Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(19): 2011
The Bacterial of Prevalence and the Production
of Bacteriocin by group B streptococcus in
neonatal sepsis
Abeer Thaher AL-Hassnawi
Collage of medicine-Babylon university-Hilla Iraq.
Abstract
Neonatal bacterial sepsis is one of the major cause of morbidity and mortality in neonates
.This study was performed to determine the prevalence of bacterial sepsis with focus on gram positive
and gram negative in neonates admitted at Karbala Teaching Hospital for Children , during the period ,
from November 2008 to April 2009.The neonates ranged from (1-30) days old .The early onset
neonates are more susceptible for neonatal sepsis (60%) than late onset sepsis (40%) and males are
more affected than females. Blood culture was performed on all neonates with risk factors or sings of
suggestive sepsis . Blood samples were cultured using brain heart infusion broth according to standard
method . From the 100 neonates (42%) had positive blood culture for group B streptococcus , (30%)
for E. coli , (16%) forPseudomonas aeruginosa , (8%) for Klebsiella pneumoniae and Staphylococcus
aureus (4%) .
Group B streptococcus bacteria represents the high percentage of bacteria in this study ,
therefore , we investigated the bacteriocin production properties of GBS isolates . (76%) of GBS
isolates produced bacteriocin while (24%) of GBS isolates didn't produce bacteriocine . E.coli ,
Pseudomonas and Klebsiella showed a high degree of resistance to commonly used antibiotic
(ampicillin , gentamicin) as well as third generation cephalosporins .
‫الخالصة‬
‫هذه الدراسة أنجزت من‬
. ‫يعتبر خمج الدم البكتيري لحديثي الوالدة السبب الرئيسي للهالكات واألصابات في حديثي الوالدة‬
‫أجل تحديد معدل األصابة بخمج الدم البكتيري لحديثي الوالدة الوافدين الى مستشفى األطفال التعليمي في محافظة كربالء للفترة من‬
. 2009 ‫ الى نيسان‬2008 ‫تشرين الثاني‬
‫ وقد تركزت االصابة في الفئة العمرية المصابة بخمج الدم المبكر‬. ‫) يوم‬30-1( ‫كان معدل أعمار حديثي الوالدة يتراوح بين‬
‫ تم سحب دم من كل‬. ‫) وكان الذكور أكثر أصابة من األناث‬%40( ‫) أكثر من الفئة العمرية المصابة بخمج الدم المتأخر‬%60(
‫ طفل حديثي الوالدة وتم عزل بكتيريا‬100 ‫ أجريت هذه الدراسة لـ‬. ‫مريض لغرض أجراء الزرع البكتيري على الوسط الزرعي المالئم‬
Pseudomonas , E.coli ‫) كما وتم عزل أنواع أخرى من البكتيريا مثل‬%42( ‫ بأعلى نسبة حوالي‬Group B Streptococcus
.‫) على التوالي‬%4(,)%8( ,)%16( ,)%30( ‫ بنسب‬Staphylococcus aureus , Klebsiella pneumoniae , aeruginosa
.‫ أعلى نسبة في هذه الدراسة ولذا فقد تم دراسة خواص أنتاج البكتريوسين لهذه البكتريا‬Group B Streptococcus ‫سجلت‬
‫ كما وأجري‬.‫) من العزالت غير منتجة للبكتيريوسين‬%24( 10 ‫) من العزالت منتجة للبكتيريوسين بينما‬%76( 32 ‫تبين من النتائج بأن‬
‫ وكانت أعلى درجة مقاومة‬Klebsiella pneumoniae, Pseudomonas aeruginosa , E.coli ‫أختبار الحساسية على بكتريا‬
.‫للمضادات الحياتية في األمبسلين والجنتامايسين والسيفالوسبورين في أختبار الحساسية الذي أجري على هذه األنواع البكتيرية الثالثة‬
Introduction
Neonatal sepsis is any infection involving an infant during the first 28 days of
life . Neonatal sepsis is also known as "sepsis neonatorum" . The infection may
involve the infant globally or may be limited to just one organ (such as the lung with
pneumonia). It may be acquired prior to birth (intrauterine sepsis) or after birth
(extrauterine sepsis). Viral (such as herpes, rubella{German measles}, bacterial (such
as group B streptococcus) and more rarely fungal (such as candida) causes may be
implicated (Mersch, 2009).
Neonatal bacterial sepsis (NBS) remains as an important cause of mortality
and morbidity among infants. Its incidence varies with geographical area and may
change in the same area with time. NBS has been classified as either early onset (0-7
455
day of age) or late onset (7-28 day of age). Early onset infection is thought to occur
during passage of the fetus through the birth canal. The principal organisms that
account for early onset infection include predominatly group B streptococcus, as well
as Escherichia coli (E. coli) in the reminder (Ali, 2004).
Early onset sepsis is an important cause of illness and death among infants
with very low birth weight (less than 1500g) (Klein, 2001; Philip, 1994; Berger, et
al., 1998). Antibiotic are used increasingly during labor to decrease the risk of
neonatal group B streptococcus infection and to reduce risk of neonatal illness after
preterm rupture of the membranes (Schrag, etal., 2000; Hager, etal., 2000; Schuchat,
etal., 2000; Benitz, etal., 1999). However there is concern that increased use of
antibiotics might result in a change in the spectrum of organisms, their susceptibility
to antibiotics or both (Isaacs and Royle, 1999; Mercer, etal., 1999; Towers, etal .,
1998). Treatment of the mother with antibiotics against GBS has resulted in a
decreased incidence of GBS, induced neonatal sepsis, and a corresponding increase
in sepsis from other organisms, in particular Gram-negative organism
including E. coli and Klebsiella species (Stoll, etal., 2002).
Late onset infection is thought to occur well beyond the early delivery
phase, and as such reflects organism that may be acquired in the hospital setting or at
home. As such, infants with late onset neonatal sepsis develop infections from
common hospital pathogens, including bacteria (coagulase negative staphylococcal
species). Viruses, and fungi(candida species) (Mara, 2007). Abnormal bacterial
colonization of the rectum and anus during pregnancy may create an abnormal vaginal
and cervical microbial environment. More than 2 decades ago, rectovaginal
colonization with GBS during pregnancy was found to be associated with this GBS
infection of the fetus on newborn (Sherman, 2009).
The most common etiology of neonatal bacterial sepsis is GBS. Nine
serotypes exist. Types I, II and III are commonly associated with neonatal GBS
infection (Anderson, 2008).
Studies of the bacteriocins of streptococci go back to the 1960 (Ingolf, etal.,
2007). The more recent focus in the isolation and characterization of streptococcal
bacteriocins has been on pathogenic streptococci. Lantibiotics are the most prevalent
peptide bacteriocins in streptococci, and the majority belong to the elongated cationic
type A lantibiotics (Ross, etal., 1993). Most bacteriocins in gram positive bacteria are
small and heat stable (peptide bacteriocins), and their antimicrobial activities are
directed against a broader spectrum of bacteria than is seen for bacteriocins of gram
negative bacteria (Chatterjee, etal., 2005).
The aims of the study
1.Investigate the causative organisms of neonatal sepsis with focus on gram positive
and gram negative organisms.
2.Determination of the bacteriocin production from isolated group B streptococcus.
3.Identification of the best measures for controlling of neonatal sepsis.
Material & Methods
A total of 100 neonates with clinical diagnosis of septicemia who admitted
at the Karbala Teaching Hospital for Children through November 2008 to April 2009
were included in this study. Blood cultures were performed routinely on all neonates
with clinical signs suggestive of sepsis(poor feeding, respiratory distress, fever and
hypothermia) or whose mothers had a history of prolonged rupture of membranes,
maternal fever and premature labor. Blood was cultured using brain heart infusion
456
Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(19): 2011
broth according to standard methods. Subcultures were performed. The isolates were
identified by standard biochemical tests.
Bacteriocin production
Stab inoculate multiple strains on separate multiple brain heart infusion agar
Petri-dishes. Incubate at 37 Cº for 24hours. Remove the cell growth with a sterile
glass slide. Kill the residual cells on the agar surface by exposure to chloroform vapor
for 30 minutes. Incubate all the plates again at 30 Cº for 24hours. The presence of
bacteriocin can be detected by zones of growth inhibition around stabs (Chung, 2003).
Antibacterial resistance pattern of the isolates was studied by Kirby-Bauer disc
diffusion technique. Susceptibility of the isolates were done and interpreted according
to National Committee for Clinical Laboratory Standards (NCCLS) recommendations.
The antibiotic concentration per disk was as following. Ampicillin (10 Mg),
Amikacin(30 Mg), Gentamicin (10 Mg), Cephalexin (30 Mg) and
Ceftriaxone (30
Mg).
Results and Discussion
Table (1) shows the categorization of sepsis cases. The results of the present
study showed that early onset sepsis (60%) were more than late onset sepsis (40%).
The number of infected males accounted 64 (64%) versus 36 (36%) from infected
females.
Table (1) : Categorization of sepsis cases.
Category
Early onset
sepsis
Late onset
sepsis
Total
Numbers of
neonates
No. & %
60 (60)%
Female
Male
No. & %
20 (20)%
No. & %
40 (40)%
40 (40)%
15 (15)%
25 (25)%
100 (100)%
35 (35)%
65 (65)%
The present study agree with Movahedian (2006 ) who reported that 86
(77.5%) cases of early onset
and 25 (22.5%) of late onset sepsis. The early onset
sepsis was more common than late onset disease which is compatible with the reports
from the other developing countries (Fisher, et al., 1983; Gladstone, et al., 1990;
Martin et al., (2003) reported sepsis was more among male than female. The
incidence of bacterial sepsis is higher in males than in females (Anderson, 2008). The
result of the present study agreed with the results of these studies.
In the present study, GBS was isolated in higher ratio (42%) (table -2). Another
types of bacteria were isolated such as E. coli, Pseudomonas aeruginosa, Klebsiella
pneumoniae and Staphylococcus aureus at (30%), (16%), (8%) and (4%),
respectively.
Table ( 2) : Frequency of microorganisms isolated from blood cultures of
neonatal sepsis .
Types of bacteria
No.
457
(%)
Group B streptococcus
E. coli
Pseudomonas aeruginosa
Klebsiella pneumoniae
Staphylococcus aureus
Total
42
30
16
8
4
100
42
30
16
8
4
100
Group B streptococcus is the most common cause of neonatal sepsis in the
United States of America (USA). From 1994 to 2002, there were about 1100with
potential neonatal septicemia (Martin, et al., 2007). Keenan (1998) reported that the
early onset disease, usually manifested as sepsis or pneumonia, is diagnosed in the
first six days of life and accounts for approximately (80%) of neonatal GBS infection.
Another study from Bangladesh revealed gram negative organisms were
responsible for almost (73%) of neonatal sepsis with E. coli as the most common
cause (30%) followed by Klebsiella species (Ahmed, et al., 2002). Pseudomonas was
the most common organism (38.3%), followed by Klebsiella species (30.4%) (Joshi,
et al., 2000). The result of present study agreed with these studies.
The results of the present study were also indicated that out of 42 neonatal
sepsis GBS isolates, 32 (76%) produced bacteriocin, (table-3)
Table (3) : Bacteriocin production of GBS isolated from neonatal sepsis.
Bacteriocin Production
Positive
Negative
Total
No. (%)
32 (76)
10 (24)
42 (100)
Pinar (2004) found the GBS still plays a major role in neonatal mortality.
Stade (2004) reported that the early onset GBS infection accounts for approximately
(30%) of neonatal infection, has a high mortality rate and is acquired through vertical
transmission from colonized mothers. Various researchers have reported that the
bacteriocins produced by Streptococcus mutans are strongly inhibitory to other
Streptococcus mutans as well as to strains of many other Streptococcus species
(Balakishnan, et al., 2002). Peptide bacteriocins produced by Streptococcus mutans
were known as mutacins (Ingolf, et al., 2007).
Heng et al., (2006) reported the mutacin production in (145) strain of S.
mutans isolates from young children and their mothers. Wescombe (2006) found
(77%) of 36 S. salivarius strains were producing the bacteriocin. Tompkins and Taqq
(1987) found seven beta-hemolytic Streptococcus salivarius isolates produced
bacteriocin in deferred antagonism tests using a set of nine indicator bacteria. Thomas
et al., (2007) also reported the evidence that S. pneumoniae produces bacteriocin
directed not only against other strains of the same species but also against closely
related Streptococci, such as S. mitis, S. oralis, S. salivarius and S. pyogenes.
Table (4) showed antibiotic resistance of E. coli, Pseudomonas and Klebsiella sepsis
to ampicillin, gentamicin, cephalexin and ceftraxone.
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Journal of Babylon University/Pure and Applied Sciences/ No.(2)/ Vol.(19): 2011
Table (4) : Antibiotic sensitivity test of gram negative bacteria.
Antibiotic
E. coli
Pseudomonas
aeruginosa
Klebsiella
pneumoniae
Ampicillin
S
0
25
0
R 100
75
100
Gentamicin
S
12
52
30
R
88
48
70
Amikacin
S
R
Cephalexin
S
R
Ceftriaxone
S
R
S: Sensitive , R: Resistant
100
0
0
100
35
65
72
28
20
80
18
82
60
40
28
72
36
64
Stoll (2002) showed that 28 of 33 isolates (85%) were resistant to ampicillin
and also the proportion of infants with E. coli sepsis was higher among those whose
mothers recived ampicillin with in 72 hours befor delivery than among those whose
mothers did not. Gram negative organisms have both innate resistance to antibiotics
and the ability to acquire resistance through new mechanisms that may be transferred
from other pathogens (Waterer and Wunderink, 2001).
Joseph et al., (1998) and Friedman et al., (2000) reported the 85% of E. coli
isolates were resistant to ampicillin. Gentamicin is considered by some studies as a
suitable aminoglycoside antidrug resistant P. aeruginosa (Kettner, et al., 1995; Jones,
et al., 1997). Cephalothin and ceftriaxone should not be considered effective agents
for P. aeruginosa sepsis treatment, due to the high resistance rates detected during the
study period (Sader, 2000; Kettner, et al., 1995).
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