Comparative Analysis of Virulence Factors of Escherichia coli from Non-enteric Infections
Reaz Mohammad Mazumdar,
Khanjada Shahnewaj Bin Mannan
There is correlation between certain properties of E. coli strains and capacity of the strain to cause non-enteric infections. The aim of this study was to compare Escherichia coli strains isolated from non-enteric infection with those from faeces of normal healthy individuals, for their possession of haemolysin, mannose- resistant-haemagglutinin, colicin, protease, cell surface hydrophobicity and antibiotic susceptibility. Source wise distribution of the haemolytic strains were 60% from urine, 41.7% from blood, 33.3% from peritoneal fluid, 50% from pus and 26.7% from stool. Colicinogeny was found to be a common property of both clinical and faecal E. coli and only a few of the urine and blood isolates (52 and 41.7%, respectively) exhibited colicin V activity. Mannose Resistant Haemagglutinin (MRHA) test showed positive (MRHA+) reaction for 47.7% of the clinical E. coli isolates, while this value was only 26.7% for the controls. A significant association between haemolysin production and MRHA of human type O erythrocytes was found, as 75.86% of the Hly+ E. coli strains were also MRHA+. The data obtained in this study suggested that haemolysin production, MRHA of human type O erythrocytes and hydrophobic cell surface might be important for E. coli strains to initiate and sustain infection at non-enteric sites.
June 12, 2012; Accepted: June 21, 2012;
Published: August 11, 2012
Escherichia coli is a commensal organism inhabiting human and animal
intestinal tract. It can cause variety of non-enteric infections when enters
into unnatural sites (Sharma et al., 2007; Frederick,
2011). Several virulence factors help E. coli isolates to survive
under adverse conditions present at the non-enteric sites and contribute to
the ability of E. coli to cause extra intestinal infections (Banu
et al., 2011). Non-enteric or Extra-intestinal pathogenic E. coli
(ExPEC) is a group of E. coli strains that induce extra-intestinal
diseases (Russo and Johnson, 2000). ExPEC has a great
impact on public health in terms of both morbidity and mortality, with an economic
cost of several billion dollars annually (Russo and Johnson,
Considerable amount of information has been accumulated in recent years on
E. coli virulence markers and their role in infections. Production of
enterotoxins, hemolysins, colicins, haemagglutinins, proteases, colonization
factors, cell surface hydrophobicity etc is some virulence-associated factors
of E. coli (Kausar et al., 2009). Secretory
proteases are common virulence factors in many bacterial and nonbacterial pathogens
including E. coli (Dezfulian et al., 2003).
Haemolysin is one of the most important virulence factors and according to some
previous studies, approximately 50% of E. coli isolates causing extra-intestinal
infections in humans are haemolytic (May et al.,
2000). Cell surface hydrophobicity has been identified to be important in
cell adhesion and pathogenicity of E. coli (Najar
et al., 2007). Mannose Resistant Haemagglutination (MRHA) are adhesive
factors, which are important in the establishment of pathogenic strains of E.
coli to various host tissue (Drews et al., 2005).
Colicins are toxic proteins produced by E. coli and active against related
bacteria and Phenotypic expression of colicin V (Col V) have been described
as an indicator of complement resistance (Riley, 1993).
As E. coli infection involving the urinary tract, peritoneum, blood
and meninges occur frequently in our country, a comprehensive study on the virulence
factors of these bacterial strains is urgent. Development of resistance against
antibiotics is making treatment of E. coli infections difficult (Mathur
et al., 2002). Practices of self-medication, drug abuse and indiscriminate
misuse of antibiotics among the general people favored the emergence of drug
resistant strains (Manikandan et al., 2011).
Considering the above facts in mind, knowledge on antibiotic susceptibility
pattern of extra-intestinal pathogenic E. coli is necessary to select
correct antibiotic(s) for proper treatment of the infections caused by them
(Sharma et al., 2007).
The aim of this study was to compare E. coli strains isolated from urinary tract infection, septicemia, peritonitis and abscess with those from faeces of normal healthy individuals, for their possession of haemolysin, mannose-resistant-haemagglutinin, colicin V, protease, hydrophobic cell surface and antibiotic susceptibility. This would provide an opportunity to determine the importance of individual characteristics of E. coli strain in different clinical situations.
MATERIALS AND METHODS
Source of isolates and strains: A total of 65 clinical isolates of Escherichia coli strains, of which 25 were from infected urine, 12 from peritoneal fluid, 12 from blood, 10 from pus and 6 from Cerebrospinal Fluid (CSF) were included in the study. Five E. coli strains isolated from stool of healthy individuals were also included. Streptococcus pyogenes, Nonpathogenic E. coli ATCC-35218, E. coli ATCC-25922, E. coli K-12 (Col-), E. coli K-12 Col V+, Pseudomonas aeruginosa ATCC-10145 were also included in the study as control. All the isolates were identified and preserved by stab culture in soft agar base and stored at 4-8°C.
Sample collection: Urine, blood, pus, peritoneal fluid, stool and Cerebrospinal Fluid (CSF) was collected through standard procedures. A midstream specimen of urine was collected in a sterile wide-mouthed bottle. Five milliliter of blood was drawn from the cephalic vein with a sterile disposable syringe. 0.2 mL pus was drawn from the abscess using a sterile disposable syringe. Immediately after abdomen was opened by the surgeon, 2 mL of peritoneal fluid was collected in a sterile disposable syringe. In case of stool sample, stool was touched with a sterile cotton swab which was returned immediately to a sterile cotton-plugged test tube. In case of cerebrospinal fluid, about 0.5 mL of CSF was collected in a sterile tube aseptically by lumber puncture.
Isolation of strains: Immediately after collection, the samples of urine,
blood, pus, CSF, peritoneal fluid and stool were directly inoculated onto MacConkey
agar and blood agar plates by spread plate technique. Plates were incubated
for 24 h at 37±0.5°C. Isolated colonies were streaked on nutrient
agar plates for pure culture and for presumptive identification using biochemical
tests (Adzitey et al., 2012).
Identification of strains: The shape and type of Gram reaction are microscopically
studied using 18 h culture from agar plate. The biochemical tests involved Klingler's
Iron (KIA) Agar, Simmon's Citrate agar, Motility Indole Urease (MIU), Lysine
Iron Agar (LIA), Urea broth, Peptone water, Methyl Red (MR), Voges Proskauer
(VP), Nutrient Nitrate Broth (NB), Gelatin liquefaction, Eijkman test, carbohydrate
fermentation test was done for lactose, sucrose, glucose and starch, Oxidase
and Catalase tests (Apun et al., 2008). Identification
of isolates obtained in pure culture was based on Gram staining, biochemical
characteristics and growth pattern on selective and differential media and according
to the procedures recommended in the Bergeys Manual of Determinative Bacteriology
(Holt, 2005; Edwards and Ewing, 1986).
Detection of haemolytic strains: The haemolytic activity was observed
on washed blood agar plates according to Sharma et al.
(2007) and Subashkumar et al. (2006). Sixty
five E. coli clinical isolates and 15 E. coli fecal isolates were
screened for haemolytic property. Streptococcus pyogenes was used as
Colicin production test: The colicin production was determined by the
method described by Fernandez-Beros et al. (1990).
The colicin negative E. coli K-12 and colicin V positive E. coli
K-12 Col V+ strains were used as control.
Haemagglutinin test: Slide haemagglutination of erythrocytes was performed
as described by Kausar et al. (2009) and Peerayeh
et al. (2008).
Mannose sensitivity test: The haemagglutination positive strains were
used for mannose-sensitivity assay. The ability of D-mannose to inhibit haemagglutination
was tested by using this sugar to pre-treat either human type O erythrocyte
or bacteria (Najar et al., 2007; Mansouri
et al., 2011).
Measurement of bacterial cell surface hydrophobicity: Salt Agglutination
Test (SAT) was used to measure the bacterial cell surface hydrophobicity (Nalina
and Rahim, 2006). An E. coli strain with a SAT value of 3M was used
as negative control.
Protease production: Protease production by E. coli was tested
by observing hydrolysis of casein when grown on milk agar medium (Paniagua
et al., 1990; Shumi et al., 2004).
Pseudomonas aeruginosa NCTC-6750 was used as positive control strains.
Antibiotic susceptibility testing: All the clinical isolates of E.
coli were tested for antibiotic resistance by the standard agar disc diffusion
technique described by Bauer et al. (1966) on
Mueller-Hinton agar using commercial discs (Oxoid, UK). The following antibiotics
with the disc strength in parentheses were used: Tetracycline (Tet, 30 μg),
Streptomycin (Str, 10 μg), Cefotaxime (Cep, 30 μg), Ceftriaxone (Cef,
30 μg), Trimethoprim-Sulfamethoxazole (Tms, 25 μg), Ampicillin (Amp,
25 μg), Chloramphenicol (Clr, 20 μg), Cefradine (Cef, 30 μg),
Gentamicin (Gen, 30 μg), Penicillin (Pen, 10 μg), Nitrofurantoin (Nit,
100 μg), Ceftazidime (Caz, 30 μg) , Polymyxin B (Pol, 300 IU) and
Nalidixic acid (Nal, 30 μg). A control strain of E. coli ATCC 25922
was included in each plate. Antimicrobial breakpoints and interpretation were
taken from the CLSI standards (CLSI, 2006).
Statistical analysis: Analysis was performed by employing statistical
package for social science (SPSS version 16) software and excel office program
for the statistical analysis of this study. To compare mean values between groups
t-test was done as a test of significance (Shahina et
RESULTS AND DISCUSSION
Identification of E. coli: The identification of the isolates of E. coli was confirmed by plating on them onto MacConkey and EMB agar. All of the isolates showed typical characteristics of E. coli. All the isolates were gram-negative, non-sporing and mostly motile. All the strains were oxidase negative, indole positive, urease negative, did not produce H2S in KIA media, catalase positive, methyl-red positive, Voges-Proskauer negative, reduced nitrate to nitrite, did not utilize citrate or liquefy gelatin.
Haemolysin production: E. coli strains were grown overnight on
sheep and human blood agar plates at 37±0.5°C. The strain that gave
clear zone of haemolysis larger than the overlying colony was considered as
positive reaction. However, the strains that produced haemolysis on two consecutive
days were labeled as haemolytic E. coli. The strain that produced haemolysis
on sheep blood agar was also found haemolytic for human blood agar. The control
(Streptococcus pyogenes) produced haemolysis on all the blood agar plates.
It was found that 29 (44.6%) clinical isolates of E. coli were haemolytic.
Among the clinical E. coli isolates from urine, blood, pus and peritoneal
fluid, 15 (60.0%), 5 (41.67%), 4 (33.3%) and 5 (50.0%) strains, respectively,
||Haemolysin production by E. coli isolates from different
While only four of the 15 (26.67%) faecal E. coli strains produced
haemolysin. Haemolysin production by E. coli from different sources are
shown in Fig. 1.
Colicin biosynthesis: Of the 65 clinical isolates of E. coli, 23 strains (35.3%) showed colicin activity when grown on trypticase soy agar (+0.6% yeast extract) medium. Of the colicin positive strains, 13 (52.0%) were isolated from urine, 6 (50.0%) from blood and 4 (33.3%) from peritoneal fluid. The colicin positive E. coli strains were further tested for colicin V biosynthesis. Among the clinical E. coli isolates, only the urinary and blood isolates produced colicin V; 6 (24.0%) urinary strains and 2 (16.7%) blood isolate showed colicin V activity. 7 (46.0%) of the control strains produced colicin, of which none was colicin V producer. Colicin production by E. coli isolates is shown in Fig. 2.
Mannose-resistant Haemagglutination (MRHA) test: All the E. coli strains were screened for their possession of mannose-resistant-haemagglutinins by using human type O erythrocytes in the slide agglutination test. The strains that gave positive reactions on two successive days were labeled as MRHA positive E. coli. The tests showed 31 (47.7%) clinical isolates of E. coli were MRHA positive, compared to 4 (26.7%) strains positive among the controls. Among the clinical E. coli isolates, 13 (52.0%) urinary strains and 5 (41.7%) blood strains were MRHA positive, whereas 26.7% E. coli strain of faecal origin were MRHA positive (Fig. 3). None of the E. coli strains isolated from peritoneal fluid and pus gave MRHA positive reaction. In total 50 strains produced either hemolysin or MRHA or both. Of these, 22 (44%) strains produced both haemolysin and MRHA, 15 (30%) strains produced only haemolysin and 13 (26%) strains were MRHA positive but haemolysin negative (Fig. 4).
|| Colicin production by E. coli isolates
||Haemagglutinin (MRHA) production by E. coli isolates
||Relationship between haemolysin production and MRHA of human
type O erythrocytes by E. coil strains
Measurement of cell-surface hydrophobicity: Salt Aggregation Test (SAT) showed that 32 (49.2%) clinical isolates of E. coli aggregated with ammonium-sulphate solution of ≤2.0 M concentration, whereas, 9 out of 15 (60%) of control strains had SAT value ≤2.0 M. Source-wise analysis showed that 18 (72%) urine isolates; 5 (41.7%) peritoneal isolates and 9 (75%) blood isolates had SAT value ≤2.0 M.
A total of 29 strains had SAT value >2.0 M. whereas, 4 out of 15 (20%) of
control strains had SAT value >2.0 M. Source-wise analysis showed that 14
(56%) urine isolates; 7 (58.7%) peritoneal isolates and 8 (66.7%) blood isolates
had SAT value >2.0 M.
A total of 24 strains had SAT value ≤1.0 M. whereas, 7 out of 15 (46.7%) of control strains had SAT value ≤1.0 M. Source-wise analysis showed that 15 (60%) urine isolates; 4 (33.3%) peritoneal isolates and 5 (41.7%) blood isolates had SAT value ≤1.0 M. Comparison of cell surface hydrophobicity of E. coli isolates from different sources is shown in Fig. 5.
Protease production: All of the 65 E. coli strains isolated from
different pathological samples were screened for protease production by cultivating
in 2% milk-agar plates. At the end of 5 days of incubation at 37±0.5°C,
6 (9.2%) clinical isolates of E. coli were protease positive, compared
to 3 (20.0%) strains positive among the controls.
||Comparison of SAT value of E. coli isolates from different
|| Protease production by E. coli isolates
Among the clinical E. coli isolates, 2 (13.3%) urinary strains, 1 (8.33%)
peritoneal strains, 1 (10%) pus strains and 2 (16.7%) blood strains were protease
positive. The control (P. aeruginosa NCTC-6750) always gave positive
result on the same medium. Production of protease by E. coli isolates
from different sources is shown in Fig. 6.
Analysis of virulence factors of E. coli isolates: The results showed that isolates of E. coli from various sources possess several virulence factors that solely or collectively contribute to their virulence. Of the 25 E. coli isolates from urine, 60% produced haemolysin, 52% produced Mannose-Resistant Haemagglutinin (MRHA), 52% produced colicin, 24% produced colicin V, 13.3% produced protease and 69% had cell surface hydrophobicity. Comparison between E. coli isolates from different sources with respect to their virulence factors have been summarized in Fig. 7.
Antimicrobial resistance: The clinical isolates of E. coli were tested for their susceptibility to 14 different antibiotics. It was found that none of the E. coli strain was susceptible to all of the antibiotics. Forty-one (63.07%) strains were resistant to 2 or more of the most commonly clinically used antibiotics. 85% strains was resistant to ampicillin, 73% strains were resistant to tetracycline, 77% strains were resistant to streptomycin, 69% strains were resistant to penicillin, while resistance to sulfamethoxazole-trimethoprim and chloramphenicol were 59.0 and 44.5%, respectively. The third-generation cephalosporin (ceftriaxone, ceftazidime and cefotaxime) and polymyxin B showed most effectiveness. Other drugs that appeared to be clinically useful were the first-generation cephalosporin (cephradine), nalidixic acid, gentamicin and nitrofurantoin. The percentage of resistance to the antibiotics is shown in Fig. 8.
|| Comparison of virulence factors of E. coli isolates
from different sources
|| Antibiotic resistance pattern of clinical isolates of E.
Discovering virulence factors is important to understand bacterial pathogenesis
and interactions of them with the host. Understanding virulence properties may
also aid to select novel targets in drug and vaccine development (Banu
et al., 2011). Capacity of E. coli to produce multiple virulence
factors may contribute to its pathogenicity in extra-intestinal infections (Sharma
et al., 2007). These virulence factors make competent some members
of the normal flora to cause an infection by overcoming the host defence mechanisms
(Emody et al., 2003). Production of haemolysin
usually associated with pathogenicity of E. coli, especially responsible
for more severe forms of infections (Johnson, 1991).
In this study, 44.6% extra-intestinal E. coli isolates were haemolytic
and 60% (15/25) of the E. coli strains isolated from urine were haemolytic
(p<0.001) indicating the importance of haemolysin in the pathogenesis of
urinary tract infection (Najar et al., 2007).
Haemolytic E. coli strains may also at an advantage in producing septicemia
as in this study 41.6% (5/12) of the septicemic E. coli strains were
haemolytic (p>0.001). Production of haemolysin was found statistically significant
only in urinary and blood isolates (p<0.001 and p>0.001, respectively).
Few strains from pus and peritoneal fluid was also haemolytic indicating it
may contribute to tissue injury (Sharma et al., 2007).
Among the extra-intestinal pathogenic E. coli isolates, only the urinary and blood isolates produced colicin V. 6 (24%) urinary strains and 2 (16.7%) blood isolates showed colicin V activity. Production of colicin was found statistically not significant in urinary, blood and peritoneal isolates (p>0.05, p>0.5 and p<0.5, respectively).
None of the E. coli strains isolated from peritoneal fluid gave MRHA
positive reaction. Possession of mannose resistant haemagglutinin was found
significant for the urinary isolates (p<0.001) while, for the blood isolates
the p value was p>0.05. Similar results were found in the study of Soleimani
and Nejad (1994).
Surface hydrophobicity is an important virulence factor of E. coli
that causes extra-intestinal infections. E. coli strain with SAT value
≤2.0 M was described as hydrophobic while those with ≥3.0 M as less hydrophobic.
Source wise analysis of the clinical isolates of E. coli strains indicated
that the urinary and the peritoneal isolates were the most hydrophobic which
is in accordance with results of Suman et al. (2001).
Blood isolates have relatively high SAT values justifying the minor role of
cell surface hydrophobicity in pathogenesis of septicemia. Another interesting
finding was all the MRHA positive E. coli strains had SAT value ≤1.0
In this study very few of the clinical or faecal isolates of E. coli produced extracellular protease and hence it can be presumed to be a minor virulence factor for E. coli infection. Some of the extra-intestinal E. coli isolates did not possess any of the virulence factors studied yet causing infections. May be these isolates induced infections in immuno-compromised hosts or they might possess properties different from those including in this study.
This study also revealed expression of multiple virulence factors by extra-intestinal
E. coli. Most of the haemolysin and MRHA positive E. coli were
also hydrophobic which is in accordance with Hughes et
al. (1982). It is difficult to accurately predict virulence of an organism
on the basis of the virulence phenotype, expression of multiple virulence factors
contribute synergistically in overcoming normal host defence mechanisms (Sharma
et al., 2007).
Antibiotic susceptibility pattern was studied for all isolates of E. coli.
Resistance was observed to commonly used antibiotics such as ampicillin, tetracycline,
penicillin and streptomycin. The greater prevalence of resistance to common
antibiotics has also been reported by other workers (Chitnis
et al., 2003; Weiner et al., 1999).
The presence of multidrug resistance may be related to the dissemination of
antibiotic resistance among hospital isolates of E. coli. Maximum number
of isolates (85%) were resistant to ampicillin and the lowest (7.8%) to ceftazidime.
From the data obtained in this study, it seemed probable that haemolysin production, MRHA of human type O erythrocytes and hydrophobic cell surface might be the important characteristics that enabled E. coli strains to cause extra-intestinal infections. Study on other characteristics, such as resistance to human serum, cytotoxin production, possession of aerobactin iron-acquisition system and adherence to uroepithelium might reveal the virulence determinants of the strains that were negative for all the characteristics studied. Further study involving larger number of E. coli strains from septicemia, peritonitis, abscess and meningitis is necessary before any factor could be implicated for the virulence of E. coli infection at those extra-intestinal sites and for better understanding of interaction of different virulence factors at molecular level.
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