raja kai mai, Deepak rani, Bettadhayam,
Navara, Karikandaga, Anadatumba, Ratnachudi, Mulabhattha, Marasumallige, Ambamohir, Kongalli,
Navalasaale, Raja mani, Sondakar, Coimatorsanna, Mara bhatha, Allure sanna.
Aadribhattha, Gowrisanna, Sannamundaga, Pusasughandhi, Ujagunda, Karimundaga, Kempudoddi,
Kushiaditam, Punkar,
Kaduvalli, Intan 81212, Narikela, Balaji, Solari, Dehlibasumati, Karangaravat, Jopuvadlu, Doddataikallu
(Sehgal et al., 2012). Initially, it was noticed only in
aerobic condition. Since 2011, it has been observed in
anaerobic condition also and appearing in all types of rice
cultivating situations. The present investigation was
undertaken to know the performance of rice landraces
against M. graminicola under in-vivo condition in the
Organic Farming Research Centre of Zonal Agricultural
Research Station (ZAHRS), Navile, Shimoga during
kharif-2013.
MATERIALS AND METHODS
The study was conducted in nematode sick soil of Zonal
Agricultural Research Station, Navile, Shimoga during kharif 2013.
135 rice landraces were screened for resistance to rice root-knot
nematode under natural condition. Observations were recorded on
30 days old seedlings. Three seedlings were pulled out carefully
from the field, roots were washed free of soil, clipped off and were
observed for total number of galls present and rated for their
1
2
3
4
5
resistant/ susceptibility as per the 0-5 rating scale (Taylor and
Sasser, 1978) (Table 1).
RESULTS AND DISCUSSION
This experiment was laid out in order to screen the
promising landraces having desired phenotype
characters for tolerance / resistance against rice root-knot
nematode (Table 2, Figure 1, Plates 1 and 2). Out of 135
landraces screened, 32 recorded least root-knot index of
1 and they were found to be highly resistant, while, 45
landraces showed root-knot indices of 2 and read to be
resistant, 40 landraces evaluated to be moderately
resistant and 9 landraces showed susceptible and highly
susceptible reactions. The present investigation is in
conformity with those of Gitanjali et al. (2007) who
screened 8 rice varieties, screening rice varieties for
resistance against root-knot nematode (M. graminicola).
Yik and Birchfield (1979) observed that out of 26
1130
Afr. J. Microbiol. Res.
Figure 1. Response of 135 landraces of rice to Meloidogyne graminicola under in-vivo condition.
Plate 1. General view of the experimental plot.
Plate 2. Infected rice root with M. graminicola.
Ravindra et al.
cultivars, 21 cultivars showed resistance to the rice rootknot nematode. Simon (2009) evaluated the susceptibility
of 53 rice genotypes to M. graminicola in field and pot
experiments and observed that 13 cultivars were highly
resistant to this nematode. Evaluation of advanced
backcross populations developed for water stress
environment revealed that Teqing and the donarscvc
Type 3, Zihui 100, ShweThwe Yin Hyv were resistant to
the nematode (Prasad et al., 2006). Das et al. (2011)
reported that O. glaberrima accessions CG 14 and TOG
5674, traditional cultivars WAB 638-1 and IRAT 216 and
aerobic rice genotype IR 81426-B-B-186-4 and IR81449B-B-51-4 were resistant to M. graminicola.
Conflict of interest
The authors did not declare any conflict of interest.
REFERENCES
Abodolereza A, Racionzer P (2009). Food outlook: Global market
analysis. pp. 23-27.
Amoussou PL, Ashurt J, Green J, Jones M, Koyama M, Snape JTW,
Atkinson H (2004). broadly based resistance to nematodes in the rice
and potato crops of subsistence farmers. DFID Plant Sciences
ResearchProgramme Annual Report, pp. 9-14.
Das K, Zhao D, Waele DD, Tiwari RKS, Shrivastava DK, Arvinda K
(2011). Reaction of traditional upland and aerobic rice genotypes to
rice root-knot nematode (Meloidogyne graminicola). J. Plant Breed.
Crop Sci. 3:131-137.
Gitanjali Devi NS, Azad T (2007). Screening of Rice
Germplasm/Varieties for resistance against root-knot nematode
(Meloidogyne graminicola). Ind. J. Nematol. 37:1
Jairajpuri MS, Baqri QH (1991). Nematode pests of rice, Oxford and
IBH publisher, New Delhi, India, p. 66.
Krishnappa K, Reddy BMR, Ravichandra NG, Ravindra H (2001).
Incidence of root-knot nematode on rice in Karnataka in: National
congress on Centenary of Nematology in India appraisal and future
plans. pp. 85.
Lorenzana OJ, Matamis PP, Mallinin CB, Jose OL, De-leon DS (1998).
Cultural management practices to control rice root knot nematode.
Philippine Council for Agriculture, Forestry and Natural Resources
Research and Development, Los Banos, Laguna (Philippines). 120 p.
Macgowan JB (1989). Rice root-knot nematode Meloidogyne
graminicola Golden and Birchfield 1965. Fla. Dept. of Agric. And
consumer Serv. Div. Plant Ind., Nematology Circular No. 166, June,
1989.
Padgham JL, Duxbury JM, Mazid AM, Abawi GS, Hossain M (2004).
Yield loss caused by Meloidogyne graminicola on low land rainfed
rice in Bangladesh. J. Nematol. 36:42-48.
Port JC, Matias DM (1995). Effect of water regime on the distribution of
Meloidogyne graminicola. And other root-parasitic nematodes in a
rice field toposequence and pathogenicity ofM. graminicola on rice on
rice cultivar UPLR 15. Nematol. 41:219-228.
1131
Prasad JS, Panwar MS, Rao YS (1985). Occurance of root-knot
nematode, Meloidogyne graminicola in semi-deep water rice. Curr.
Sci. 54:387-388.
Prasad JS, Panwar MS, Rao YS (1986). Screening of some rice
cultivars against the root-knot nematode Meloidogyne graminicola,
Ind. J. Nematol. 16:112-113.
Prasad JS, Panwar MS, Rao YS (1987). Nematode problems of rice in
india. Int. J. T. Pest Manag.33:127-136
Prasad JS, Vijayakumar CHM, Sankar M, Varaprasad KS, Srinivasa
PM, Kondala RY (2006). Root-knot nematode resistance in advanced
back cross populations of rice developed for water stress conditions.
Nematol. Medit. 34:3-8.
Rao KV, Singh SP, Sureka K, Muthuraman P (2010). Site specific
integrated nutrient management in rice and rice based cropping
system. Indian Agric. Res. Directorate Rice Res. pp. 1-2.
Simon LSD (2009). Screening of rice germplasm against root knot
nematode Meloidogynegraminicola. Ind. Phytopathol. 62: 131-132.
Taylor AL, Sasser JN (1978). Biology, identification and control of rootknot nematodes (Meloidogyne spp.). Corporative publication,
Department of Plant Pathology, NC5U and U5AID, Raleigh, North
Carolina, p. 111.
Yik CP, Birchfield W (1979). Host studies and reaction of rice cultivars
to Meloidogyne graminicola. Phytopathology 49:497-499.
Vol. 9(16), pp. 1132-1138, 22 April, 2015
DOI: 10.5897/AJMR2015.7402
Article Number: A08C05252659
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Performance evaluation of laboratory professionals on
tuberculosis microscopy at Hawassa Town,
Southern Ethiopia
Mengistu Hailemariam*, Abebe Minuta, Gezahegn Bewoket, Tadesse Alehegn,
Yismake Worku and Moges Desta
Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Hawassa University, Ethiopia.
Received 28 January, 2015; Accepted 13 April, 2015
Microscopic diagnosis of Ziehl-Neelsen stained sputum by microscopists has remained the best routine
laboratory method for the diagnosis of tuberculosis (TB). However, detection and identification of TB
require skilled laboratory personnel. The aim of the study was to assess the performance of laboratory
professionals in detecting TB bacilli at Hawassa town health institutions. A cross-sectional study design
was employed among a total of 81 laboratory professionals working in public and private health
facilities. A standardized pre-validated slide panel and questionnaires were distributed to laboratory
professionals. Agreement in detection of TB bacilli sensitivity, specificity and predictive values of
readings were assessed using SPSS version 16.0. Among the 81 participant, 11(13.6%) correctly
reported all panel slides, 70 (86.4%) missed at least one slides. A total of 29.75% (241/810) error was
reported that include major errors of 2.22% (13 HFN; 5 HFP) and minor errors of 27.5% (25 LFN; 60 LFP
and138 QE). The sensitivity and specificity of participants in detecting TB bacilli as compared to the
reference reading were 91.97, 80.00, 87.30 and 86.92%, respectively. Overall agreement of participants
with the reference reading on TB detection was 95.18% (Kappa = 0.73). Agreement of the participants
with reference reading in the detection of TB bacilli was good. Even though the study revealed only
2.22% major error, the laboratory professionals need continuous supervision and remedial actions on
time for successful TB control programs.
Key words: Tuberculosis microscopy, performance test, laboratory professionals, southern Ethiopia.
INTRODUCTION
According to WHO tuberculosis (TB) report 2011,
Ethiopia ranks 7th in the list of the world‟s 22 high burden
countries for TB with incidence estimated at 379/100,000
for all forms of TB and 168/100,000 for smear positive
tuberculosis (Boulahbal et al., 1976). Direct sputum
smear microscopy remains the most cost effective tool for
diagnosing patients with infectious tuberculosis and for
monitoring progress of treatment although the limited
diagnostic capacity for TB in the country remains a
challenge to improving case detection rate (World Health
*Corresponding author. E-mail:mengemariam@yahoo.com. Tel: +251-913-641103. Fax: +251-462-205421.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
Hailemariam et al.
Organization, 2011; Ethiopian Health and Nutrition
Research Institute, 2008).
Case detection through quality assured laboratories is
an essential element of the WHO STOP TB Strategy
(Ethiopian Health and Nutrition Research Institute, 2008;
World Health Organization, 2006). The WHO for
tuberculosis control (DOTS) relies on a network of
laboratories that provide acid fast bacilli (AFB) sputum
smear microscopy. But, if the laboratory diagnosis is
unreliable, all other activities will be affected. It is known
that microscopy errors are likely to result in failure to
detect persons with infectious TB who will then continue
to spread infection in the community, or unnecessary
treatment for non-cases. On the other hand, errors in
reading follow up smears can result in patients being
placed on prolonged treatment, or in treatment
discontinued prematurely. Therefore, quality assurance of
AFB sputum smear microscopy is essential to reduce
such type of problems (Ethiopian Health and Nutrition
Research Institute, 2009; World Health Organization,
2011).
Quality of AFB smear microscopy is dependent on
national programs that monitor and perform proficiency
testing of individual laboratories (Jakes and Joyce, 2001).
Proficiency testing is a system in which „reference
material‟ of known but undisclosed content are introduced
into the laboratory and examined by the staff using the
same procedures as would normally be used to examine
patients‟ specimen of the same type (Ethiopian Health
and Nutrition Research Institute, 2008; Jakes and Joyce,
2001).
This study intends to assess the proficiency level in
detecting Mycobacterium tuberculosis from sputum
smears by the microscopists at Hawassa health
institution laboratories.
MATERIALS AND METHODS
A prospective cross-sectional study was conducted to evaluate the
quality of TB smear microscopic examination from April 23 to June
26 2012. The study was conducted at Hawassa City: two
governmental hospital, two private hospital, two governmental
health center and one non-governmental health institution
laboratories. The study involves 81 laboratory professionals, all
laboratories were provided TB microscopy on daily basis. The
professionals were selected based on convenient availability at time
of data collection.
Panel slide preparation and distribution
Experts who have been qualified and certified on TB microscopy at
the Hawassa regional laboratory had prepared and validated the
panel slides. Both TB positive and TB negative sputum were used.
Further concentration as well as dilution of bacilli was done after
bleach concentration techniques. Experts interpreted the prepared
AFB smears using investigative criteria for the presence or absence
of TB bacilli and also quantification of bacilli number.
For this study, ten slides per set, covering the full range from
negative to strongly positive were used according to WHO manual
for preparation of proficiency test slides. The composition of test
1133
panels standardized according to WHO manual and Ethiopian
Health & Nutrition Research Institutes guide line (National
Tuberculosis and Leprosy Control Programme, 1999; Ethiopian
Health and Nutrition Research Institute, 2009). Each panel of
slides include four with negative slide and six with different bacterial
density (3 with 1-9 AFB/100 fields (trace), one with 1+, one with 2+
and one with 3+). The total number of slides per panel was ten.
Groups of standardized panels with respect to the characteristics of
the positive (mycobacterium and level of bacteria) as well as
negative slides were used so that the results of the assessment by
different laboratories could be compared.
Next to preparation and validation, the slides were arranged in ten
sets and then packed for distribution to the participant. The
reporting formats and orders of how to perform the tests were
packed separately.
A structured questionnaire including information on the
participating facilities and professionals was distributed. The
questionnaire includes the socio-demographic characteristics,
educational background and service of the professionals.
The result of TB diagnosis reported by the participants was
evaluated using different parameter. Sensitivity was determined as
the ability of participants to diagnose positive TB slides whereas;
specificity was calculated for their ability to diagnose negative TB
slides. Inter-rater agreement is the degree of agreement between
participants and expertise reader. It is calculated by computing the
sum of true positives and true negatives and then divided by the
total.
“Major error” was defined as incorrect diagnosis of TB, that is,
reporting “negative” in the case of a sample with TB bacilli (HFN);
and falsely reporting “positive” in the absence of TB bacilli in the
sample (HFP). “Minor error” was defined as quantification error(QE)
and reporting negative with the correct report was 1-9AFB/100
fields and vice versa (Table 1). The distinction between a minor
and a major error was based on the effect the error could potentially
have on the patient's diagnosis and clinical management (2, 6
Ethiopian Health and Nutrition Research Institute, 2008 and 2009).
Regarding the score, in a set of 10 panel testing slides, each
slide carries 10 points, total possible score of 100. Committing
major error (HFP and HFN) result in a score of 0 where as minor
error (LFP, LFN and QE (QE = 2 grades difference)) result in
scores of 5 points. In general, assessment of performance was
based on Table 1. Passing score was 80 points and poor
performance was <80%. Interpretation of results was done based
on International Union against Tuberculosis and Lung Disease
(IUATLD) WHO recommended grading of sputum smear
microscopy results using World Health Organization (2002).
The laboratory personals in the selected laboratory reported the
results of examined panel slides along with the principal
investigator as compared to the results against the expertise
reading. Data was entered and analyzed using SPSS statistical
soft ware version 16.0 at a statistical significance of p < 0.05
and 95% confidence intervals. Error rates of p ar t ic ip a n t s were
statistic ally tested using logistic regression analysis. The level of
agreement among various diagnostic levels was measured using
Kappa.
The study was presented to the research committee of the
department of Medical Laboratory Science and endorsed by the
department commission then ethically cleared by the College
Research and publication committee. Official letter was written to
the participating facilities. In addition, participants had the right not
to participate or to withdraw from the study at any time.
RESULTS
A total of 81 laboratory professionals responded to the
questionnaires with a response rate of 100%. Forty eight
1134
Afr. J. Microbiol. Res.
Table 1. Evaluation and interpretation of errors between expertise and participant
technicians.
Result of technician
Negative
1-9AFB/100f
1+
2+
3+
Negative
Correct
LFP
HFP
HFP
HFP
Result of expertise
1-9AFB/100f
1+
2+
LFN
HFN
HFN
Correct
Correct
QE
Correct
Correct Correct
QE
Correct Correct
QE
QE
Correct
3+
HFN
QE
QE
Correct
Correct
1. QE = Quantification error, minor error; 2, LFN = low false negative, minor error; 3, LFP =
low false positive, minor error; 4, HFN = high false negative, major error; 5, HFP = high
false positive, major error.
(59.3%) of them were from government health institution
(31 from hospital and 17 from health center), 29(35.8%)
were from three private hospital and 4 (4.9%) were from
nongovernmental clinic. More than 75% of participant
responded that they got TB microscopy in-service training
and almost 50% of participant examined showed greater
than 10 TB case per day. Fifty two (64.2%) of participant
were BSc. degree holder and 37 (45.7%) were serving for
less than two years. The males were 45 (55.6%), mean
age of the participants was 28 (SD = 2.4) years (Table 2).
There were no problems with the functionality of
microscopes and accessibility of reagents in any of the
laboratories.
There was no statistically significant association
between the proportion of errors made by the
participants in the detection of TB bacilli and their sex,
experience, number of TB case examined per day, inservice training and EQA involvement (Table 3).
Of 81 participants, 11 (13.6%) correctly interpreted all
panel slides, 19 (23.5%) made two incorrect reading out
of the ten panel slides and 70 (86.4%) committed at least
one error among 10 slides. Fifty one (63.0%) participants
correctly reported all four negative slides, 20 (24.7%) had
at least one error among 4 negative slides and 10
(12.3%) misread all of the 4 negative slides. Eighteen
(22.2%) of the participants reported all positive slides
correctly, 22 (27.2%) had two error among 6 positive
slides and 3 (3.7%) reported incorrectly five of the 6
positive slides (Figure 1).
Overall agreement of participants with the reference
reading on TB detection was 95.18% (Kappa = 0.73).
Comparison across institutions showed that agreement in
detection was higher in NGO health institutions (95%)
(Kappa = 0.89). The lowest agreement on detection was
found among working in private hospital with an
agreement of 80.68% with reference reading
(Kappa = 0.59) (Table 4).
Overall, the sensitivity, specificity, positive predictive
values (PPV) and negative predictive value (NPV) of
participants in detecting TB bacilli as compared to the
reference reading were 91.97, 80.00, 87.30 and 86.92%,
Table 2. Demographic characteristics of laboratory professionals,
Hawassa Town, Southern Ethiopia, 201 (N=81).
Variable
Sex
Male
Female
Age in year
20-30
31-40
>40
Place of work
Government hospital
Private hospital
Government health center
NGO clinic
Educational qualification
Diploma
degree
Work experience
<2 years
2-5 years
>5 years
EQA involvement
Yes with feed back
Yes without feed back
No
TB microscopy in-service training
Yes
No
TB case examined /day
<5
5-10
>10
Frequency
(%)
45(55.6)
36(44.4)
48(59.3)
26(32.1)
7(8.6)
31(38.3)
29(35.8)
17(21)
4(4.9)
29(35.8)
52(64.2)
37(45.7)
29(35.8)
15(18.5)
36(44.4)
43(53.1)
2(2.5)
63(77.8)
18(22.2)
23(28.4)
19(23.5)
39(48.1)
respectively. Agreement with reference was 95.18%
(Kappa = 0.73) on detection of TB bacilli. Assessment
Hailemariam et al.
1135
Table 3. Relationship between score of participant with selected demographic characteristics, Hawassa Town,
Southern Ethiopia, 2012 (N=81).
Variable
Passed
≥ 80/100 (%)
Sex
Male
26(57.8)
Female
19(52.8)
Place of work
Government hospital
20(64.5)
Private hospital
16(55.1)
Government health center
14(82.4)
NGO clinic
2(50)
Work experience
<2 years
17(46)
2-5 years
15(51.7)
>5 years
4(26.7)
EQA involvement
Yes with feed back
15(41.7)
Yes without feed back
18(41.8)
no
0(0)
TB microscopy in-service training
Yes
27(42.9)
No
9(50)
Failed
<80/100 (%)
Chi-squire
Degree of freedom
P-value
19(42.2)
17(47.2)
0.126
1
0.72
11(35.5)
13(44.9)
3(17.6)
2(50)
0.73
3
0.68
2.048
2
0.36
21(58.3)
25(58.2)
2(100)
0.033
1
0.86
36(57.1)
9(50)
0.004
1
0.95
20(54)
14(48.3)
11(73.3)
Figure 1. Distribution of error in detection of TB bacilli among participants, Hawassa town, southern Ethiopia (n= 81), 2012.
across institutions showed lower sensitivity, specificity,
PPV and NPV 89, 68, 80.7 and 80.6% detected in those
working in private hospital with an agreement of 80.68%
(Kappa = 0.59) with reference reading (Table 4).
Out of 81 panel tested laboratory professionals,
29.75% (241/810) were reported wrong that includes
major errors of 2.22% (13HFN; 5 HFP) and minor errors
of 27.5% (25 LFN; 60 LFP and 138 QE). Among the 4
negative slides 3 (3.7%) of the participants made major
errors (HFP) and among the 6 positive slides, 15 (18.5%)
of participants made major error (HFN). On the other
hand, 33.3% of participants made minor errors (LFP) on
4 negative slides. On all positive slides, 71.6% minor
error (QE) was reported. A low number (6.2%) of
participant made minor errors (LFN) on the 1-9AFB/100
field slides (Table 5).
According to IUATLD/WHO recommended grading of
sputum smear microscopy results, 45 (55.5%) of the
1136
Afr. J. Microbiol. Res.
Table 4. Overall sensitivity, specificity, predictive value and agreement of participants in detecting TB bacilli by health institution.
Reference reader
Positive
Negative
174
21
12
103
186
124
Health
institution
Participant
reader
Government
hospital
Positive
Negative
Total
Private
hospital
Positive
Negative
Total
155
19
174
37
79
116
192
98
290
Government
health center
Positive
Negative
Total
94
8
102
5
63
68
99
71
170
NGO clinic
Positive
Negative
Total
24
0
24
2
14
16
26
14
40
Total
Positive
Negative
Total
447
39
486
65
259
324
512
298
810
Total
Sensitivity (%)
Specificity (%)
PPV*
NPV**
Agreement
Kappa
93.54
83.06
89.2
89.6
89.35
0.78
89.08
68.10
80.72
80.61
80.68
0.59
92.15
92.64
94.94
88.73
92.35
0.83
100
87.50
92.30
100
95.00
0.89
91.97
80.00
87.30
86.92
95.18
0.73
195
115
310
*Positive predictive value; **negative predictive value.
Table 5. Type of errors of participant in detecting TB bacilli by health institution, (N=81).
Health institution
Gov. hospitals (n=31)
Private hospitals (n=27)
Gov. health center (n=17)
NGO clinic (n=4)
Total
Major error
HFP
HFN
No (%) No (%)
2(0.65) 1(0.32)
2(0.69)
7(2.4)
1(0.59)
5(2.9)
0(0)
0(0)
5(0.62) 13(1.60)
18(2.22)
LFP
No (%)
19(6.13)
35(12.06)
4(2.35)
2(5)
60(7.40)
Miner error
LFN
QE
No (%)
No (%)
11(3.55) 55(17.74)
11(3.80) 50(17.24)
3(1.77)
25(14.70)
0(0)
8(20)
25(3.08) 138(17.03)
223(27.5)
Total
error
no. (%)
88(28.38)
105(36.20)
38(22.35)
10(25.00)
241(29.75)
Hailemariam et al.
participants were rated as passed, 36 (44.5%) failed.
Among 31 participants who worked at government
hospital, 20 (64.8%) passed, 11 (35.5%) failed. Among
participants who had TB microscopy in-service training,
27 (42.9%) passed, 36(57.1%) failed. However, there
was no statistically significant difference in level of
agreement based on training (Table 3).
DISCUSSION
Sputum smear examination is the most important test to
diagnose a person infected with pulmonary TB.
Although new diagnostic technologies
were
available, still microscopic examination of sputum
smear was used in Ethiopia. Therefore, the skill of
laboratory personals on AFB examination, seriously
affect the case management of TB. Consequently,
proficiency testing in sputum smear microscopy was
essential for a successful TB control program
(Ethiopian Health and Nutrition Research Institute, 2009).
Quality of AFB microscopy relies on national programs
that support, train and monitor the testing performance of
individual laboratories. It is well known that grave
deficiencies can occur in the laboratory when insufficient
attention is given to the quality of the work product. Many
countries including Ethiopia, however, have no complete
laboratory EQA program or do not provide sufficient
administrative support and attention (John, 2002).
In this study, the overall sensitivity, specificity, PPV and
NPV of participants in detecting TB bacilli were 91.97,
80.00, 87.30 and 86.92%, respectively. These findings
were in agreement with a study conducted in northern
Ethiopia, and elsewhere in the world (Mekete et al., 2011;
Mundy et al., 2002; Rieder et al., 1997; Nguyen et al.,
1999; Boulahbal et al., 1976; Selvakumar et al., 2005;
Fadizilah et al., 2010). Low specificity in detection of TB
bacilli indicates that there were many false positive
results, that is, false diagnoses of uninfected individual.
This can lead to exposure and wastage of treatment,
unnecessary care as well as financial lose.
Our finding of an overall agreement on detection of TB
bacilli with expertise reading was 95.18% (kappa = 0.73)
which is defined as good agreement based on the Kappa
index interpretation (Landis and Koch, 1977). It is known
that sputum microscopy never reach 100% agreement in
reading smear even among expertise (World Health
Organization, 1998). The overall agreement in the current
study was almost similar with findings with external
quality assessment of national public health laboratories
in Africa 2002–2009 (Mundy et al., 2002) also with study
in Ethiopia (Mekete et al., 2011; Estifanos et al., 2005),
98% (Kappa = 0.85) and 88.2% in Malaysia (Fadzilah et
al., 2013).
In the present study, major error were reported by 18
(2.22%) of participants, among these, 5 (0.62 %) were
HFP. Even though the reading is lower than the national
1137
critical cut-off point of 2% set by the NTLCP (National
Tuberculosis and Leprosy Control Programme,1999), the
possible reason could be due to lack of knowledge in
identifying AFB, work overload and carelessness of
technicians in reading of smears. Similar finding was also
reported from different study (Estifanos et al., 2005;
Nguyen et al., 1999). On the other hand, HFN 13 (1.6%)
result was also below the national threshold for corrective
action (1.6 vs. 5%) (National Tuberculosis and Leprosy
Control Programme, 1999). HFN indicate misclassifications that fundamentally change the disease classifycation and management of a patient. The main drive of
an EQA programme is to identify HFN which suggests
that patients with TB case were not treated on time
because of diagnosis error. HFN result in suffering due to
the disease; further spread of TB and elevated
transmitted rate in general population, in addition,
patients may lose confidence in the health services or a
particular laboratory.
This finding was similar with
Malaysia (Fadzilah et al., 2013). Even though the major
error in relative term was little, but it does not mean the
finding was insignificant; as the problem was major public
health important in the area, further attention need to be
paid (World Health Organization, 2002). Especially, HFN
results were serious because positive persons went to
the community without treatment so that it will increase
the transmission of the disease drastically.
In this study, 27.5% minor error (LFP 7.4%, LFN 3.08%
and QE 17.03%) rates were reported. This suggested
that microscopist fail to detect slides with low AFB count
(Trace and 1+ slide). Because of this, the dominant errors
committed among technicians were QE and LFP. Even
though quantification errors are minor importance, it does
not influence case management. But this type of error
can distinguish performances of laboratory personals.
The possible reason for miss might be the laboratory
personals not performing reading in all parts of the fields.
On the other hand, as one of the minor error, QE of 17%
in this study indicates possibility of microscopists not
following the standard procedure for reading the smears.
In the case of LFN, under-reading of number of AFB can
give indication of problem areas in the diagnostic
process. As a result, patients with bacillary disease may
perhaps be misdiagnosed as negative results in AFB
microscopy. In additions such type of error led to failures
of WHOs strategy, so patients will not receive treatment
on time, resulting in further community spread and failure
in diagnosis of pulmonary TB. The consequence of LFP
results in the beginning of treatment is unnecessary, also
anti-tuberculosis drugs are wasted (John et al., 2012).
The limitation of this study is that we only used
proficiency testing of slide reading using known panels to
evaluate the skill of laboratory professionals under
optimal conditions, rather than routine or day to day
performance in the diagnosis of TB. Besides, we did not
evaluate the performance of the laboratory personnel
with regards to smearing, staining qualities and the post
1138
Afr. J. Microbiol. Res.
analytical prospects like documentation of data. Findings
and interpretations from this study were only applicable to
the microscopists in the study area.
Conclusion
Even though the study revealed only 2.22% major error,
the overall 29.75% error made due to minor errors are a
great concern for countries like Ethiopia where most of
the suspected TB cases may have miss diagnosed at the
onset of disease. The country now have improved health
facilities and better awareness for patients with
suspected TB to look for treatment early, however it
might not be identified by sputum smear microscopy if
such errors are not solved on time.
Conflict of interests
The authors did not declare any conflict of interest.
REFERENCES
Boulahbal F, Mazouni L, Chaulet P (1976). Prospective study of the
organization and supervision of the bacteriologic diagnosis of
pulmonary tuberculosis in a case finding network in Algeria. Bull. Int.
Union Tuberc. 51: 313–321.
Estifanos B, Mohammed A, Bernt L (2005). Quality control of sputum
microscopic examinations for acid fast bacilli in Southern Ethiopia.
Ethiop. J. Health Dev. 19 (2):104-108.
Ethiopian Health & Nutrition Research Institute (EHNRI) (2009).
Laboratory Network Guidelines for Quality Assurance of Smear
Microscopy for Tuberculosis Diagnosis. Addis Ababa, Ethiopia.
Ethiopian Health and Nutrition Research Institute and Ministry Of Health
(2008). Ethiopian Guideline of quality assurance of smear microscopy
for TB diagnosis. Addis Ababa, Ethiopia.
Fadzilah K, Ganeswrie R, Loe YK, Teh CH, Suzana H (2013). The
Proficiency Level of Microscopists Detecting Mycobacterium
tuberculosis at Government Health Clinics in Three Selected States
of Malaysia, 2009-2010. OSIR 6(3):11-17.
Jakes R, Joyce M (2001). Guideline for Implementing Proficiency
testing for TB smear microscopy in the Northern province, South
Africa;ISBN:1-919743-62-6;October
2001,
available
at
www.hst.org.za
John F, Olga P, Vivian F et al. (2012). External quality assessment of
national public health laboratories in Africa, 2002–2009. Bull World
Health Organ 2012; 90:191–199A | doi:10.2471/BLT.11.091876
John R (2002). Public Health Practice Program Office Centers for
Disease Control and Prevention EQA for AFB smear microscopy.
Landis GR, Koch GG (1977). A one way component of variance model
for categorical data. Biometrics 33:671-679.
Mekete M, Kassu D, Getachew B, Ibrahim A (2011). Quality
performance Evaluation of Laboratories on AFB smears Microscopy
in Eastern Amhara Region, Ethiopia. Addis Ababa University
Libraries Electronic Thesis and Dissertations: AAU-ETD.
Mundy C, Harries A, Banerjee A, Salaniponi F, Gilks C, Squire S
(2002). Quality assessment of sputum transportation, smear
preparation and AFB microscopy in a rural district in Malawi. Int. J.
Tuberc. Lung Dis. 6 (1):47-54.
National Tuberculosis and Leprosy Control Programme (1999). Manual
for Laboratory Technicians. NTLCP, Addis Ababa, Ethiopia.
Nguyen TNL, Wells CD, Binkin NJ, Pham DL, Nguyen VC (1999).The
importance of quality control of sputum smear microscopy: the effect
of reading errors on treatment decisions and outcomes. Int. J.
Tuberc. Lung Dis. 3: 483-487.
Rieder H, Arnadottir T, Tardencilla Guttierrez A (1997). Evaluation of a
standardized recording tool for sputum smear microscopy for acidfast bacilli under routine conditions in low income countries. Int. J.
Tuberc. Lung Dis. 1:339-345.
Selvakumar N, Vanaja K, Gopi Sivagamasundari PGS, Prabhakaran E,
Samuel V, Narayanan PR (2005). Proficiency to read sputum AFB
smears by senior tuberculosis laboratory supervisors under training
at a reference laboratory in India. Indian J. Tuberc. 52:11-14
World Health Organization (1998). Laboratoty service in tuberculosis
control
part
1;
organization
and
management.
http://whqlibdoc.who.int/hq/1998/8.258_(part1).pdf
World Health Organization (2006). Strategic approach for strengthening
of laboratory services for tuberculosis control, 2006-2009. Geneva,
Switzerland: World Health organization.
World Health Organization (2011). Global tuberculosis control report.
WHO
report
2011.
Geneva,
Switzerland:
WHO.
WHO/HTM/TB/201116.
World Health Organization (WHO) (2002). Association of Public Health
Laboratory(APHL), Center for Disease Control (CDC), international
union Against Tuberculosis and Lung Disease: External quality
assessment for AFB smear microscopy Washington DC: association
of Public health laboratories.
World Health Organization: Stop TB strategy: building on and
enhancing DOTS to meet the TB -related millenium development
goals. Geneva, Switzerland: World Health organization; 2006.
Vol. 9(16), pp. 1139-1149, 22 April, 2015
DOI: 10.5897/AJMR2015.7456
Article Number: 7E820E052661
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Biosynthesis of indole-3-acetic acid by plant growth
promoting rhizobacteria, Klebsiella pneumonia,
Bacillus amyloliquefaciens and Bacillus subtilis
Vijendra Kumar Mishra* and Ashok Kumar
Microbial Biotechnology Unit, School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005,
(U.P), India.
Received 28 February, 2015; Accepted 13 April, 2015
Indole-3-acetic acid biosynthetic activity of Klebsiella pneumonia, strain MR-M1, Bacillus
amyloliquefaciens strain MR-AI, four Bacillus sp. strain MR-SP, RR-R2, WR-W2 and MR-Z1 were
investigated. Here, the authors demonstrated the effect of carbon sources, amino acids, vitamins and
abiotic stress on the indole-3-acetic acid (IAA) production level under in vitro condition by the six
strains. The culture medium was exogenously supplemented with L-tryptophan (200 μg/ml) and
incubated for 96 h. IAA biosynthesis was further confirmed by indole-3-pyruvate decarboxylase
encoding ipdc gene amplification. Succinate followed by acetate followed by malate was observed as
the most preferred carbon source for IAA production. Stimulation of IAA production at pH 6.0, 0.1%
salinity and 32°C temperature after 96 h of growth in presence of L-tryptophan was observed. The
highest amount of IAA production was observed in strain MR-M1 followed by WR-W2. Decreasing trend
of IAA levels was observed in the presence of vitamins and amino acids as compared to the control. The
amplicon of 250 bp was observed in all the six rhizospheric strains. Taken together, the result shows
that L-tryptophan stimulates IAA production and strain MR-M1 was observed as the most efficient IAA
producing rhizospheric bacteria.
Key words: L-tryptophan, indole-3-acetic acid, plant growth promoting rhizobacteria, indole pyruvate
decarboxylase.
INTRODUCTION
Rhizospheric colonization of cereal crop plants and plant
growth promoting activity involving indole-3-acetic acid
(IAA) production by Klebsiella sp. and Bacillus sp. has
been well documented (El-khawas and Adachi, 1999;
Mishra and Kumar, 2012; Saharan and Nehra, 2011).
Phytohormone, especially auxin indole-3-acetic acid
plays important role in the plant growth regulation as cell
enlargement, cell division, cell elongation and root
initiation by tryptophan dependent and independent
pathways (Woodward and Bartel, 2005). Rhizosphere is
a site with complex interactions between the root and
associated microorganisms with high microbial diversity
(Saharan and Nehra, 2011). Microbial population in
rhizospheric soil is physiologically more active, as
*Corresponding author. E-mail: vijendrakmishra@gmail.com. Tel: +91- 0542-2368331/6701593. Fax: +91- 0542-2368693/236817.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
1140
Afr. J. Microbiol. Res.
the nutritional substance is released by the root and its
surfaces. Rhizospheric microorganisms are considered
as labile source of nutrients such as B-vitamins, amino
acids, tryptamine, soluble root exudates, carbon sources
and organic matter (Saharan and Nehra, 2011; Sainio et
al., 1996; Ahemad et al., 2013; Barraquio et al., 2000).
The role and metabolism of IAA in Gram-negative
bacteria is well documented, but little is known about IAA
biosynthesis and regulation in Gram-positive bacteria
(Saharan and Nehra, 2011; Sainio et al., 1996; Ahemad
et al., 2013,). L-tryptophan serves as a physiological
precursor for biosynthesis of IAA in plants and in
microbes (Woodward and Bartel, 2005). IAA is a
secondary metabolite produced in early stationary phase
of growth. Synthesis of IAA depends on environmental
conditions, availability, uptake and rate of deamination of
precursor, the types of soil nutrients, metabolites
released and the preferred pathways involved (Spaepen
et al., 2007; Woodward and Bartel, 2005). Soil pH,
temperature and salinity condition are crucial for
attachment and spread of the microbes (Strzelczyk et al.,
1994; Dobbereinere et al., 2001). Moreover, L-tryptophan
dependent biosynthesis of IAA involving ipdc, indole-3acetamide (iam) and indole-3-acetonitrile (ian) gene has
been well documented in plant growth promoting
rhizobacteria (PGPR) (Duca et al., 2014). Although, the
complete mechanism involved in the biosynthesis of IAA
is poorly understood.
Reports suggest that most plant associated microorganism might have cellulase activity for adoption or
establishment of a plant microbe interaction. Cellulase
activities have seen in many N2 fixing bacteria such as
Bacillus spharricus, Bacillus circulans, Paenibacillus
azotofixans and Azospirillum (Emtiazi et al., 2007).
Moreover, recent study reveals cellulase production by
the Bacillus subtilis (Femi-Ola and Aderibigbe, 2008).
Indole pyruvate decarboxylase encoding ipdc gene acts
as key modulator for IAA production, which catalyses
formation of indole-3-acetaldehyde from indole-3-pyruvic
acid in Klebsiella pneumonia and Bacillus sp. (El-khawas
and Adachi, 1999; Idris et al., 2002). The 16S rRNA gene
(1.5 Kb) sequence involves identification of bacteria upto
genus level on the basis of highly conserved sequences
(Elgaml et al., 2013). Literature showed that the
hypervariable regions V2 and V3 contain the maximum
degree of nucleotide heterogeneity and the maximum
discriminatory power for the bacterial species
identification (Baker et al., 2003; Chakravorty et al.,
2007). After thorough review of literature, it appears that
the rhizospheric bacteria are of great relevance in terms
of agricultural productivity. The present study was aimed
to demonstrate IAA production capability of most efficient
isolates under various physiological conditions. To
replace costlier and hazardous chemically synthesized
fertilizers, application of efficient PGPR as biofertilizer
can bridge the gap. Many workers have acknowledged
the role of PGPR and significant advancements have
been made in this interesting area of research but there
are still gaps left especially on molecular aspect, which
needs further study.
MATERIALS AND METHODS
Sample collection, culture media and incubation condition
To isolate most efficient IAA producing PGPR, soil samples were
collected from rice, (RR-R2) wheat (WR-W2) and maize (MR-M1,
MR-SP, MR-MZ and MR-AI) agricultural field of Banaras Hindu
University, Varanasi, Uttar Pradesh, India. 1 kg rhizospheric soil
sample were brought to the laboratory in the sterile polythene bags.
Isolation of bacteria from rhizospheric soil was performed, following
the standard microbiological methods of Barraquio et al. (2000).
One gram of rhizospheric soil of each sample were suspended in
10 ml sterilized double distilled water (DDW) separately and serially
diluted in 50 ml Borosil glass tubes, up to 10 dilutions with three
replicates. The soil suspension obtained was used to pour onto
dextrose, yeast, glutamate medium (DYGS), agar-agar solid plate
containing dextrose 1.0 g/L, yeast extract 2.0 g/L, glutamate 1.5
g/L, peptone 1.5 g/L, K2HPO4 0.5 g/L, MgSO4.7H2O 0.5 g/L, pH 6.0
(Kirchhof et al., 2001). 100 μl inoculums were taken from 10-1 to 1010
dilutions and plated onto both solid culture plates in duplicate set,
incubated at 32°C for 24 h. Pure culture was isolated from rice,
wheat and maize soil samples by streaking three to four times
repeated subculture on the fresh slightly modified Johanna Nitrogen
fixing bacteria (JNFb) agar-agar medium containing malic acid 5.0
g/L, K2HPO4 0.60 g/L, KH2 PO4 1.80 g/L, MgSO47H2O 0.20 g/L, NaCl
0.10 g/L, CaCl2.2H2O 0.20G g/L, Na2 MoO4.2H2O 0.002 g/L, KOH
4.5 g/L, Fe.-EDTA (1.4%) 4.0 ml and NH4Cl, 2.5 mM at pH 5.8
(Dobereiner, 1995). pH was maintained at 5.8 with the help of KOH
and HCl by cyberscan ph ion 510 bench pH/ion/mv emter (Eutech
instruments Pvt Ltd, Singapore). Six most efficient IAA producing
rhizospheric bacteria were selected for plant growth promoting
activity study namely MR-M1, MR-SP, MR-Z1, MR-AI, WR-W2 and
RR-R2. All the selected strains were repeatedly subcultured and
maintained onto JNFb solid plate.
Quantitative estimation of IAA
Quantitative estimation of IAA was performed according to the
colorimetric assay of Gordon and Weber (1951). Culture was grown
in JNFb liquid medium additionally supplemented with tryptophan
(200 µg/ml). Equal volume of inoculation was made (1/10th) in
inoculums coming from the same medium grown culture and
incubated in shaking conditions at 100 rev/min for 72-96 h. 1.5 ml
culture was harvested via centrifugation at 8000 rpm for 5 min. 1.0
ml supernatant were collected and added with 2 ml IAA test
reagent, that is, Salkowski reagent containing 1.0 ml of 0.5 M of
FeCl3 in 50 ml of 35% HClO4 (Yamada et al., 1990). Appearance of
pink color indicates presence of IAA and absorbance was recorded
at 530 nm in UV spectrophotometer. Culture medium without
tryptophan was maintained as control for each isolate. The
concentration of IAA produced was estimated using a standard
prepared separately with pure IAA (Loba, India). Estimation of IAA
was carried out after the incubation of 96 h at 32°C in triplicate set.
Effect of carbon sources on IAA production
Estimation of IAA production was checked in JNFb medium, where
malate was replaced by eleven different carbon source such as
ribose (0.1%), mannitol (1%), malate (0.5%), sucrose (0.5%),
glucose (0.5%), succinate (0.5%), sorbitol (1.0%), maltose (0.5%),
fructose (1.0%), sodium acetate (0.5%), dextrose (0.5%) exoge-
Mishra and Kumar
nously supplemented with tryptophan (200 µg/ml). In control set,
malate (0.5%) was used as carbon source. Quantity of IAA was
estimated after the incubation of 96 h at 32°C in triplicate set.
Effect of vitamins on IAA production
IAA production in the presence of five vitamins such as vitamin B6
(Pyredoxin: 100 µg/ml), vitamin B5 (nicotinic acid: 100 µg/ml),
vitamin B2 (riboflavin: 100 µg/ml), vitamin B12 (cyanocobalamin: 50
µg/ml) and vitamin H (biotin: 100 µg/ml) were checked. Isolates
were grown in JNFb medium exogenously supplemented with
tryptophan (200 µg/ml). Control set was prepared without
tryptophan for each isolate.
1141
The upper acetate phase was collected and the extraction
procedure was repeated thrice. Furthermore, the ethyl acetate
phase was evaporated by air-drying. The dried pellet was collected
in 1.5 ml methanol and again evaporated to the 0.5 ml of IAA in
methanol. 10.0 µl of above collected supernatant were loaded onto
thin layer plate. The running solvent used was: N-butanol: glacial
acetic acid: DDW in ratio 12:3:5. After complete running, the TLC
plate was activated at 60°C in oven followed by spraying with
Salkowski reagent. After 10 min, appearance of pink spot clearly
indicates the production of IAA, as same color of spot also shown
by the standard IAA (Sigma, India). Rf value was calculated as the
distance traveled by the compound divided by the distance traveled
by the solvent.
Effect of amino acids on IAA production
Determination of dry weight
IAA producing capacity were checked in the presence of five amino
acids like L-phenylalanine (50 µg/ml), L-glutamic acid (100 µg/ml),
L-tyrosine (100 µg/ml), L-serine (100 µg/ml) and L-lysine (100
µg/ml) in JNFb medium with tryptophan (200 µg/ml). Control set
lack tryptophan for each test strains. On the other hand, culture
tube containing tryptophan (200 µg/ml) only without amino acids
was also maintained for each isolate.
1.0 ml of grown culture was harvested at 10,000x rpm for 5 min,
pellets were washed with phosphate buffer saline (PBS) thrice and
suspended in the 500 µl PBS and filtered through 0.45 µM (Millipore
Intertech Inc., Bedford, MA, USA) filter paper employing vacuum
filtration instrument. Thus, all the cells were carefully collected onto
filter paper and allowed to dry in hot air oven for 1 h. Therefore, the
weight of cells containing filter paper was measured and previously
recorded weight of fresh filter paper was deducted and expressed
in mg dry wt.
Time course study of IAA production
To further examine the effect of time duration, all the six strains
were grown in JNFb culture medium added with L-tryptophan (200
µg/ml) and incubated for 0, 24, 48, 72 and 96 h.
Effect of abiotic stress on IAA production
To explore the effect of abiotic stress on the bioproduction of IAA,
strains were grown in JNFb medium containing 0, 0.1, 1.0 and 5.0%
sodium chloride added with L-tryptophan (200 µg/ml). To check the
effect of pH on the production of IAA level, strains were cultured in
JNFb medium exogenously supplemented with L-tryptophan (200
µg/ml). pH of the culture test tubes were adjusted with KOH and
HCl upto 6, 7, 8 and 9 separately for each strain. To study the effect
of temperature on the production level of IAA, strains were grown in
JNFb medium with L-tryptophan (200 µg/ml) and incubated at 20,
30 and 40°C separately for each strains. All the quantitative
estimations were performed after 96 h of growth.
Extra cellular cellulase assay
Extra cellular cellulase assay was carried out according to the
method adopted by Park et al. (1997) with slight modification. In
brief,celluloase production was determined in JNFb agar-agar solid
medium amended with carboxy methyl cellulose (0.4%). 10 μl of 48
h grown JNFb cultures of the 6 isolates were used to spot inoculation
onto solid plate. Qualitative estimation plate based cellulase activity
was performed after 4 days of incubation at 32°C. The plates were
stained with 0.1% Congo red solution for 30 min, rinsed with DDW,
washed twice with 1 M NaCl and then stained with 0.1 N HCl.
Thin layer chromatography of IAA
Extraction of IAA from each strain was performed according to the
modified method of Manulis et al. (1994) Briefly, 10 ml of stationary
phase grown and tryptophan added (200 µg/ml) (Loba, India)
culture was harvested via centrifugation followed by the addition of
equal volume of ethyl acetate to the collected supernatant. The
above-prepared solution were mixed properly and incubated for 1 h.
Amplification of IPDC gene
The PCR primers used to amplify the ipdc gene were designed by
Patten and Glick (2002), forward primer: 5`-GAA GGA TCC CTG
TTA TGC GAA CC-3` and reverse primer: 5`-CTG GGG ATC CGA
CAA GTA ATC AGG C-3`. Amplification condition used was
denaturation at 94°C for 30 s, annealing at 45°C for 30 s elongation
at 72°C for 2 min and final extension was kept 72°C for 5 min for 35
cycles.
Genomic DNA isolation and polymerase chain reaction of 16S
rDNA
Whole cell genomic DNA was extracted following the standard
protocol of Sambrook et al. (1989). PCR amplification of 16S rDNA
was performed following the method of Eckert et al. (2001) with
some modification using universal primer in a final volume of 50 μl.
The PCR reaction mix included; 1.5 U of Taq DNA polymerase
(Banglore Genei, India), 1X PCR assay buffer, 25 mM MgCl2, 20
pmol each forward and reverse primers (Integrated DNA
Technologies, Inc, CA, USA), each dNTPs:200 μM (Banglore
Genei, India) template DNA:50 ng. Primer pair was forward 5’-AGA
GTT TGA TYM TGG CTC AG-3’ and reverse 5`-CTA CGG CTA
CCT TGT TAC GA-3`). Amplification was performed in PTC-100
Thermal Cycler (MJ Research, Inc, Walthon, MA, USA), using initial
denaturation at 94°C for 30 s, annealing at 57°C for 1 min,
elongation at 72°C and final extension at 72°C for 5 min and finally,
storage at 4°C. 5 μl of amplified reaction mixture was analyzed by
agarose (2% w/v) gel electrophoresis in TAE buffer (40 mM Tris, 1
mM EDTA, pH -8.0). After, running at 50 V for 3 h, the gel was
stained with ethidium bromide (0.5 μg/ml) and photograph was
taken in Gel-documentation system (Bio-Rad Laboratories,
Hercules, CA, USA).
Molecular identification
The 16S rDNA insert was sequenced by the dideoxy-chain
1142
Afr. J. Microbiol. Res.
IAA (µg/mg Dry wt.)
25
20
15
10
5
0
MR-M1 MR-SP
RR-R2
MR-Z1
MR-AI WR-W2
Figure 1. Quantitative estimation of IAA production.
40
IAA (µg/mg Dry wt.)
35
30
25
20
15
10
5
MR-Z1
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M
al
S
D
ex
t
ro
s
to
s
e
e
e
MR-M1
Fr
uc
to
s
M
al
co
se
G
lu
uc
r
S
R
ib
o
se
os
e
0
MR-SP
MR-AI
WR-W2
RR-R2
Figure 2. Test of IAA production in the presence of different carbon sources.
termination method using an automated DNA sequencer (ABI
Prism; Model 3100). To identify on genus level, PCR amplification
and partial sequencing of 1.5 Kb gene sequence and 400 bp of
hypervariable region between V2-V3 regions of 16S rRNA of the six
strains were carried out. Highest percent similarity sequences
obtained were searched through online available Basic Local
Alignment Search tool (BLAST) tool of National Center for
Biotechnology Information (NCBI). The 16S rRNA gene sequences
were
submitted
to
the
NCBI
gene
bank
on
http://www.ncbi.nlm.nih.gov.
Statistical analysis
Values were expressed as means of ± SD for triplicate samples.
Differences were considered to be significant at the P<0.05 level.
RESULTS AND DISCUSSION
Result presented in Figure 1 shows that the highest
amount of IAA was produced by MR-M1 (19.36 μg/mg
dry wt.) followed by WR-W2 (15.84 μg/mg dry wt.).
Isolate RR-R2 shows poor amount of IAA producing
ability (5 μg/mg dry wt). On the other hand, isolate MRSP, MR-AI and MR-Z1 shows more or less similar
amount of IAA producing activity. Literature survey show
that the highest accumulation of IAA occurs in the
presence of L-tryptophan after 96 h of growth
(Bhattacharya and Basu, 1992), which is in accordance
with the present study. It is evident from data presented
Mishra and Kumar
1143
Table 1. Effect on pH during IAA production in the presence of various carbon
sources.
Carbon source
Sucrose
Glucose
Maltose
Fructose
Dextrose
Succinate
Malate
Acetate
MR-M1
3.42±3
3.62±2
3.73±4
3.16±3
3.31±3
8.80±2
9.18±2
3.25±3
MR-Z1
3.82±2
3.32±3
3.63±2
3.45±2
3.54±3
8.85±3
8.62±3
3.64±4
in Figure 2 that isolate MR-M1 proved to be the most
efficient strain considering its capability to produce IAA,
utilizing a wide range of carbon sources, whereas others
showed variable preferences to carbon sources. Malate,
succinate and acetate were found to be the best carbon
source. On the other hand sucrose, glucose, mannitol,
sorbitol, maltose and furctose were found to be
intermediate. However, dextrose and ribose were
identified as poor carbon sources for the IAA production.
In the presence of sucrose, glucose, mannitol and
sorbitol, more or less similar amount of IAA production
was observed. The highest amount of IAA production
was observed in MR-M1, that is, 32 μg/gm dry wt. in the
presence of succinate. IAA production was not observed
without tryptophan. Effect of various carbon compounds
on IAA production has been well documented in Rhizobia
(Bhowmik and Basu, 1984, Frankenberger and Arshad,
1995). To our knowledge, this is the first report
demonstrating highest amount of IAA production, when
organic acid salts succinate, acetate and malate acts as
a sole carbon source in the strains studied. Previous
studies suggest malate as the best carbon source for
microorganism mediate IAA production (Bhowmik and
Basu, 1984). In the present experiment, the change in
pH after 96 h of growth was recorded to study whether
IAA production is accompanied with the production of
other organic acid in the culture medium. It is evident
from the result presented in Table 1 that a continuous
decrease in pH 5.8 to 3.0 to 4.0 takes place, which
showed the production of organic acid. However, more or
less similar pattern of pH change was observed.
Interestingly, when malate and succinate was used as
carbon source a continuous increase in pH was observed
from 5.8 to 9.18 and 8.88, respectively. The pH has a
significant effect on the amount of IAA produced
(Kirchhof et al., 2001). Since pH of the culture medium
directly influences the growth of the strain, studies
suggest either release of IAA under in vitro condition is
the major cause of decrease in pH of the culture medium
or accumulation of IAA is directly proportional to
decrease in pH. However, increase in pH up to 8 to 9
pH
MR-SP MR-AI
3.20±3 4.00±3
3.02±3 2.54±3
2.43±3 2.60±3
4.02±2 3.00±3
3.96±2 4.01±3
9.02±4 8.95±3
8.73±3 8.51±3
2.50±2 4.10±3
WR-W2
3.60±3
4.42±3
3.75±3
2.95±2
3.68±2
8.60±2
8.76±2
2.91±2
RR-R2
4.14±2
4.02±2
3.74±2
4.16±2
3.21±2
8.74±2
8.98±2
3.75±2
after 96 h of growth was observed when malate and
succinate were used as sole carbon source. The increase
in pH might be due to release of other alkaline
metabolites or other unknown compounds. Therefore,
decrease in the culture medium pH could not always be
correlated with accumulation of IAA. However, the drop in
pH and IAA content in the tryptophan supplemented
medium at different time varies according to the type and
genotype of the strain. Result presented in Figure 3
shows differential IAA production efficiency in response
to various amino acids. Tryptophan followed by glutamic
acid proved to the most suitable amino acid considering
its capability to produce IAA. The production of IAA was
inhibited in the presence of L-phenylalanine and L-lysine
as compared to control. Production of IAA was not
observed in presence of L-tyrosine (100 µg/ml), L-serine
(100µg/ml) and L-methionine (25 µg/ml) even in the
presence of tryptophan, which might be due to the fact
that, these amino acids may act as competitive inhibitor
of tryptophan. Reports shown indole and serine are
involved in biosynthesis of tryptophan (Tatum and
Bonner, 1944). (data not shown). Therefore, L-tyrosine,
L-serine and L-methionine could be considered as
inhibitor of IAA biosynthesis. Biosynthesis of IAA was
also checked in the presence of 5 vitamins namely,
vitamine B12, B5, B6, B2 and biotin. All the vitamins
studied were found to inhibit the IAA production by all the
isolates in the presence of tryptophan (Figure 4).
Vitamins showed differential influence on IAA production
by different isolates. Biotin showed weak inhibitory effect
whereas B2 and B12 showed strong inhibition of IAA
production. IAA production was not observed in the
presence of vitamins without tryptophan. There were also
productions of IAA in above vitamins containing (without
tryptophan) JNFb medium. Effect of vitamin and amino
acids on IAA production has been demonstrated by
Azospirillum brasilense and various other microbes
(Zakharova et al., 2000). Vitamins and amino acids may
play a key role in the production of IAA by microorganism. Although, the literature related to the PGPR
mediated IAA production in presence of vitamins and
1144
Afr. J. Microbiol. Res.
IAA (µg/mg Dry wt.)
25
20
15
10
5
0
MR-M1 MR-Z1 MR-SP
Control
Phenylalanine
MR-AI WR-W2 RR-R2
Glutamic acid
Lysine
Figure 3. Test of IAA production in the presence of different amino acids.
IAA (µg/mg Dry wt.)
25
20
15
10
5
0
MR-M1
Control
MR-Z1
Vit. B6
MR-SP
Vit. B5
MR-AI
WR-W2
RR-R2
Vit. B2 Vit. B12 Biotin
Figure 4. Test of IAA production in the presence of various vitamins.
amino acids are limited. Suppression of IAA production in
presence of vitamin and amino acids as compared to
control, might be due its high molecular weight, which
make it inaccessible to microorganisms, even in the
presence of tryptophan also. Production of IAA was not
observed in the presence of L-tyrosine, L-serine and Lmethionine, even in the presence of tryptophan.
Appearance of single pink spot after spray of Salkowaski
reagent on the TLC plate shows release of IAA in the
culture medium. The Rf value of MR-M1: 0.919; MR-SP:
0.957; RR-R2: MR-Z1 and MR-AI: 0.987 and WR-W2
was 0.979 which corresponded to the standard IAA
(0.953) (data not shown). The highest accumulation of
IAA level was observed after 96 h of growth by the six
strains as shown in Figure 5.
Biosynthesis of IAA was observed with the beginning of
exponential phase of the growth. In the present
experiment, the change in pH after 0, 24, 48 72 and 96 h
of growth was recorded. The continuous increase in pH
was observed from initial pH 5.8 to 9.11 as presented in
Table 2 which clearly shows release of alkaline
compounds or formation of any byproduct reaction
complex and require further study. Results of this study
showed that pH 5.8-6.0, (Figure 6.) 0.1% salinity
condition (Figure 7) and 30±2°C temperature (Figure 8)
are the most optimal condition for IAA bioproduction.
Poor or no growth of the isolates was observed at pH
above 9.0 or below pH 3.0 and hence no IAA production
was observed. Similarly, in the presence of 7.5% or
higher sodium chloride concentration, growth of the
Mishra and Kumar
IAA (µg/mg Dry wt.)
25
20
15
10
5
0
MR-M1
MR-SP
RR-R2
24 h
48 h
MR-Z1
72 h
MR-AI
WR-W2
96 h
Figure 5. Time course study of the IAA production.
Table 2. Effect on pH during time course study of IAA production.
Isolate
MR-M1
MR-SP
RR-R2
MR-Z1
MR-AI
WR-W2
0h
5.96±2
5.95±2
5.97±2
5.95±2
5.94±2
5.94±2
6h
7.23±3
6.06±3
6.91±3
6.49±3
6.42±3
6.17±3
18 h
8.25±3
7.42±3
7.81±3
6.74±3
6.73±3
6.59±3
Time
24h
8.35±2
7.62±2
8.29±2
7.16±2
6.80±2
7.08±2
48h
8.62±3
8.39±3
8.71±3
7.59±3
7.07±3
8.43±3
72 h
8.91±2
8.77±2
8.98±2
8.64±2
7.30±2
8.84±2
96 h
8.97±3
8.86±3
9.11±3
8.80±3
7.35±3
8.91±3
25
IAA (μg/mg Dry wt.)
20
15
10
5
0
MR-M1
WR-W2
Figure 6. Effect of pH on IAA production.
MR-Z1
5
6
MR-SP
7
8
MR-AI
9
RR-R2
1145
Afr. J. Microbiol. Res.
25
20
IAA (μg/mg Dry wt.)
1146
15
10
5
0
MR-M1
MR-SP
0%
RR-R2
0.10%
MR-Z1
MR-AI
1.00%
5.00%
WR-W2
Figure 7. Effect of salinity (NaCl) on IAA production.
Figure 8. Effect of temperatures on IAA production.
M 1
2 3
4
5 6
200bp
Figure 9. Amplification of ipdc gene of
the six isolates where 1.MR-M1, 2.
MR-SP 3. RR-R2 4. MR-Z1.5.MR-AI
6.WR-W2.
strains were not observed and hence IAA production was
not observed. Strain MR-M1 proved to be most efficient
strains in terms of IAA production under abiotic stress
conditions. Similarly, buffered (pH 6.9 with 21 mM
K2HPO4 and 11 mM KH2PO4) rich L.B medium containing
no salt was used to study of Escherichia coli and B.
subtilis osmoregulation (Boylan et al., 1993). Poor growth
of rhizobacteria and decreased level of IAA production
under salinity stress condition was reported in Rhizobium
sp. by Ikeda et al. (1989). Environmental stress some
time leads to hormonal changes to PGPR (Strzelczyk et
al., 1994). Cellulase production was determined in
nitrogen free medium amended with carboxy methyl
cellulose (CMC) (0.4%). Cellulose, a β-1,4-linked polymer
of glucose, represents approximately half of the dry
weight of plant cell walls, which also contain xyloglucan
and xylan (Coughlan and Mayer, 1992). Moreover,
Figure. 9. Amplification of ipdc gene of the six isolates where 1.MR-M1, 2. MR-SP
RR-R2
4. MR- 1147
Mishra3.
and
Kumar
Z1.5.MR-AI 6.WR-W2
5
1
3
4
6
2
Figure 10. Halo zone production by extra cellular cellulase activity, where 1. MR-M1, 2. RR-R2, 3.
MR-Z1,4. MR-SP, 5. MR-AI and 6. WR-W2.
Figure.10. Halo zone production by extra cellular cellulase activity, where 1.MR-M1, 2.RR-R2, 3.MRZ1,4.MR-SP, 5.MR-AI, 6.WR-W2
Diameter in Centimeter
2.5
2
1.5
1
0.5
0
MR-M1 MR-SP
RR-R2
MR-Z1
Halo Zone Diameter
MR-AI WR-W2
Colony Diameter
Figure 11. Colony and halozone diameter (cm) of cellulose assay.
cellulolytic enzymes released by rhizospheric bacteria are
the important factors involved in enzymatic hydrolysis of
fungal cell walls and in preventing plant pathogens.
Transient appearance of yellow/orange halo zone of
various diameters onto CMC added agar-agar plate and
against a dark/blue background ensures biosynthesis of
cellulase. Cellulase production was observed in all the six
strains as shown in Figure 10. On the basis of halo zone
production, isolate MR-AI followed by MR-Z1 followed by
MR-SP was observed to be the most efficient cellulase
producer. On the other hand, strain MR-M1 followed by
RR-R2. Colony diameter and halo zone diameter
production was measures as represented in Figure 11.
Diverse level of cellulose activity has been demonstrated
in the six strains. To further examine IAA production by
the six isolates, amplification of ipdc gene was performed
(Figure 9). Indole pyruvate decarboxylase, a key enzyme
for IAA biosynthesis, was observed in all the isolates. The
enzyme catalyzes the decarboxylation of indole-3-pyruvic
acid to yield indole-3-acetaldehyde and carbon dioxide.
Moreover, Koga et al. (1991) firstly reported the
identification, purification and characterization of
indolepyruvate decarboxylase, ipdc which is a novel
enzyme for IAA biosynthesis in Enterobacter cloacae.
Amplicons of 200 bp was observed in all the six isolates.
Ipdc gene was identified as a key gene involved in IAA
biosynthetic pathway in the decarboxylation step of the
indole-3-acetaldehyde as shown in Bacillus sp. (Idris et
al., 2002). Sequence analysis of the 16S ribosomal RNA
(rRNA) gene has been widely used to identify bacterial
1148
Afr. J. Microbiol. Res.
species and to perform taxonomic studies (Chakravorty
et al., 2007). Nucleotide sequence accession numbers of
16S rRNA gene sequences of the six strains are MR-AI
(1460 bp), identified as Bacillus amyloliquefaciens
[FJ222551]. Strain MR-M1 (1468 bp) was revealed as K.
pneumonia, [FJ222552]; Strain WR-W2, (1473 bp)
identified as B. subtilis [FJ222553]; Strain MR-Z1 (1459
bp) identified as B. subtilis [FJ269243]; Strain RR-2 (393
bp) identified as B. subtilis [EU327502] and Strain MR-SP
(384 bp) identified as B. subtilis [EU327504]. Reports
suggest that human pathogenic bacteria are inhabitants
of plants, which may be due to beneficial relationship
between each other (Tyler and Triplett, 2008).
Conclusion
The present research investigations proved that the
strains could serve as an excellent model to study the
physiological and biochemical mechanism of IAA
production and provide tremendous opportunities in
environmentally sustainable approach to increase crop
production. The present study need further research so
that the strains could be directly applied to crop fields in
different formulations for sustainable agriculture.
Conflict of interests
The authors did not declare any conflict of interest.
REFERENCES
Ahemad M, Kibret K (2013). Mechanisms and applications of plant
growth promoting rhizobacteria: Current perspective. J. King Saud
Univ. Sci. 26:1-20.
Baker GC, Smith JJ, Cowan DA (2003). Review and re-analysis of
domain-specific 16S primers. J. Microbiol. Methods 55:541-555.
Barraquio WL, Segubre EM, Gonzalez MS, Verma SC, James EK,
Ladha JK, Tripathi AK (2000). Diazotrophic Enterobacteria: what is
their role in rhizosphere of rice? In: J.K. Ladha and P.M. Reddy,
Editors, The Quest for Nitrogen Fixation in Rice, Los Banos,
Philippines. pp. 93-118.
Bhattacharya RN, Basu PS (1992). Bioproduction of indole acetic acid
by a Rhizobium sp from root nodules of a leguminous climer,
Psophocarpus Tetragonolobus dc. Indian J. Exper. Biol. 30:632-635.
Bhowmik PK, Basu PS (1994). Contents of hormones and IAA
metabolism in root nodule of Erythrina indica Lamk., S. grandiflora
Pers. and Pterocarpus santalinus Linn. Biochemica Physioligie-derpflanzen. 179:455-462.
Boylan SA, Redfield AR, Brody MS, Price CW (1993) Stress-Induced
B
Activation of the σ Transcription Factor of Bacillus subtilis. J.
Bacteriol. 175(24):7931-7937.
Chakravorty S, Helb D, Burday M, Connell N, Alland D (2007). A
detailed analysis of 16S ribosomal RNA gene segments for the
diagnosis of pathogenic bacteria. J. Microbiol Methods 69:330-339.
Coughlan MP, Mayer F (1992). The cellulose-decomposing bacteria
and their enzyme systems. In: Balows A, Truper HG, Dworkin M,
Harder W, Schleifer K, editors. The prokaryotes. A handbook on the
biology of bacteria. ecophysiology, isolation, identification,
applications, vol. I. 2nd ed. Berlin, Germany: Springer-Verlag. pp.
460-516.
Dobbereinere S A, Croonenborghs M, Thys A, Ptacek D, Vanderleyden
J (2001). Response of agronomically important crops to inoculation
with Azospirillum. Australian J. Plant Physiol. 28:871-879.
Dobereiner J (1995). Isolation and identification of aerobic N 2-fixing
bacteria from soil and plants. In: Methods in Applied Soil Microbiology
and Biochemistry, (K. Alef and P. Nannipieri Eds.) Academic Press,
London. pp: 134-141.
Duca D, Lorv J, Patten CL, Rose D, Glick BR (2014). Indole-3-acetic
acid in plant-microbe interactions. Antonie Leeuwenhoek 106(1):85125.
Eckert B, Weber OB, Kirchhof G, Halbritter A, Stoffels M, Hartmann A
(2001). Azospirillum doebereinerae sp. A nitrogen-fixing bacterium
associated with the C4-grass Miscanthus. Int. J. Syst. Evol. Microbiol.
51:17-26.
Elgaml A, Hassan R, Barwa R, Shokralla S, El-Naggar W (2013).
Analysis of 16S ribosomal RNA gene segments for the diagnosis of
Gram negative pathogenc bacteria isolated from urinary tract
infections. Afr. J. Microbiol. Res. 7(23):2862-2869.
El-khawas H, Adachi K (1999). Identification and quantification of auxins
in culture media of Azospirillum and Klebsiella and their effect on rice
roots. Biol. Fertil. Soil 28:377-381.
Emtiazi G, Pooyan M, Shamalnasab M (2007). Cellulase Activities in
Nitrogen Fixing Paenibacillus Isolated from Soil in N-free Media.
World J. Agric. Sci. 5: 602-608.
Femi-Ola TO, Aderibigbe EY (2008). Studies on the effect of some
wood extracts on growth and celluase production by strains of
Bacillus subtilis. Asian J. Plant Sci. 1-3.
Frankenberger WT, Arshad M (1995). Phytohormones in soils: Microbial
production and function. Marcel Dekker Inc. N Y. 503.
Gordon SA, Weber RP (1951). Colorimetric estimation of indole acetic
acid. Plant Physiol. 26:192-195.
Idris EE, Makarewicz O, Farouk A, Rosner K, Greiner R, Bochow H,
Richter T, Borriss R (2002). Extracellular phytase activity of Bacillus
amyloliquefaciens FZB45 contributes to its plant-growth-promoting
effect. Microbiology 148: 2097-2109.
Ikeda Z, Kobavashi T, Takahashi H, Jyunichi I, Kobayasi M, Tkashashi
E (1989). The effect of salt stress on growth, IAA productivity and
extracellular polysaccharides productivity of Rhizobium meliloti and
other Rhizobium sp. Jpn. J. soil. Sci Sci. Soil Manure Plant Nutr.
60:41-46.
Kirchhof G, Eckert B, Stoffels M, Baldani JI, Reis VM, Hartmann A
(2001). Herbaspirillum frisingense sp. nov., a new nitrogen-fixing
bacterial species that occurs in C4-fibre plants. Int. J. Syst. Evol.
Microbiol. 51:157-168.
Koga J, Adachi T, Hiaka H (1991). IAA biosynthetic pathway from
tryptophan via indole-3-pyruvic acid in Enterobacter cloacae. Agric.
Biol. Chem. 55:701-706.
Manulis S, Shafrir H, Epstein E, Lichter A, Barash I (1994). Biosynthesis
of IAA via the indole-3-acetamide pathway in Streptomyces spp.
Microbiology 140:1045-1050.
Mishra VK, Kumar A (2012). Plant growth promoting and
phytostimulatory potential of Bacillus subtilis and Bacillus
amyloliquefaciens. J. Agric. Biol. Sci. 7 (7): 509-512.
Park YW, Lim ST, Cho SJ, Dae H, Yun HD (1997). Characterization of
Erwinia carotovora subsp. Carotovora LY34 endo-1,4-b-Glucanase
Genes and Rapid Identification of Their Gene Products. Biochem.
Biophys. Res. Commun. 241:636-641.
Patten CL, Glick BR (2002). Role of Pseudomonas putida Indole acetic
acid in development of the host plant root system. Appl. Environ.
Microbiol. 68(8):3795-3801.
Saharan BS, Nehra V (2011). Plant growth promoting rhizobacteria: A
Critical Review. Life Sci. Med. Res. 2011(21):1-30.
Sainio EL, Pulkki K, Young SN (1996). L-Tryptophan: Biochemical,
nutritional and pharmacological aspects. Amino Acids 10:21-47.
Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: A
Laboratory Manual Cold Spring Harbour Laboratory, N Y.
Spaepen S, Vanderleyden J, Remans R (2007). Indole-3-acetic acid in
microbial and microorganism-plant signaling. FEMS Microbiol. Rev.
1-24.
Strzelczyk E, Kampert M, Pachlewski R (1994). The influence of pH and
temperature on ethylene production by mycorrhizal fungi of pine.
Mycorrhiza 4:193-196.
Tatum EL, Bonner DM (1944). Indole and serine in the biosynthesis and
breakdown of tryptophan, Proc. Nat. Acad. Sci. USA. 30:30-37.
Tyler HL, Triplett EW (2008). Plants as a habitat for beneficial and/or
Mishra and Kumar
human pathogenic bacteria. Ann. Rev. Phytopathol. 46: 53-73.
Woodward AW, Bartel B (2005). Auxin: Regulation, Action, and
Interaction. Ann. Bot. (Lond) 95:707-735.
Yamada T, Tsukamopto H, Shrraishi T, Nomura T, Oku H (1990).
Detection of IAA biosynthesis in some species of taphrina causing
hyperplasic diseases in plants. Ann. Phytopathol. Soc. Jpn. 56:532540.
1149
Zakharova EA, Iosipenko AD, Ignatov VV (2000). Effect of water-soluble
vitamins on the production of indole-3-acetic acid by Azospirillum
brasilense. Microbiol. Res. 155:209-214.
Vol. 9(16), pp. 1150-1161, 22 April, 2015
DOI: 10.5897/AJMR2013.5909
Article Number: 7D46FAD52663
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Assessment of phenol biodegradation capacity of
indigenous bacteria isolated from sewage
treatment plant
N. R. Das1* and N. Kumar2
1
Centre for Environment Science and Climate Resilient Agriculture (CESCRA), Indian Agricultural Research Institute
(IARI), Pusa, New Delhi,110012, India.
2
Centre for Environmental Science, School of Earth, Biological and Life Sciences, Central University of Bihar, Patna,
Bihar, 800014, India.
Received 30 April, 2013; Accepted 25 March, 2015
Among different xenobiotics, phenol is a man made as well as a naturally occurring aromatic compound
and an important intermediate in the biodegradation of natural and industrial aromatic compounds. The
investigation was undertaken to isolate, characterize and exploit phenol degrading bacteria from
sewage treatment plant (STP) (artificial ecosystem having diverse group of bacteria which are adapted
to different aromatic pollutant and capable to degrade xenobiotic aromatic organic pollutants). Out of
five different phenol degrading bacteria, one potent strain (IS-3) was identified as Citrobacter freundii
with maximum degradation capacity of 1000 ppm phenol in three days under in vitro studies. Phenol
degradation performance was greatly influenced by different physical factors like incubation
temperature, supplemented glucose, nitrogen source, NaCl and pH of the growth medium. The
maximum phenol degradation was observed at incubation temperature of 33°C, 7.5 pH of the medium,
0.1 gl-1 of NaCl, 0.25 gl-1 of glucose and 0.25 gl-1 of ammonium sulphate. We report in this study that the
identified potential strain (IS-3) can be used for treatment of phenol contaminated waste water
maintaining above the mentioned optimum factors for faster degradation of the phenol contaminated
industrial effluents.
Key words: Xenobiotics, phenol, bio-degradation.
INTRODUCTION
With the advent of modern technology and
industrialization, contamination of the natural resources
by chemicals has become a serious concern. A huge
quantity of xenobiotic compounds especially phenolic
compounds are introduced into the ecosystem every day
due to the production of agricultural, industrial and
pharmaceuticals products. These phenolic compounds
are often found in waste water from coal-gasification,
*Corresponding author. E-mail: soilnami@gmail.com/namitadas@iari.res.in. Tel: +91-9711375220.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
Das and Kumar
coke-oven batteries, refinery and petrochemical plants
and other industries, such as synthetic chemicals,
herbicides, pesticides, antioxidants, pulp and paper,
photo developing chemicals, etc. (Kavita and Palanivelu,
2004; Jayachandran and Kunhi, 2008). Phenols released
into the environment from different industries are of high
concern because of their potential toxicity. Many of the
phenolic compounds are carcinogenic and are included in
the U.S. Environmental Protection Agency’s priority
pollutant lists (Zhang and Wiegel, 1990; Neumaan et al.,
2004) and these toxic and xenobiotic chemicals cause
challenging problems for the environment due to
recalcitrant in nature. According to WHO (1994), the
concentration of phenol has been prescribed as 1 µgl -1 in
drinking water. Therefore, the removal of such chemicals
from industrial effluents is of great concern. The annual
production of phenol is around 8.9 million tonnes in 2012
which is again expected to be increased to 10.7 million
tonnes in 2016 (Anonymous, 2015). Phenol and their
derivatives are aromatic organic compound and resistant
to natural biodegradation and persist in the environment
for long period (Juang et al., 2006). Current methods for
removing phenols from wastewater include hybrid
process (Bodalo et al., 2008) like electro catalytic
degradation (Wang et al., 2009), adsorption on to
different matrices, chemical oxidation, solvent extraction
or irradiation (Spiker et al., 1992). Most of these
processes are expensive and also generate other toxic
byproducts (Ajay et al., 2004). One of the cheapest
possible solutions to resolve phenol contamination
problem is bioremediation using vast and diverse
microbes (Basak et al., 2014). Biodegradation is a viable
bioremediation technology for organic pollutants by using
microorganisms to remove or detoxify toxic or unwanted
chemicals in an environment. A goal of bioremediation is
to transform organic pollutants into harmless metabolites
or mineralize the pollutants into carbon dioxide and water
(Rittman and McCarty, 2001). Work on phenol biodegradation has been reported by using pure and mixed
cultures of bacteria which are able to thrive on high
concentration of phenol (Gonzalez et al., 2001; Shen et
al., 2009; Basak et al., 2014). Wang et al. (2007) isolated
a new phenol-degrading bacterium Acinetobacter sp.
strain PD12 from activated sludge and reported that there
still need to isolate new phenol-degrading bacteria that
can grow at elevated concentration of phenol. Besides
these, the other optimum physical conditions like temperature, pH, additional substrate supplementation on
biodegradation efficiency of naturally occurring microbial
strains can also provide further insight into the bioremediation process. Therefore, the present investigation
was carried out to isolate and to characterize phenol
degrading indigenous bacteria from sewage treatment
plant (STP) and to study degradation kinetics of phenol
degradation under different growth conditions (pH,
incubation temperature, additional nutrient sources, salt
1151
amendment and additional different carbon sources).
MATERIAL AND METHODS
Site description and sample collection
To isolate potent bacteria with phenol degrading capacity, sewage
water sample was collected from sewage treatment plant of
Saidpur, Patna, Bihar, India (latitude 25o34’ 31” N and longitude
85o05’52” E). The initial phenol concentration of the sewage water
(before treatment) was 453 ppm. The waste water was stored at
4C in a closed and previously sterile bottle for further analysis.
Growth medium and isolation of phenol degrading bacteria
Indigenous bacteria of sewage waste water were isolated in mineral
salt agar medium supplemented with phenol concentration of 200
mg l-1. The compositions of mineral salt medium (MSM) in g l-1 were
KH2 PO4 (0.42), K2HPO4 (0.375), (NH4)2SO4 (0.244), NaCl (0.015),
CaCl2.2H2O (0.015), MgSO4.7H2 O (0.05) and FeCl3.6H2 O (0.054).
Sewage water sample was serially diluted, spread plated and
incubated at 30C.
Morphological characterization
The isolated colonies of each pure strain were streaked upon solid
agar plates for detailed morphological characterization. The colonial
shape, color, texture and opacity were recorded following standard
protocol (Pelczar and Reid, 1958). Based on morphological
difference, five different bacterial strains were isolated. Gram nature
of isolates has been carried out using Gram staining and individual
cellular morphology in presence and absence of phenol were
studied using scanning electron microscope (Eichorst et al., 2007).
The pure cultures were maintained in serum bottles in mineral salt
medium (MM1) containing 200 ppm phenol.
Screening of potential phenol degrading bacteria
In this method, best phenol degrading isolate was enriched by
continuously supplying small portions of phenol to the medium. All
the five isolates were spread and grown in the MM1 media plates
enriched with 200, 500, 800 and 1000 ppm of phenol. The isolate
which survived in the maximum concentration of phenol exposure
was selected for further study.
Characterization and identification of the phenol resistant
isolate
The isolates were characterized based on their morphology by
following Bergey’s Manual of Systematic Bacteriology (2001),
biochemical characteristics, metabolic versatility for different carbon
substrate utilization and Gram’s staining. Motility was assessed by
direct microscopic observation during growth and by testing the
ability of the strains to migrate from the point of inoculation through
semisolid agar (0.3%) plates containing 20 mM succinate (Adler,
1966). The isolates were verified by using BIOLOG plates
(BIOLOG, Hayward, CA, USA), Analytical Profile Index (API)
scheme for characterization.
BIOLOG identification of bacteria
Bacteria demonstrating consistent biodegradation capacity for
1152
Afr. J. Microbiol. Res.
Figure 1a. Biolog MicroTitre Ecoplate for bacterial identification.
phenol were selected for confirmation of its identification using the
MicrologTM system (BIOLOG, Inc., Hayward, CA). Single colonies
were obtained by streaking on media and the following steps were
performed in the process of identification: (i) bacteria were streaked
onto BIOLOG universal growth (BUG) agar medium (BIOLOG, Inc.);
(ii) approximate bacterial number was quantified by serial dilution
and plate technique and 150 µl of the bacterial solution were
pipetted into each of the 96 wells in the BIOLOG microplates
(Figure 1a); (iii) the plates were incubated at 30C for 16–24 h and
then read with an automated ELISA plate reader (BIOLOG, Inc.)
and also assessed visually and identified to species level.
Estimation of phenol biodegradation
Residual phenol concentration was measured following 4aminoantipyrine colorimetric method based on the standard
methods for the examination of water and wastewater (APHA,
1998) with little modifications (wavelength 500 nm). The analytical
procedure included the following chemicals viz. 0.5 N sodium
bicarbonate, 0.6% (w/v) 4-aminoantipyrine, 2.4% (w/v) potassium
ferrocynate. For quantification of phenol in broth cultures, 2 ml broth
culture was withdrawn in a micro-centrifuge tube and centrifuged at
12000 rpm for 3 min. 20 µl of this supernatant was added with 80 µl
distilled water to make 1:5 dilution. These were then used for
phenol estimation by antipyrine method (APHA, 1998).
Optimization of physical factors for phenol degradation by the
isolates and data analysis
The optimum physical factors such as pH of the medium,
temperature, carbon source, NaCl and ammonium sulphate (as
nutrient source) were evaluated for maximum phenol degradation.
Phenol degradation at incubation temperature from 22 to 40C with
constant initial concentration of phenol (200 mg l-1) and neutral pH
in absence of carbon was carried out. Similarly, other parameters
were kept constant. To see the effect of different pH level on phenol
biodegradation, five different pH level (6, 6.5, 7, 7.5 and 8) was
maintained. For optimization of glucose as a carbon source, the
culture was kept at 30C with neutral pH and four different glucose
concentrations (no glucose, 0.25% glucose, 0.5% of glucose and
1% glucose). The residual phenol concentration was measured at
time slots of 24, 48, 72 and 96 h after inoculation with pure isolates.
Each level of factors was replicated three times. Data generated
from the experiment was analysed statistically using statistical
software INDOSTAT (version 8.0).
RESULTS AND DISCUSSION
Characterization and screening of phenol degrading
isolates
In our study, the treated sewage water and sediment of
Saidpur STP of Patna, India was used for isolation of
bacterial strain. Initially, a total of ten strains were
isolated among which five isolates (IS-6, IS-7, IS-8, IS-9
and IS-10) showed identical characteristics. The
remaining five isolates (IS-1, IS-2, IS-3, IS-4 and IS-5)
showed distinct colony morphology and preliminarily
chosen for phenol degrader (Table 1). These five
bacterial strains were found capable to use phenol as a
sole source of carbon and energy. All the isolates except
IS-3 were found as single celled, mono-cocci. IS-3 was
found as single celled, rod shaped Gram-negative bacilli.
Scanning Electron Microscopic (SEM) view confirmed
that cell size of IS-3 varied from 2-5 µM in length. During
exponential phase, the cells of IS-3 were found motile by
means of several peritrichous flagella (Figure 1b). Motility
of IS-3 was least to negligible in the cultures at stationary
phase. IS-3 was found as most efficient in terms of
phenol degradation as compared to other strains (IS-2,
IS-1, IS-4 and IS-5). The phenol degradation rate of IS-3
(18 mgl-1h1) was almost thrice than IS-1 and IS-2 and
twice than IS-4 and IS-5 (Figure 2). As IS-3 is a native
and indigenous bacterial strain isolated from phenol
polluted sewage waste water, hence, higher degradation
capacity for xenobiotic compounds corresponds to earlier
Das and Kumar
1153
Table 1. Characterization of phenol degrading isolates of sewage treatment plant waste water (Saidpur, Patna, Bihar, India).
Colony morphology (in MM1
media)
Orangish, circular
Cell
morphology
Mono-cocci
polar
Optimum
pH
6.5
Phenol tolerance
(ppm)
1000
Generation time
(h)
12
IS-2
Light creemish, circular,
transperaent
Mono-cocci
Polar
7.0
1000
12
IS-3
Dirty creemish, circular,
transparent
Rod
Peritrichus
7.5
1800
9.5
IS-4
White, circular, wrinkled,
opaque
Mono-cocci
Peritrichus
7.0
1200
11
IS-5
Light orangish, small circular,
opaque
Mono-cocci
Polar
7.0
1500
12
Strain
IS-1
Flagella
Figure 1b. Scanning electron microscopic (SEM) view of IS-3 (Citrobacter freundii).
Figure 2. Degradation kinetics of phenol (initial enrichment of 200 ppm) by five
different isolates (All the results are average of 3 replications).
1154
Afr. J. Microbiol. Res.
Table 2.
Metabolic versatility for different carbon substrate
utilization pattern (foot print) of the isolates studied by BIOLOG.
Carbon substrates
Pyruvic acid methyl ester
Tween 40
Tween 80
α-Cyclodextrin
Glycozen
D-Cellobiose
α -D-lactose
β- Methyl-D-Glucoside
D-Xylose
i-Erythritol
D-Mannitol
N-Acetyl-D-Glucosamine
D-Glucosamine Acid
Glucose-1-Phosphate
D,L-α-Glycerol Phosphate
D-Galactonic acid γ-Lactone
D-Galacturonic Acid
2-Hydroxy Benzoic Acid
4-Hydroxy Benzoic Acid
γ-Hydroxybuteric Acid
Itaconic Acid
α-Ketobutyric Acid
D-Malic Acid
L-Arganine
L-Asparagine
L-Phenylalanine
L-Serine
L-Threonine
Glycyl-L-Glutamic Acid
Phenylethyl-amine
Putrescine
IS-1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
IS-2
+
+
+
+
+
+
+
-
Isolates
IS-3 IS-4
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
IS-5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Symbols: +, positive reaction; - negative reaction.
adaptation to phenolic compounds present in the waste
water. Buitron and Gonzalelez (1996) also reported that
previously adapted microbes perform faster degradation
than the others. As sewage treatment plant is an artificial
ecosystem where diverse group of bacteria are found and
which have an exposure for diverse group of xenobiotics
are capable to degrade a large group of xenobiotic
aromatic organic pollutants. Hence, in treated sewage
waste water, chances for presence of already adapted
and acclimatized phenol degrading bacteria are quite
more. Even on similar assumptions, the presence of
indigenous microorganisms in contaminated soil (with
industrial oil) was chosen as due to high probability of the
presence of and acclimatized microorganisms (Nuhoglu
Table 3. Metabolic versatility for different aromatic compound
utilization for carbon and energy by the isolates under aerobic
conditions.
Aromatic carbon substrates
Phenol
p-Cresol
4-OH benzylalcohol
4-OH benzylaldehyde
4-OH benzoate
Protocatechuate
Benzoate
Benzyl alcohol
Benzylaldehyde
Phenylacetate
4-OH phenylacetate
Cinnamic acid
Hydrocinnamic acid
Phenylalanine
Tyrosine
2-Aminobenzoate
Toluene
Benzene
Tryptophan
2-aminophenol
4-aminophenol
IS1
+
+
+
+
+
+
+
+
+
+
+
+
_
+
+
IS2
+
+
+
+
+
+
+
+
+
Strains
IS3 IS4
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
IS5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
All compounds were tested at a concentration of 200 ppm under
aerobic conditions, +, positive reaction; -, negative reaction.
and Yalcin, 2005).
Metabolic versatility
As IS-3 showed good performance in terms of phenol
degradation in comparison to other isolates, this strain
was tested for their metabolic versatility by judging their
ability to utilize different carbon substrates by using
BIOLOG GN-III plates to identify the isolate at species
level (Tables 2 and 3). Biolog’s powerful carbon source
utilization technology accurately identifies environmental
and pathogenic microorganisms by producing a
characteristic pattern or “metabolic fingerprint” from
discrete test reactions performed within a 96 well
microplate. The scope of the 96 assay reactions, coupled
with sophisticated interpretation software, delivers a high
level of accuracy that is comparable to molecular
methods. Stefanowicz et al. (2006) also reported the
efficiency for identification of bacterial strains up to
species level. Results of metabolic foot print for utilization
of simple sugar to complex aromatics carbon substrates
are summarized in Table 2. Among the five isolates, IS-3
had shown more catabolic versatility in terms of utilization
of more diverse and complex carbon substrate utilization.
Das and Kumar
1155
Figure 3. Degradation kinetics of residual phenol (initial enrichment of 500 ppm)
by STP isolates (All the results are average of 3 replications).
Carbon substrate utilization foot print was matched with
the inventory and IS-3 was confirmed as C. freundii.
According to Bergey’s manual of systematic Bacteriology
(2001), 96% of results showed the similarity in
characteristics with C. freundii. Various phenol degrading
micro-organisms including bacteria, yeast, fungi and
algae have been reported previously (Yang and
Humphrey, 1975; Alcocer et al., 2007; Dong et al., 2008;
Banerjee and Ghosal, 2010). Among them, bacteria are
of specific importance. Bacteria (Pseudomonas sp.,
Acinetobacter sp.), yeast (Pleurotutus ostreatus, Candida
tropicalis, Trichosporon cutaneum and Phanerochaete
chrysosporium) and fungi (Fusarium flucciferum and
Aspergillus fumigates) can degrade phenol and among
algae, Ochromonas danica can degrade phenol (Ariana
et al., 2004). Although, there are several report about
diverse group of microorganisms belong to several
genera for phenol degradation (Kennes and Lema, 1994;
Bandyopadhyay et al., 1998; Annadurai et al., 2007;
Varma and Gaikwad, 2009; Basak et al., 2014) but there
are no published report on Citrobacter sp. for phenol
degradation. While the isolated strains were tested for
their ability to mineralize or transform a large variety of
simple aromatic compounds under aerobic conditions,
similar trends was noticed for IS-3 (Table 3).
Degradation kinetics of phenol and screening of
potent phenol degrading isolate
There were five distinct colonies identified after serial
dilution. Their morphological features are presented in
Table 1. Although, all of the five isolates were found
capable of degrading phenol but IS-3 was found
significantly different in phenol degradation capacity.
Responses of all of the isolates were significantly
different from each other while tested in 200 ppm of initial
enrichment of phenol in the media. Out of different
isolates, IS-3 degraded 200 ppm of initial enrichment of
phenol completely after 72 h of inoculation [Critical
difference (CD) =4.09] (Figure 2). The IS-2 was also able
to degrade completely within 96 hrs. At 500 ppm of initial
enrichment of phenol, IS-3 was able to degrade it
completely in 96 h (Figure 3). After 5 days of inoculation
of IS-2 in MM1 broth containing 500 ppm of phenol,
residual phenol concentration of 40 ppm was found. After
5 days of inoculation, IS-1 and IS-4 in MM1 broth
containing 800 ppm of initial enrichment of phenol had
residual phenol concentration between 100 to 150 ppm
(CD=3.93; Figure 4). At 1000 ppm of initial enrichment of
phenol, IS-3 was able to degrade it completely within 5
days and the rest of the isolate had shown residual
concentration of phenol in between 400 to 600 ppm
(CD=3.16; Figure 5). Among all isolates these, IS-3 was
found very much potent to degrade phenol at all initial
enrichment irrespective of lower to higher concentration
(200-1000 ppm). Overall, IS-3 was found faster and most
efficient degrader which can be exploited for further
bioremediation
studies.
Acclimatization
of
the
microorganisms and the bacteria which are adaptive in
general overcomes the substrate inhibition problems that
normally occurred in phenol biodegradation at high
concentration (Lob and Tar, 2000).
Optimization of physical factors for efficient
degradation of contaminated phenol present in waste
water
Effect of incubation temperature
biodegradation by Citrobacter freundii
on
phenol
C. freundii was able to grow and degrade at temperature
ranging from 22-40C (Figure 6a). However, maximum
1156
Afr. J. Microbiol. Res.
Figure 4. Degradation kinetics of residual phenol (initial enrichment of 800 ppm) by
STP isolates.
Figure 5. Degradation kinetics of residual phenol (initial enrichment of 1000 ppm)
by isolates.
phenol degradation (420 ppm) was noticed at 33C and
24 h after inoculation. Even at 35 and 37C, biodegradation was significantly (p<0.05) higher while
compared with biodegradation rate with lower incubation
temperature (22, 25, 28 and 30°C). As incubation
temperature increased from 33°C onwards, a declining
trend in the degradation of phenol was noticed. Although
in all incubation temperature except 33°C, complete
phenol (initial enrichment of 500 ppm) was utilized as
sole source of carbon and energy within five days after
incubation, but degradation kinetics was quite slower in
both lower and higher temperature regimes (except at
optimum point) while after 72 h at 22 and 40°C the
residual concentration of phenol was 252.57 and 180.98
ppm, respectively. This result corroborates with previous
report of phenol degradation by Pseudomonas sp.
(Polymenakou and Stephanou, 2005) but contradict the
findings of Rosa et al. (2004) where 30C was optimum
for biodegradation of phenol. At elevated temperature
exposure than the optimum one, showed decreasing
trend in the biodegradation capacity. Similar trends had
been reported by Gurusamy et al. (2007) for
Pseudomonas pictorum.
Effect of growth medium pH
biodegradation by Citrobacter freundii
on
phenol
To see the effect of pH on phenol degradation by IS-3
(Citrobacter freundii), five different pH level (6, 6.5, 7, 7.5
Das and Kumar
1157
Figure 6a. Effect of incubation temperature on phenol degradation by Citrobacter freundii
(IS-3). Initial pH of the growth medium was maintained 7 and medium was enriched with
500 ppm of phenol (T1: 22C; T2: 25C; T3: 28C; T4: 30°C; T5: 33C; T6: 35C; T7:
37°C; T8: 40C).
.
Figure 6b. Effect of pH of the growth medium on phenol degradation by Citrobacter
freundii (IS-3). Incubation temperature for the culture was maintained at 30C and
medium was enriched with 500 ppm of phenol (P1:6; P2:6.5; P3:7; P4:7.5; P5:8).
and 8) were adjusted. Phenol degradation was greatly
influenced by initial culture pH (Figure 6b). Out of 500
ppm, maximum phenol degradation (440 ppm) was
noticed at pH level of 7.5 which later on was confirmed as
the optimum pH level for highest biomass or cell growth
for Citrobacter freundii under 30C and absorbance was
taken at 600 nm. Citrobacter belongs from
Enterobacteriaceae family and the strain which was
isolated from STP waste water, favoured slightly alkaline
pH range. Although in neutral pH range a significant and
good degradation (380 ppm in 24 h after inoculation) rate
was noticed but was significantly less (p<0.05) than the
degradation observed at 7.5 pH level. Though growth of
the isolate was not suppressed completely at pH 6 but
the level of degradation (180 ppm at 24 h after
inoculation) was significantly lower than pH 7, 7.5 and 8.
However, phenol degradation rate was found statistically
at par for pH 6 and 6.5. Suhaila et al. (2010) also
reported highest phenol degradation, which was
associated with the highest growth of Rhodococcus
1158
Afr. J. Microbiol. Res.
Figure 6c. Effect of augmentation of the growth media with different concentration of
Ammonium Sulphate as extra nutrient source on phenol degradation by Citrobacter
freundii (IS-3). Initial temperature and pH of the growth medium was 30 C and 7,
respectively and medium was enriched with 500 ppm of phenol (A1:0 g/l); A2:0.10 g/l;
A3:0.25 g/l; A4:0.50 g/l;A5:0.75 g/l; A6:1 g/l).
UKM-P at neutral pH range of 7 to 7.5, where all the
phenol present in the culture (0.5 gl-1) was degraded.
Higher pH (>8) inhibited phenol degradation. At pH higher
than 8, growth of Rhodococcus UKM-P was slightly
inhibited, which resulted into incomplete degradation of
phenol present in the culture (Suhaila et al., 2010).
Similarly for Citrobactor, growth was slightly inhibited
from pH 8 onwards.
Effect of nitrogen source on phenol biodegradation
by Citrobacter freundii
To see the effect of external nutrient source of nitrogen
on phenol degradation by C. freundii, ammonium
sulphate was amended at different level and results are
presented in Figure 6c. Among different amendment
-1
level, 0.25 gl of ammonium sulphate depicted highest
(456 ppm) degradation of phenol out of 500 ppm.
Although, further higher level of amendment encouraged
a good growth of bacterial biomass, but significantly
suppressed phenol degradation. At 1 gl-1 of enrichment
with ammonium sulphate had contributed only 56 ppm
phenol degradation. This trend was almost comparable
with 0.5 and 0.75 gl-1 enrichment with ammonium
sulphate as nitrogen source. Our result contradict the
findings of Suhaila et al. (2010) where highest
degradation of phenol by Rhodococcus erythropolis was
-1
obtained at 0.4 and 0.8 gl of amendments of ammonium
sulphate. This may be because of the fact that our
isolated bacteria was different from the one reported by
Suhailia et al. (2010). The microbes are versatile in
nature. Even similar species isolated from two different
habitats may behave differently in their metabolic activity
(Martin dos Santos et al., 2008).
Effect of glucose concentration
biodegradation by Citrobacter freundii
on
phenol
Phenol degradation capacity by isolated C. freundii was
examined at different level (no glucose, 0.25, 0.5 and 1
gl-1) of glucose amended in the growth media and
residual phenol was estimated at 24 hr after inoculation
and incubated at 30°C (Figure 6d). The result envisages
highest phenol degradation (426 ppm out of 500 ppm) at
0.25 gl-1 of glucose. The degradation rate had dropped
down to 32% at 0.5 gl-1 of glucose concentration. This
drastic decrease in the degradation rate may be due to
presence of glucose in the media which is a simple sugar
and easily available to the inoculum. When easily
available substrates are present bacteria try to avoid
oxidation and utilization for complex substrates like
phenol present in media. Similarly, further increase in the
glucose concentration suppressed phenol degradation
kinetics and only 67 ppm out of 500 ppm of phenol was
degraded at 24 h after inoculation. In earlier study, Kar et
al. (1996) showed the effect of glucose on phenol
degradation and the results indicate that when a mixed
substrate (phenol and glucose) was used, phenol
acclimatized population showed initial preference for
phenol to glucose concentration. But our results are in
contradiction with the report of Kar et al. (1996). From
this study it is clear that higher concentration of glucose
curtailed down utilization of phenol as a sole source of
carbon and energy. This result corroborates with earlier
works done by Suhaila et al. (2010). A glucose
concentration of 0.5% repressed the induction of phenol
Das and Kumar
1159
Figure 6d. Effect of amendment with different concentration of glucose on phenol
degradation by Citrobacter freundii (IS-3). Initial temperature and pH of the growth
medium was 30C and 7, respectively and medium was enriched with 500 ppm of phenol
(G1:0 g/l; G2:0.25 g/l; G3: 0.50 g/l; G4:1 g/l).
Figure 6e. Effect of amendment with different concentration of NaCl on phenol
degradation by Citrobacter freundii (IS-3). Initial temperature and pH of the growth
medium was 30o C and 7, respectively and medium was enriched with 500 ppm of
phenol (N1 0 g/l; N2 0.1 g/l; N3:0.25 g/l; N4: 0.50 g/l; N5:0.75 g/l; N6:1 g/l)
oxidation though glucose and did not fully repress
utilization of phenol. Similar kind of results was obtained
by Khaled (2006).
Effect of different level of NaCl concentration on
phenol biodegradation by Citrobacter freundii
A result pertaining to effects of different NaCl
concentration on phenol degradation is presented in
Figure 6e. The highest degradation of phenol (449 ppm
out of 500 ppm that is 89%) was noticed in 0.1% NaCl
level. A little increase in NaCl level (0.25 gl -1) had
revealed a significant suppression in the degradation
rate. Similar kinds of trends were also reported by
Suhaila et al. (2010). Higher phenol degradation at lower
concentration of NaCl may be attributed towards
encouraged bacterial growth with small NaCl
amendment. Further increase in the NaCl level upto 1 gl -1
had suppressed phenol degradation to a negligible
status. This repression in the degradation may be
attributed to inhibitory effect on bacterial growth as NaCl
1160
Afr. J. Microbiol. Res.
at higher concentration poses preservative or
antimicrobial effect. Even though the effects of other
factors on phenol biodegradation was studied vastly
(Chakraborty et al., 2010) but we report here the effects
of NaCl amendment on phenol degradation by phenolacclimatized pure culture.
Conclusion
The study reveals that IS-3 (C. freundii) was highly
capable of degrading phenol when the initial enrichment
of phenol varied from lower (200 ppm) to higher (1000
ppm) concentration and moreover this strain showed
tolerance for phenol at higher level (1800 ppm). This
strain degrades effectively and rapidly the phenol present
in the water sample. Phenol degradation performance
was greatly influenced by different physical factors like
incubation temperature, supplemented glucose, nitrogen
source, NaCl and growth medium pH. From this
investigation, optimum factors were observed as 33C
(incubation temperature), 7.5 (pH of the medium), 0.1 gl-1
of NaCl, 0.25 gl-1 of glucose and 0.25 gl-1 of ammonium
sulphate as external amendment for bio augmentation.
Members of the genus Citrobacter are known pollutant
degrader and from this study it is clearly revealed that a
slight manipulation or alteration or optimization of the
physical factors may enhance the phenol degradation
capacity greatly. Hence, this strain (IS-3) is a potent
candidate for utilization in the waste water treatment for
phenol contaminated effluent from different industries as
well as bioremediation and restoration of a degraded site.
Conflict of interests
The authors did not declare any conflict of interest.
ACKNOWLEDGEMENT
Authors are thankful to the Central University of Bihar
(CUB), Patna, India for the financial support and
providing laboratory facilities for conducting this work.
REFERENCES
Adler J (1966). Chemotaxis in bacteria. Sci. 153: 708-716.
Ajay KJ, Vinod KG, Subhi J, Suhas (2004). Removal of chlorophenol
using industrial wastes. Environ. Sci. Technol. 38: 1195-1200.
Alcocer AS, Ordaz NR, Ramírez CJ, Mayer JG (2007). Continuous
biodegradation of single and mixed chlorophenols by a mixed
microbial culture constituted by Burkholderia sp. Microbacterium
phyllosphaerae, and Candida tropicalis. Biochem. Eng. J. 37:201211.
Annadurai G, Ling LY, Fwulee J (2007). Biodegradation of phenol by
Pseudomonas pictorum on immobilized with chitin. Afr. J. Biotechnol.
6(3):296-303.
Anonymous (2015). Phenol: 2015 World Market Outlook and Forecast
up to 2019. http://mcgroup.co.uk/researches/phenol.
APHA (1998): Standard methods for the examination of water and
waste water.Clesceri LS, Greenberg AE, Eaton AD (Eds.),
Washington DC.
Ariana F, Elke B, Thomas B (2004). Rapid monitoring of the
biodegradation of phenol-like compounds by the Candida maltosa
using BOD measurements. Int. Biodete. Biodegr. 54: 69-76.
Bandyopadhyay K, Das D, Maiti BR (1998). Kinetics of phenol
degradation using Pseudomonas putida MTCC 1194. Bioprocess
Eng. 18: 373-377.
Banerjee A, Goshal AK (2010). Phenol degradation by Bacillus cerces.
Pathway and Kinetic modeling. Bioesour. Technol. 101: 5501-5507.
Basak SP, Sarkar P, Pal P (2014). Isolation and characterization of
phenol utilizing bacteria from industrial effluent-contaminated soil and
kinetic evaluation of their biodegradation potential. J. Environ. Sci.
Health. Part A: Toxic/Hazardous Sub. Environ. Eng. 49:66-77.
Bergey’s Manual of Systematic Bacteriology (2001). Boone
nd
DR, Castenholz RW. (Eds.), 2 Edition, Springer Science+Business
Media, New York.
Bodalo A, Gomez JL, Gomez M, Leon G, Hidalgom AM, Ruiz MA
(2008). Phenol removal from water by hybrid processes: Study of the
membrane process step. Desalination, 223: 323-329.
Buitron G, Gonzalez A (1996). Characterization of the microorganisms
from an acclimated activated sludge degrading phenolic compound.
Water Sci. Technol. 34: 289-294.
Chakraborty S, Bhattacharya T, Patel TN, Tiwari KK (2010).
Biodegradation of phenol by native microorganisms isolated from
coke processing wastewater. J. Env. Biol. 31:293-296.
Dong X, Hong Q, He L, Jiang X, Li S (2008). Characterization of phenoldegrading bacterial strains isolated from natural soil. Inter.
Biodeterior. Biodegrad. 62: 257-262.
Eichorst SA, Breznak JA, Schmidt TM (2007). Isolation and
characterisation of soil bacteria that define Terriglobus gen. nov., in
Phylum Acidobacteria. Appl. Environ. Microbiol. 73: 2708-2717.
Gonzalez G, Herrera G, Garcia MT, Pena M (2001). Biodegradation of
phenolic industrial wastewater in a fluidized bed bioreactor with
immobilized cells of Pseudomonas putida. Bioresource Technol.
80(2): 137-42.
Gurusamy A, Lai YL, Jiunn FL (2007). Biodegradation of phenol by
Pseudomonas pictorum on immobilized with chitin. Afr. J. Biotechnol.
6:296-303.
Jayachandran VP, Kunhi AAM (2008). Degradation of 3-chlorobenzoate
and phenol singly and in mixture by a mixed culture of two ortho
pathways following Pseudomonas strains. J. Indus. Microbiol.
Biotechnol. 5: 120-122.
Juang RS, Tsai SY (2006). Growth kinetics of Pseudomonas putida in
the biodegradation of single and mixed phenol and sodium salicylate.
Biochem. Eng. J. 31:130-140.
Kar S, Swaminathan T, Baradarajan A (1996). Studies on
biodegradation of a mixture of toxic and non-toxic pollutant using
Arthrobacter species.-Bioproc. Biosys. Eng. 15: 195-199.
Kavita V, Palanivelu K (2004). The role of ferrous ion in Fenton and
photo Fenton process for the degradation of phenol. Chemosphere,
55:1235-1243.
Kennes C, Lema JM (1994). Simultaneous biodegradation of p-cresol
and phenol by the basidiomycete Phanerochaete chrysosporium. J.
Ind. Microbiol. 13: 311-314.
Khaled MK (2006). Biodegradation of phenol by Ewingella americana:
Effect of carbon starvation and some growth conditions. Proc.
Biochem. 41:2010-2016.
Lob KC, Tar PP (2000). Effect of additional carbon source on
biodegradation of phenol. Bull. Environ. Contam. Toxicol. 64:756767.
Martins dos Santos VAP, Yakimov MM, Timmis KN, Golyshin PN
(2008). Genomic Insights into oil biodegradation in marine systems.
In Microbial Biodegradation: Genomics and Molecular Biology (Díaz
E Eds.). Caister Academic Press, Wareham Road, U.K.
Das and Kumar
Nuhoglu A, Yalcin B (2005). Modeling of phenol removal in a batch
reactor. Process Biochem. 40: 1233-1239.
Neumaan G, Teras R, Manson L, Kivisaar M, Schayer F, Heipieper HJ
(2004). Simultaneous degradation of atrazine and phenol by
Pseudomons sp. ADP: Effects of toxicity and adaptation. Appl.
Environ. Microbiol. 70: 1907-1912.
Pelczar MJJ, Reid RD (1958). Pure cultures and growth characteristics.
In: Microbiology; McGraw-Hill Book Company: New York. pp. 76-84.
Polymenakou PN, Stephanou EG (2005). Effect of temperature and
additional carbon sources on phenol degradation by an indigenous
soil Pseudomonad. Biodegradation 16:403-413.
Rittman BE, McCarty PL (2001). Environmental Biotechnology: Principle
and applications McGraw-Hill International. pp. 695-713.
Rosa M, Peter S, Cathrin S, Anne MG (2004). Arthrobacter
psychrophenolicus sp. isolated from an alpine ice cave. Int. J. Syst.
Evol. Microbiol. 54:2067-2072.
Shen J, He R, Yu H, Wang L, Zhang J, Sun X, Li J, Han W, Xu L
(2009). Biodegradation of 2, 4, 6-trinitrophenol (picric acid) in a
biological aerated filter (BAF). Bioresour. Technol. 11:1922-1930.
Spiker K, Crawford L, Thiel C (1992). Oxidation of phenolic and nonphenolic substrates by the lignin peroxidase of Streptomyces
viridosporous T7A. Appl. Microbiol. Biotechnol. 37:518-523.
Stefanowicz A (2006). The biolog plates technique as a tool in
ecological studies of microbial communities. Polish J. Environ.
Studies. 15:669-676.
Suhaila NY, Ariff A,, Rosfarizan M, Abdul L, Ahmad SA,, Norazah MN,
Shukor MYA (2010). Optimization of parameters for phenol
degradation by Rhodococcus UKM-P in shake flask culture.
Proceedings of the world congress on engineering, June 30 - July 2,
2010, London, U.K. I: 601-604.
1161
Varma RJ, Gaikwad BG (2009). Biodegradation and phenol tolerance
by recycled cells of Candida tropicalis NCIM 3556. Int. Biodeterior.
Biodegrad. 63(4): 539-542.
Wang Y, Gu B, Xu W (2009). Electro-catalytic degradation of phenol on
several metal-oxide anodes. J. Hazard. Mat. 162: 1159-1164.
Wang Y, Tian Y, Han B, Zhaw HB, Bi J N and Cai BL. (2007).
Biodegradation of phenol by free and immobilized Acinetobacter sp.
strain PD12. J. Environ. Sci. 19: 222–225.
WHO (1994). Phenol: Environmental Health Criteria-EHC 161: Author:
Geneva, Switzerland.
Yang RD, Humphrey AE (1975). Dynamic and steady state studies of
phenol biodegradation in pure and mixed cultures. Biotechnol.
Bioeng. 17: 1211-1235.
Zhang X, Wiegel J (1990). Sequential anaerobic degradation of 2, 4dichlorophenol in freshwater sediments. Appl. Env. Microbiol. 56:
1119-1127.
Vol. 9(16), pp. 1162-1164, 22 April, 2015
DOI: 10.5897/AJMR2014.7344
Article Number: 16C978552665
ISSN 1996-0808
Copyright © 2015
Author(s) retain the copyright of this article
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Short Communication
Resistance of heavy metals on some pathogenic
bacterial species
Aditi Singh*, Maitreyi Mishra, Parul Tripathi and Shweta Sachan
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Malhaur, Gomti Nagar Extension,
Lucknow- 226028, India.
Received 19 December, 2014; Accepted 20 March, 2015
Microorganisms are known to be highly sensitive to the presence of heavy metals and some of the early
attempts to control microorganisms had used copper sulphate as plant fungicide and mercury salts for
some infectious diseases; but, the order of toxicity varies among different organisms and in general
mercury and silver are more toxic than manganese and zinc. It has been seen that responses of
organisms to heavy metal occur at concentration considerably below those at which they response to
alkali and alkaline earth metal occur. Here an attempt has been made to study the susceptibility and
resistance pattern of three common pathogenic bacteria, Klebsiella pneumonia, Escherichia coli and
Staphylococcus aureus against heavy metals. The inhibitory effect of different concentrations of five
metal salts, namely chromium, nickel, iron, cobalt and zinc on microbial growth were studied using gel
diffusion method. Results show that all three study organisms were completely resistant for all
concentrations of chromium and iron salts. E. coli and S. aureus were most susceptible for zinc and
nickel salts as compared to K. pneumonia. In all salts, zones of inhibition were increased along with
increasing concentrations of salts and maximum inhibition was seen at 150 mM concentration. All the
three microbes were highly susceptible for zinc.
Key words: Chromium, iron, metal salts, resistant.
INTRODUCTION
Microorganisms are ubiquitous in nature and involved in
almost all biological processes of life. Heavy metals have
been found in increasing proportions in microbial habitats
because of rapid urbanization and natural processes
(Issazadeh et al., 2013). Metals like nickel, copper
(Clausen, 2000), cobalt and zinc have been playing a
major role both directly or indirectly in almost all
metabolic processes, growth and development of
microorganisms (Tamer et al., 2013). However,
increasing concentrations of metals beyond tolerance
levels have forced these organisms to adapt to various
biological mechanisms to cope with this condition
(Nikaido, 2009). Some mechanisms like metal efflux
systems, complexation, reduction of metal ions or
utilization of the metal as a terminal electron acceptor in
anaerobic respiration helps microbes to tolerate heavy
metal accumulation (Nageswaran et al., 2012). Bacteria
that
is
resistant
to
such
heavy
metals
*Corresponding author. E-mail: asingh3@lko.amity.edu. Tel +91 9839009661; +91 522 2399553.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
Singh et al.
1163
Figure 1. Inhibition of test organisms by metal salts at 10mM concentration. The X-axis represents the
test organisms and the Y-axis represents the diameter of Zone of inhibition, measured in mm. Total
resistance of organisms towards chromium and ferrous was observed, whereas only Klebsiella was
inhibited by 10 mM CoCl2).
(Narasimhulu et al., 2010) and have the ability to grow in
high concentrations of these metals play an important
role in their biological cycling which has great potential in
bioremediation of poorly cultivable soil high in heavy
metal content (Nyamboya et al., 2013). The present study
is formulated to evaluate the effect of increasing metal
salt concentration on growth of bacteria. Five metals such
as cobalt chloride, zinc sulphate, chromium oxide, ferric
chloride and nickel sulphate were used for metal
tolerance tests against three strains (K. pneumonia, E.
coli and S. aureus) in which K. pneumonia and E. coli are
Gram negative whereas S.aureus is Gram positive.
MATERIALS AND METHODS
Test organisms
Three common organisms S. aureus (ATCC25923), E. coli
(ATCC25922) and K. pneumonia (ATCC700603) were included in
the study. The organisms were made as stock by mixing 100 µl of
suspension in 10 ml of sterile nutrient broth and grown overnight.
The organisms were maintained by subculturing them on nutrient
agar at regular intervals and used throughout the study.
Preparation of metal salts
Five metal salts such as cobalt chloride, zinc sulphate, chromium
oxide, ferric chloride and nickel sulphate were used in this study.
One molar stock solution of metal salts was prepared, from which
dilutions of different molarities (10, 50, 100 and 150 mM) were
prepared.
Testing of microbial susceptibility to metal salts
The microbial susceptibility test was done by gel diffusion method.
After preparing Nutrient Agar plates, four wells of 0.5 mm width and
0.5 mm depth were made at equal distance on each plate
aseptically. Separate plates were inoculated with 50 µl of E. coli, K.
pneumonia and S. aureus. For each test organism, 50 µl of salt
solutions of different molarities was put in the wells and plates were
incubated at 37C for 24 and 48 h. Antimicrobial activity was
expressed in terms of zone of inhibition (mm). Each experiment was
repeated thrice and average was taken.
RESULTS AND DISCUSSION
Though many metals are essential for growth, some can
be harmful for living organisms (Reilly, 1991). This is
mainly due to the fact that heavy metals form complexes
with protein molecules and inactivate them (Shanker et
al., 2004). Three different microbes were taken in this
study and their susceptibility patterns were studied
against heavy metal salts. The organisms responded in a
variety of patterns. Zones of inhibition were observed at
different concentrations of salts and the results are
demonstrated in Figure 1.
For cobalt chloride, K. pneumonia was found to be
more susceptible than the other two. Figure 1 shows that
CoCl2 at 10 mM concentration was highly susceptible for
K. pneumonia strain while the other microbes have
demonstrated resistance against the provided concentration of metals. At increasing concentrations, all three
organisms were inhibited and showed no resistance.
There was no inhibition on growth for any of the test
organisms with CrO2 and FeSO4 salt solution and all
microbes showed complete resistance till the highest
concentration used; that is, 150 mM.
The results for nickel sulphate (NiSO4) demonstrated
that S. aureus and E. coli had a high susceptibility and
1164
Afr. J. Microbiol. Res.
less resistance to the metal. K. pneumonia was resistant
at the lowest used concentration; that is, 10 mM; however
it was inhibited by increasing the concentration of NiSO4.
E. coli has shown largest zone of inhibition at all
concentrations of NiSO4, indicating more susceptibility for
the salt when compared with the other two.
All the test organisms showed a strong susceptibility
towards the ZnSO4 salt and growth was inhibited for all
the test bacteria with K. pneumonia being least affected.
A number of studies have shown that microorganisms
have the capacity to resist antibiotics and heavy metals,
which may be extremely harmful to human being and
animals (Samanta et al., 2012; Monchy et al., 2003;
Silver, 1996). The current study also demonstrates
effective inhibition of growth of the microbes by salts. In
future, the work can be extended to some other salts,
some other strains of bacteria and at lesser and higher
concentrations. Much more studies in this regard are
required to be done. The metal tolerant nature of bacteria
has tremendous potential in the bioremediation of heavy
metal accumulation in soil and water (Karthikeyan and
Kulakow, 2003) and also in the treatment of sewage and
toxic wastes (McIntyre, 2003).
Conflict of interests
The authors did not declare any conflict of interest.
REFERENCES
Clausen CA (2000). Isolating metal-tolerant bacteria capable of
removing copper, chromium, and arsenic from treated wood. Waste
Manag. Res. 18:264-268.
Issazadeh K, Jahanpour N, Pourghorbanali F, Raeisi G, Faekhondeh J
(2013). Heavy metals resistance by bacterial strains. Ann. Biol. Res.
4(2):60-63.
Karthikeyan R, Kulakow PA (2003). Soil plant microbe interactions in
phytoremediation. Adv. Biochem. Eng. Biotechnol. 78:51-74.
McIntyre T (2003). Phytoremediation of heavy metals from soils. Adv.
Biochem. Eng. Biotechnol. 78:97-123.
Monchy S, Benotmane A, Janssen P, Vallaeys T, Taghavi S, van der
Lelie D, Mergeay M (2003). Ralstonia metallidurans, a bacterium
specifically adapted to toxic metals: towards a catalogue of metal
resistance genes. FEMS Microbiol. Rev. 27(2-3):385-410.
Nageswaran N, Ramteke PW, Verma OP, Pande A (2012). Antibiotic
susceptibility and heavy metal tolerance pattern of serratia
marcescens isolated from soil and water. J.
Bioremediation
Biodegrad. 3:158.
Narasimhulu K, Sreenivasa Rao PS, Vinod AV (2010). Isolation and
identification of bacterial strains and study of their resistance to heavy
metals and antibiotics. J. Microb. Biochem. Technol. 2:74-76.
Nikaido H (2009). Multidrug resistance in Bacteria. Annu. Rev.
Biochem. 78:119-146.
Nyamboya RA, Okemo PO, Ombori O (2013). Heavy metal and
associated antibiotic resistance of fecal coliforms, fecal streptococci
and pathogens isolated from wastewaters of abattoirs in Nairobi,
Kenya. J. Appl. Biosci. 64:4858-4866.
Reilly C (1991). Metal Contamination of Food. London and New York:
Elsevier Science Publishers Ltd. 2nd ed.
Samanta A, Bera P, Khatun M, Sinha C, Pal P, Lalee A, Mandal A
(2012). An investigation on heavy metal tolerance and antibiotic
resistance properties of bacterial strain Bacillus sp. isolated from
municipal waste. J. Microbiol. Biotechnol. Res. 2(1):178-189.
Shanker AK, Djanaguiraman M, Sudhagar R, Jayaram R,
Pathmanabhan G (2004). Expression of metallothionein 3 (MT3) like
protein mRNA in Sorghum cultivars under chromium stress. Curr. Sci.
86:901-902.
Silver S (1996). Bacterial resistance to toxic metal ions – a review.
Gene 179:9-19.
Tamer A, Aysenur K, Sadik D (2013). Antibiotic levels and heavy metal
resistance in Gram-negative bacteria isolated from sea water
Iskenderun Organized Industrial zone. J. Appl. Biol. Biosci. 7(1):1014.
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