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RESEARCH ARTICLE
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Prevalence and Antimicrobial Susceptibility of Enteric Bacterial
Infection in Lao PDR, 2012
Phanthaneeya Teepruksa1,2, Phengta Vongpachanh2, Noikaseumsy Sithivong2,
Keseara Na-Bangchang1, Wanna Chaijaroenkul1
1
Chulabhorn International College of Medicine, Thammasat Univesity (Rangsit
Campus), Pathumthani 12121, Thailand
2
National Center for Laboratory and Epidemiology, Km 3 Thaduae Rd.,
Sisattanack District, Vientiane Capital, LAO PDR
.
Abstract
Diarrhea is an important health problem which is the second leading cause
of deaths among children in developing countries. The antimicrobial resistance
surveillance in Lao PDR was established in 2012. The aim of this study was to
identify bacterial pathogens, the virulence of pathogenic E. coli, and the
antimicrobial susceptibility profile of enteric pathogen from diarrhea patients in
Lao PDR. Stool and rectal swab samples were collected from diarrheal patients
during January to December 2012. The cultivation, biochemical method, multiplex
PCR and antimicrobial susceptibility were performed. Results showed that 284 out
of 711 specimens were infected with Aeromonas spp (1.7%), Plesiomonas
shigelloides (1.7%), Salmonella spp (4.9%), Shigella spp (1.4%), Vibrio spp.
(0.4%), rotavirus (0.4%), pathogenic Escherichia coli (17.8%), and mixed infection
(11.5%). The prevalence of samples positive for Aeromonas spp., Plesiomonas
shigelloides, and Vibrio spp. were significantly higher in patients aged > 5 years.
The prevalence of samples with mixed infection was however significantly higher
in children aged <5 years (p =0.020). Most samples infected with Aeromonas spp
and Plesiomonas shigelloides were susceptible (resistance prevalence < 35%) to
amoxicillin-clavulanate, chloramphenicol, ciprofloxacin, ceftriaxone, gentamicin,
trimethoprim-sulfamethoxazole, and tetracycline. Samples infected with Vibrio
spp. were sensitive to amoxicillin-clavulanate, chloramphenicol, ciprofloxacin,
ceftriaxone, gentamicin, and tetracycline. Those infected with Salmonella spp. was
resistant to ampicillin and tetracycline (resistance prevalence = 80% and 67%,
respectively). Most samples infected with Shigella spp. were resistant to ampicillin
(69%), chloramphenicol (64%), trimethoprim-sulfamethoxazole (93%), and
tetracycline (82%).
Keywords: Enteric bacteria, Antimicrobial, Lao PDR
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Introduction
Diarrheal disease is an important health problem which is the second
leading cause of deaths annually, especially children under five in developing
countries (1, 2). Diarrhea is caused by several microorganisms such as viruses,
parasites, and bacteria. The pathogenic bacteria associated with diarrhea include
Shigella, Salmonella, Vibrio, and pathogenic Escherichia coli. E. coli is the most
commonly found pathogen of acute diarrhea in developing countries. Although this
microorganism is the normal flora of the human intestine, several strains of E. coli
are pathogenic to humans (3). The virulent strains of E. coli that cause diarrheal
diseases are enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC),
enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and
enteroaggregative E. coli (EAEC) (3).
The antimicrobial resistance (AMR) is a worldwide public health concern.
A number of AMR cases have been reported in the Asia pacific region (4, 5), but
with limited reports from Lao PDR. AMR surveillance is recommended by the
World Health Organization (WHO) as an approach to control microbial infections.
To cope with the AMR issue in Lao PDR, the surveillance program of the causative
microorganisms of diarrhea has been launched in 2012 by the National Center for
Laboratory and Epidemiology. The information of surveillance program is essential
for planning and implementing disease control in Lao PDR. The aim of this study
was to identify bacterial pathogens that cause diarrhea, the virulence of pathogenic
E. coli, and the antimicrobial susceptibility profiles of enteric pathogens from
diarrhea patients in Lao PDR during 2012.
Materials and Methods
1. Study areas and samples collection
A cross sectional study was conducted in Vientiane capital as indicatorbased surveillance (IBS) in eight hospitals/health centers: Mother and Child
hospital (MC), Sisattanak district hospital (SIS), Sikottabong district hospital
(SKB), Hatsaifong district hospital (HAT), Banhom health center (SH), Mahosot
hospital (MHS), Settathilath hospital (SET), and Mittapab hospital (MTB).
Clinical samples were from all the diarrheal patients attending the Emergency
clinic, Internal department, Infectious ward and Pediatric ward of these hospitals.
The event-based surveillance (EBS) of the disease outbreak was conducted during
January to December 2012 throughout the country. The samples were collected
randomly from five diarrheal case clusters. Diarrhea was defined as the passage of
three or more liquid stools during a 24 hour period. Fresh stool, stool swab in Cary
blair transport medium, or rectal swab in Cary blair transport medium were
collected. A total of 711 stool samples were collected and transported to the
microbiology laboratory of The National Center for Laboratory and Epidemiology
(NCLE) for cultivation of the causative agents.
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2. Isolation and identification of enteric pathogen
Each clinical sample was mixed in 0.5 ml 0.85% normal saline solution to
obtain working sample. Then working sample was initially inoculated with
MacConkey agar, Salmonella-Shigella (SS-) agar, and Xylose lysine deoxycholate
(XLD) agar, at 35°C for 18 to 48 hours for observing colonies of Salmonella,
Shigella, Vibrio, Aeromonas and Plesiomonas, respectively (Figure 1). To identify
the colonies of Vibrio spp., the working sample was further inoculated in alkaline
peptone water (APW) and incubated at 35°C for 4-6 hours, and then transferred to
thiosulfate citrate bile salts sucrose (TCBS) agar and incubated at 35°C for 18-48
hours. To identify the colonies of Salmonella, all working samples were inoculated
overnight in Selenite broth at 35°C and transferred to SS-agar and XLD agar and
incubated at 35°C for an additional 18-48 hours. The suspected organisms which
grew on culture media were further identified by observing colony, gram-stain
morphology, and traditional biochemical tests. Confirmation using commercial
reagent- API identification kit (Biomerieux, France) was required for some cases.
Figure 1 Diagram of bacterial culture process from stool/rectal swab
3. Identification of the virulence of pathogenic E. coli
Polymerase chain reaction (PCR) was used for identification of the
virulence of E. coli. A loop-full E. coli-like colonies from inoculated MC agar were
swept and suspended into the extraction buffer (Tris-EDTA buffer) and DNA was
extracted by boiling method. The following target genes were used to differentiate
pathogenic E. coli: eae and bfp (EPEC), eae, stx1 and stx2 (EHEC), st and lt
(ETEC), and ipaH (EIEC). All primers sequences (Table 1) and PCR conditions
were developed and/or modified by the Department of Bacteriology I, The National
Institute of Infectious Diseases (NIID) Japan. The multiplex PCR conditions were
as follows: 96°C for 2 min, 30 cycles of 96°C for 20 sec, 55°C for 20 sec, 72°C for
1 min, and 72°C for 1 min.
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4. Antimicrobial susceptibility testing
Antimicrobial susceptibility test was performed on Mueller-Hinton agar
(Becton Dickinson, USA) using disc diffusion technique. The antimicrobials under
investigation included: ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg),
tetracycline (30 µg), gentamycin (10 µg), chloramphenicol (30 µg), ciprofloxacin
(5 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), and ceftriaxone (30 µg)
(Oxoid, UK). The colonies from non-selective media were selected and the
working bacteria suspension was prepared in 0.85% sterile normal saline solution
(0.85% NSS). Turbidity of the broth culture was equilibrated to match 0.5
McFarland standard. The surface of Mueller-Hinton agar plate was evenly
inoculated with the culture using a sterile cotton swab. The antibiotic discs were
applied to the surface of the inoculated agar. After 18–24 hours of incubation, the
diameters of growth inhibition around the discs were measured and interpreted as
sensitive, intermediate, or resistant (6). The reference strain E. coli ATCC 25922
was used for quality control.
5. Data analysis
Statistical analysis was performed using SPSS version 12. Qualitative data
are presented as number and percentage values. Comparison of the frequencies of
microorganisms among all age groups was performed using Pearson’s chi-square
or Fisher exact test. Statistical significance was set at  = 0.05.
Table 1 Primer sequences for identification of pathogenic E. coli.
Target gene
eae
Primer name
eaeA-newF
eae-newR
Sequence 5’-3’
AACGGCTATTTCCGCATGAG
CACATAAGCMGGCAAAATAGCCTG
bfp
bfpB-F
bfpB-newR
GACACCTCATTGCTGAAGTCG
GCCCAGAACACCTCCGTTAT
stx1
mMK1-1
mMK1-2
GAATTTACCTTAGACTTCTCGAC
TGTCACATATAAATTATTTCGTTCA
stx2
mMK2-1
mMK2-2
GAGTTTACGATAGACCTTTCGAC
GGCCACATATAAATTATTTTGCTC
st
ST1a-s
St1a-as
ST1b-s
St1b-as
GCAATTTTTATTTCTGTATTATCTT
GGATTACAACAAAGTTCACAG
TTTATTTTTCTTTCTGTATTGTCTT
GGATTACAACACAATTCACAG
lt
lt-Fv
lt-Rv
CTATTACAGAACTATGTTCGGAATATC
GGGGGTTTTATTATTCCATACAC
ipaH
ipaH-F,
ipaH-Rv
GTTCCTTGACCGCCTTTCCGATACCGTC
GCCGGTCAGCCACCCTCTGAGRGTAC
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Results
1. Identification of enteric pathogens
Of the 708 stool and rectal swab specimens collected in 2012, 554 were
from acute watery diarrhea (AWD) surveillance program in Vientiane capital, 132
from diarrhea and/or food poisoning outbreak investigation, and 22 from public
and private health facilities. Four hundred and sixty-seven samples were collected
from patients aged less than 5 years. Two-hundred and eighty four samples were
infected with Aeromonas spp. (1.7%), Plesiomonas shigelloides (1.7%),
Salmonella spp. (4.9%), Shigella spp. (1.4%), Vibrio spp. (0.4%), pathogenic E.
coli (17.9%), and mixed infection (11.6%) (Table 2). The prevalence of samples
infected with Aeromonas spp., Plesiomonas shigelloides, Shigella spp. and Vibrio
spp. were significantly higher in patients aged > 5 years (p = 0.007, < 0.0001,
0.042 and 0.009, respectively). In contrast, the prevalence of mixed infection with
at least one enteric pathogen was significantly lower in children aged < 5 years (p
= 0.013) (Table 3).
Table 2 Identification of pathogenic microorganisms in the clinical samples. Data
are presented as number (n) and percentage (%) values.
Bacterial identification
Aeromonas spp.
Plesiomonas shigelloides
Salmonella spp.
Shigella spp.
Vibrio spp.
Escherichia coli (E. coli)
- Enterohemorrhagic E. coli (EHEC)
- Enteroinvasive E. coli (EIEC)
- Enteropathogenic E. coli (EPEC)
- Enterotoxigenic E. coli - heat labile toxin (ETEC.LT)
- Enterotoxigenic E. coli - heat stable toxin (ETEC.ST)
Mixed infection with > 1 spp.
Negative
Total
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Number (n) Percentage (%)
12
1.7
12
1.7
35
4.9
10
1.4
3
0.4
127
17.9
20
2.8
26
3.7
58
8.2
9
1.3
14
2.0
82
11.6
427
60.3
708
100.0
2. Antimicrobial susceptibility
Antimicrobial susceptibility testing for the five microorganisms was
successfully performed in 116 samples. Most samples infected with Aeromonas
spp. and Plesiomonas shigelloides were susceptible to amoxicillin-clavulanate,
chloramphenicol, ciprofloxacin, ceftriaxone, gentamicin, trimethoprimsulfamethoxazole, and tetracycline; resistant isolates were found at the prevalence
of 0-35%. Those infected with Vibrio spp. was sensitive to amoxicillin-clavulanate,
chloramphenicol, ciprofloxacin, ceftriaxone, gentamicin, and tetracycline. Samples
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infected with Salmonella spp. were resistant to amoxicillin and tetracycline
(resistant rate = 80% and 67%, respectively). Most of the samples infected with
Shigella spp. were resistant to amoxicillin (69%), chloramphenicol (64%),
trimethoprim-sulfamethoxazole (93%), and tetracycline (82%) (Figure 2).
Table 3 The enteric pathogens identified in clinical samples classified according to
age groups. Data are presented as numbers (n).
Bacterial identification
Number of samples (n) p-value
≤ 5 years > 5 years
Aeromonas spp.
4
8
0.007*
Plesiomonas shigelloides
1
11
< 0.0001*
Salmonella spp.
26
9
0.596
Shigella spp.
4
6
0.042*
Vibrio spp.
0
3
0.009*
Escherichia coli (E. coli)
85
42
0.507
- Enterohemorrhagic E. coli (EHEC)
10
10
0.058
- Enteroinvasive E. coli (EIEC)
16
10
0.361
- Enteropathogenic E. coli (EPEC)
41
17
0.917
- Enterotoxigenic E. coli - heat labile toxin (ETEC.LT)
8
1
0.220
- Enterotoxigenic E. coli - heat stable toxin (ETEC.ST)
10
4
0.910
Mixed infection with > 1 spp.
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36
0.013*
166
115
Total
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Discussion
The prevalence of death from diarrhea in children under 5 years reported In
Lao PDR in 2004 was 16.9% (7). The outbreaks of diarrhea were reported in 1993
and 1994, and during the period 2000-2002. In 2007, the outbreak of diarrhea was
investigated and more than 50% of V. cholerae was found to be the cause of
diarrhea (8). Nevertheless, antimicrobial susceptibility of the pathogenic
microorganisms was not investigated during these outbreaks. The susceptibility
testing for E. coli was not accomplished due to technical problem at the step of
bacterial purification.
Results of the present study show that only 40% of patients infected with
bacterial pathogens and the major cause of diarrhea was E. coli (17.9%) with
comparable prevalence in both age groups (< 5 and > 5 years). Some samples with
negative bacterial infection were found to be infected with protozoas, helminthes,
or viruses.
The previous study in Vientiane, Lao PDR during 1996-1997, showed the
major cause of diarrhea were E. coli (35.8%) and Shigella spp. (16.8%) infections
(9). The highest number of E. coli infection was also found in the present study but
the Shigella spp. infection was only found 1.4%. The prevalence of
enteropathogenic E. coli was similar to that reported previously, except the
enterotoxigenic E. coli - heat stable toxin (ETEC.ST) (2.0 % vs. 17.2%) (9). It was
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noted however that the present study was conducted 15 years back and the
prevalence and patterns of infected micro-organisms may be changed.
A trend of antimicrobial resistance in Salmonella spp. and Shigella spp. was
observed in the study samples. Samples infected with Shigella spp. were resistant
(multidrug
resistant)
to
ampicillin,
chloramphenicol,
trimethoprimsulfamethoxazole, and tetracycline. High level of resistance in samples infected
with Shigella spp. has been observed in several countries (10-13). In this study,
resistance to ciprofloxacin was not found and resistance to ceftriaxone and
nalidixic acid was found at the prevalence of 20% and 35%, respectively.
Continuous monitoring of antimicrobial resistance in Lao PDR and control use of
antimicrobial drugs is required.
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Figure 1 Antimicrobial susceptibility of the clinical samples infected with
Aeromonas spp., Plesiomonas shigelloides, Salmonella spp., Shigella spp., and
Vibrio spp.
AMC: Amoxicillin-clavulanate, AM: Ampicillin, C: Chloramphenicol, CIP:
Ciprofloxacin, CRO: Ceftriaxone, NA: Nalidixic acid, GM: Gentamicin, SXT:
Trimethoprim-sulfamethoxazole, TE: Tetracycline,
Conclusion
The enteric pathogen is an important cause of diarrhea worldwide including
Lao PDR. Our results show that most (40%) diarrhea in Lao PDR is caused by the
bacterial pathogens E. coli, Aeromonas spp., Plesiomonas shigelloides, Salmonella
spp., Shigella spp., and Vibrio spp. Those infected with Shigella spp. were
multidrug resistant to several antimicrobial drugs. Continuous monitoring of
antimicrobial resistance in Lao PDR and control use of antimicrobial drugs is
required.
Acknowledgements
This study is supported by the United State Agency for International
Development (USAID) - Emerging Pandemic Threats Program (EPT) and the
European Union (EU) through the World Health Organization (WHO), Lao PDR.
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