Prevalence of Enteropathogenic Escherichia coli (EPEC) in Adult Diarrhea Cases and their Antibiotic Susceptibility Pattern

Introduction: Diarrheagenic Escherichia coli (DEC), an important etiologic agent of diarrhea is a major public health problem in developing countries. Relatively few studies have reported the role of enteropathogenic E. coli (EPEC) as etiological agent of adult diarrhea. Objective: To know the prevalence of EPEC in adults and to know their antimicrobial susceptibility patterns. Methods: Diarrheagenic stool samples (n=300), received at the department of Microbiology, Kasturba Medical College hospital, Mangalore, were cultured to isolate E. coli and other intestinal pathogens. Biochemically identified E. coli isolates were further characterized by polymerase chain reaction (PCR). Moreover, all the stool samples were subjected directly to PCR. Antibiotic susceptibility for EPEC was done by Kirby Bauer’s disk diffusion method. Results: Of the 300 stool samples processed, 61 samples showed the growth of E. coli. Four Original Research Article samples had grown Shigella flexneri , three were Vibrio cholerae and One was Aeromonas hydrophila . Among the E. coli isolates characterized by PCR, four were typical EPEC, and atypical EPEC and one isolate was found to be Enterotoxigenic E. coli (ETEC). PCR performed directly on stool samples also yielded the same result. Antibiotic susceptibility testing revealed 42% of the E. coli other than DEC to be extended spectrum beta lactamase (ESBL) producers. However, one of the atypical EPEC was an ESBL producer. Conclusions: In this study DEC, including EPEC types I and II, was found in a number of adult diarrheagenic stool samples and could be a possible cause of diarrhea in these patients. our study highlights the importance of PCR to differentiate atypical and typical EPEC. Presence of ESBL in commensal E. coli is a concern. Further characterization of these isolates from diarrheagenic individual and healthy controls is necessary to know their epidemiological significance.


INTRODUCTION
Escherichia coli is one of the most important members of the family Enterobacteriaceae. They are the commonest cause of infections of the urinary tract and central nervous system [1,2] [3,4].
EPEC enteritis is common in communities with poor hygiene where sporadic cases and frequent out breaks occur in community as well as in institutions [5]. Importance of EPEC as a cause of enteritis in adults is difficult to evaluate due to two reasons. Firstly, adults may have antibodies in their serum due to childhood infection. Hence may get only subclinical infection and may not show symptoms. Secondly, many of the clinical laboratories in India consider E. coli isolates of stool as commensal and do not characterize them further. In a recent study from western Iran 47.5% of the diarrheagenic E. coli from adult patients were found to be EPEC [6,7]. However, literature search has not revealed data on the prevalence of EPEC among adults in southern India. Hence, an attempt was made to directly detect E. coli by PCR, and isolate them by culture from diarrhoeagenic stool. Further, the E. coli isolates were characterized by PCR and their antibiotic susceptibility pattern was studied.

Specimen Collection
Over a period of one year (January to December 2012), diarrheagenic stool samples (n= 300) received at the Department of Microbiology, Kasturba Medical College Hospital, Mangalore, from adult patients who were more than18 years of age, were included in the study by following random sampling method. Individuals who were less than 18 years of age and those adults with diarrhea who were on antibiotic treatment were excluded from the study. This study was approved by the institutional ethics committee. Stool samples were transported at room temperature (25-30°C) and processed within 30 minutes of their receipt.

Microscopic Examination
Stool samples were initially screened microscopically for pus cells, red blood corpuscles (RBC)'s, ova and cysts of parasites. Two to three loops of liquid stool was placed on a clean glass slide and mixed with a drop of saline, covered with a cover slip and observed under low power objective and high power objectives to examine for pus cells, RBCs and trophozoites. Two to three loops of liquid stool was mixed with a drop of iodine solution on a glass slide, covered with a coverslip and observed under low power objective and high power objectives to detect ova and cysts of parasites.

Isolation of Enteric Pathogens
Stool samples were cultured on Sorbitol MacConkey's agar (SMAC), and MacConkey's agar. Enrichment culture was done by inoculation into selenite F broth (SFB) and alkaline peptone water (APW) and were incubated at 37°C for 6-8 hr. Enrichment broths were subcultured on Deoxycholate Citrate agar (DCA) and Thiosulphate Citrate Bile salt Sucrose agar (TCBS) respectively. All the culture plates were incubated at 37°C for 18hr. Both sorbitol fermenting and non-fermenting colonies (n=5) from SMAC and lactose fermenting and nonfermenting colonies (n=5) from MacConkey's agar, lactose non-fermenting colonies from DCA, and sucrose fermenting colonies from TCBS were picked and identified by standard biochemical tests [8]. The tests included catalase, oxidase, fermentation of lactose, glucose and sucrose using triple sugar iron agar, decarboxylation of lysine using lysine iron agar, production of indole, methyl red test, voges proskauer and utilization of citrate. The enteric pathogenic bacterial isolates and E. coli isolates were preserved at -20°C in 20% glycerol broth for further characterization. Apart from the bacterial isolates seven stool samples showed the growth of Candida spp. on MacConkeys agar plates which were not speciated further.

Antimicrobial Susceptibility Testing
Antibiotic susceptibility test was performed by Kirby Bauer's disk diffusion method. Briefly, biochemically confirmed E. coli isolates were grown in Muller Hinton broth for 6 hr at 37ºC. Turbidity was adjusted to 0.5 Mc Farland standard and Muller Hinton agar plates were seeded with culture. Different antibiotics (Himedia laboratories Ltd, Mumbai, India) like ampicillin, ceftazidime, ceftazidime / clavulanicacid, cefotaxime, ciprofloxacin, cefuroxime, cefoxitin and gentamicin were placed on the medium. Antibiotic sensitivity plates were incubated at 37°C for 24hr. E. coli ATCC25922 was used as quality control strain. Zones of clearing around the disks were measured and compared with Clinical and Laboratory Standard Institute (CLSI) standards and interpreted as either sensitive, resistant or intermediate [9].
Isolates were tested for extended spectrum beta lactamase (ESBL) production by the combination disk method using ceftazidime (30 µg) and ceftazidime /clavulanic acid (10 µg). A ≥5 mm increase in diameter of the inhibition zone of the cephalosporin-plus-clavulanate disc when compared to the cephalosporin disc alone were interpreted as phenotypic evidence of ESBL production. Klebsiella pneumonia ATCC 700603 was used as an ESBL producing control and E. coli ATCC 25922 as a negative control [9].

DNA Extraction and PCR
DNA from all stool sample was extracted by using QIA amp stool kit (Genetix Asia Pvt.Ltd., Bangalore) following the manufacturer's instructions. PCR was performed on all the stool samples as it is highly sensitive and specific test. However PCR was done on all the 61 biochemically confirmed E. coli to categorize them in to various DEC. DNA from E. coli Isolates were extracted by boiling method. Briefly, three E. coli colonies were inoculated into 200 µL of distilled water. Boiled for 15 min at 95°C in a dry bath, centrifuged at 12000 g for 5 min.1 µL supernatant is used as DNA in PCR. Table 1 were used for the detection of the virulence genes of E. coli based on the previously published reports [10]. E. coli reference strain EDL 933 was used as positive control for EPEC and STEC. Reference strain E2348/69, were used as positive control for ETEC PCR reactions. PCR was carried out for 35 cycles in the thermo cycler. The reaction conditions were: initial denaturation at 95°C for 5 min, denaturation at 95°C for 1 min, primer annealing at 60°C for 1.5 min, extension at 72°C for 1.5 min and final extension at 72°C for 5 min. Amplified products were separated by using 2% agarose gel, stained with ethidium bromide and photographed using gel documentation system [11].

RESULTS AND DISCUSSION
Of the 300 samples processed, 159 were without pus cells and RBC and did not yield any bacterial pathogens by culture or PCR. Of the 141 stool samples with pus cells, 61samples showed the growth of biochemically confirmed E. coli isolates, four samples S. flexneri, three samples V. cholerae, one sample Aeromonas hydrophila and seven samples had the growth of Candida spp. The remaining 65 samples with pus cells did not yield any bacterial pathogens. Parasitic ova and cysts were not seen in the stool samples screened.  Table 2.
Out of the 300 stool samples directly tested by PCR,148 were negative for all the DEC genes. Four stool samples were positive for both eaeA and bfpA, another four were positive for only eaeA gene, two were positive for only st gene and only one was positive for both st and lt genes as shown Table 3.
Most of the E. coli strains isolated showed resistance to the antibiotic tested. None of the strains was 100% sensitive to the antibiotic tested. Antibiotic susceptibility pattern of biochemically confirmed E. coli isolates is shown in Table 4.
The prevalence and epidemiological significance of E. coli category isolated from stool sample varies with the geographical area. In the present study, various pathogens were isolated from diarrhoeagenic stool samples of adults in addition to EPEC (n=04) atypical EPEC (n=04) and ETEC (n=01). Generally, EPEC are of two types. Type I or typical EPEC are those, which are positive for both eaeA and bfpA, genes. Type II or atypical EPCE are those which are positive for only eaeA gene and lack especially bfpA gene and other DEC genes [12,13]. In the study, we have isolated both typical and atypical EPEC as sole pathogens from stool samples and detected them in stool samples directly by PCR. Studies from India and abroad have reported an increasing trend in the isolation of atypical EPEC than typical EPEC from childhood diarrhea [1,[14][15][16][17]. It is true in the present study concentrated on adult diarrhea patients, where 6.6% of the isolates were typical and atypical EPEC. Hence these eaeA positive atypical EPEC requires further study with regard to their virulence and epidemiologic significance and serotyping. Further, only1.6% of the isolates were ETEC (positive for both st and lt genes) which is found to be lower than the previously reported data from India [2,7], which was mainly focused on pediatric age group, unlike the current study. However, two E. coli isolates were positive for only st genes and negative for lt genes. These st gene positive E. coli isolates needs further characterization for expression of colonization factor genes of ETEC. Recently, EPEC has been reported to be a commonly identified DEC strain in adult diarrhea cases in Iran [6]. However, prevalence of EPEC among adult diarrhea cases in India is not available to compare with our data.     antimicrobial resistance seen in commensal E. coli isolates could be due to indiscriminate use of antimicrobials in clinical practice and sale of antibiotics across the counter. High prevalence of antimicrobial resistance among EPEC strains was documented in different parts of the world [14,18,19]. In this study, resistance was seen more commonly in typical EPEC than in atypical strains which is in agreement with the earlier findings. However one of the atypical EPEC was ESBL producer. Hence rapid detection and differentiation of DEC from commensal E. coli from stool samples by PCR plays an important role in patient management.

CONCLUSION
Results of our study highlights the importance of characterization of all E. coli strains isolated from diarrhoeagenic stool samples by PCR. If EPEC are detected in stool samples of adults suffering from diarrhoea it should not be ignored. Further, direct detection of DEC virulence genes in stool samples by PCR would save time and also help in fast patient management. Further studies are necessary to characterize large number of typical and atypical E. coli isolates from diarrhoeagenic stool samples to know their pathogenic potential.

ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the Institutional ethics committee of Kasturba Medical College Mangalore, and have therefore been performed in accordance with the ethical standards.