Cultural Isolation and PCR Based Assay for Detection of flaA gene of Campylobacter jejuni from Acute Diarrheic Patients in Tertiary Care Hospital at Dhaka, Bangladesh

Aim: Among bacterial causes of acute diarrhea Campylobacter species is frequently responsible. Campylobacter spp . is the leading agents of bacterial gastroenteritis in developed as well as developing countries. This study was conducted to determine the frequency and antimicrobial susceptibility pattern of Campylobacter spp . isolated from stool sample of acute diarrheic patients. Study Design: This was a cross-sectional study. Place and Duration of Study: Two hundred stool samples of children of age between 6 months to 5 years with diarrhea/dysentery were taken from outpatient and inpatient department of Dhaka Medical College Hospital (DMCH) and Dhaka Shishu Hospital from the 1st January, 2011 to 31st December, 2011. The study was carried out at the department of Microbiology, Dhaka Medical College (DMC). Methodology: The samples were inoculated on Campy-Bap media and MacConkey’s agar media and were incubated at 42°C under microaerophilic condit ions. The growth after 48 hours was provisionally identified by colonial morphology, oxidase test, catalase test, Gram staining and motility. The organisms were identified to species level by hippurate hydrolysis and resistance to cephalothin. All isolates of Campylobacter jejuni by using conventional bacteriological method were also positive using the Polymerized Chain Reaction (PCR) assay by detecting flaA gene specific for Campylobacter jejuni . Susceptibilities of 27 Campylobacter isolates were determined for seven antimicrobial drugs using the disk diffusion assay. Results: Using conventional bacteriological methods, 27(13.5%) of 200 stool samples were positive for Campylobacter spp. Among isolated Campylobacter spp, Campylobacter jejuni was (88.89%); the remaining isolates were Campylobacter coli (11.11%). Peak age of children with Campylobacter spp . infection was 6-12 months. The male and female ratio was 1.5:1. Resistance to co-trimoxazole was the most common finding, followed by resistance to nalidixic acid, and ciprofloxacin.


INTRODUCTION
Diarrheal disease is the second leading cause of death and causes 1.3 million deaths every year in children under five years [1]. The causes of diarrhoea include a wide range of viruses, bacteria and parasites [2]. Campylobacter spp. are universally acknowledged as the most frequently isolated bacterial pathogens associated with human gastroenteritis, particularly in young children. Among the Campylobacter spp., C. jejuni, accounting for about 90% of infections and rest 10% infection is caused by C. coli [3]. In Bangladesh, the frequency of Campylobacter spp. causing acute childhood diarrhea varies from 12.3% to 12.9% [4,5]. In the Middle East countries, about 5% to 10% of acute diarrhea cases are caused by Campylobacter spp. [6] and in Pakistan the frequency is about 18% [7].
Various pathogenic mechanisms like production of cholera like enterotoxin and cytotoxin and ability to adhere and invade epithelial cells have been proposed to play the role in cases of enteritis [8] caused by Campylobacter spp. The prevalence of flaA gene in all the isolates indicates pathogenic potential since the flaA gene plays an important role in Campylobacter pathogenesis [9]. The flaA gene is the pathogenicity determinants of Campylobacter and a popular target of genotyping especially in epidemiological studies [10].
Antimicrobial resistance in both human and animal Campylobacter isolates has become increasingly common in developing countries [11]. Campylobacter spp. also is generally susceptible to aminoglycosides, chloramphenicol, clindamycin, nitrofurantoins and imipenem [12].
The present study was designed to estimate the frequency of C. jejuni infection among acute diarrheal children by culture and genotype detection by PCR and identify their antimicrobial susceptibility pattern. Among the bacterial causes of diarrhea, Campylobacter spp. is not an uncommon cause. But in our country this bacteria is not routinely isolated is clinical laboratories. As a result, vast part of diarrheic stool sample is reported as 'normal flora' and patients do not get treatment properly. Among them, a large number of patients become chronic carrier. Campylobacter gained more importance particularly during last 30 years as it has also been recognized as a major cause of human illnesses ranging from gastroenteritis to Guillain-Barre Syndrome [13,14]. The sequelae are autoimmune-mediated demyelinating neuropathies Guillain-Barre´ and Miller Fisher syndromes [15], reactive arthritis, hemolytic uraemic syndrome and meningitis [16]. It is necessary to determine the antibiotic susceptibilities of these enteropathogens in order to pinpoint the best mode of therapy [17]. In Bangladesh, few studies have been done for detection of C. jejuni by PCR [18]. Identification to species level is hindered by variations in methodology and the subjective interpretation of biochemical test results. There are also isolates with atypical phenotypes. For example, the differentiation of C. jejuni from C. coli relies on the ability of C. jejuni to hydrolyze hippurate [19] but certain atypical C. jejuni strains fail to do so [20], rendering identification based on this single test unreliable. In some instances, serotyping alone may be insufficient, cross reactivity may occur and expensive to characterize C. jejuni and C. coli but may be useful in conjunction with other typing schemes such as PCR [21].

MATERIALS AND METHODS
This cross sectional study was performed on two hundred stool samples of children of age between 6 months to 5 years with diarrhea/dysentery in outpatient and inpatient department of Dhaka Medical College Hospital (DMCH) and Dhaka Shishu Hospital from the 1 st January 2011 to 31 st December, 2011. Stool of acute watery diarrhoea or dysentery mixed with or without blood or mucus were collected in a clean, dry, wide mouth, leak proof container. Before collection the containers were labelled with full name of patient, age, sex, serial number, date and time of collection. Then stool samples were carried in ice box to Department of Microbiology, DMC within 1 to 2 hours for further procedures. Chi-square (X 2 ) test was carried out to determine the relative importance of various variables. The Ethical Review Committee (ERC) approved the protocol. Informed written consent was taken from guardian before collection of sample.

Microbiological Study
The samples were inoculated in Campy-Bap media containing plates and kept in candle jar and incubated at 42°C (microaerophilic condition) for at least 48 hours. If no growth after 48 hours, then the culture plates were reincubated for next 24 hrs.
The growth was identified after 48 hours by colony morphology, Gram staining, motility test, catalase test, oxidase test (old DC, 1996), and cephalothin sensitivity test [22,23]. All suspected colonies of Campylobacter spp. were resistant to cephalothin. The organisms were identified to species level by hippurate hydrolysis. C. jejuni gave positive reaction while C. coli gave negative reaction.

Molecular Detection
Colonies of Campylobacter spp. were taken from blood agar media and bacterial pellets were formed after incubation the colony at 42°C in sterile eppendorf tube. Then 300 µl distilled water was mixed with bacterial pellet and was vortexed until mixed well. The eppendorf tube was kept in block heater (DAIHA Scientific, Seoul, Korea) at 100°C for 10 minutes for boiling. After boiling the tube was immediately kept on ice. Then the tube was centrifuged at 4°C at 14000 rpm for 10 minutes. Finally supernatant containing extracted DNA was taken using micropipette. Amplification was performed in a final reaction volume of 25 µl. The 2 µl extracted DNA from Campylobacter spp. was mixed in 12.5 µl master mix -PCR buffer, dNTP, Taq polymerase enzyme, MgCl 2 and loaded dye (Promega Corporation, USA), together with 4 µl primer of flaA gene (forward and reverse). The primer sequence of flaA gene C16S-F 5´CTAGCTTGCTAGAACTTAGA3´ and C16-R 5´GTCCACACCTTCCTCCTC3´ and the size of amplified product is 155bp [24]. Volume of the reaction mixture was adjusted by adding 6.5 µl filtered deionized water (nuclease free water). After a brief vortex, the tubes were centrifuged in a microcentrifuge machine for few seconds.
PCR assays were performed in a DNA thermal cycler (Eppendorf AG, Mastercycler gradient, Hamburg, Germany). Each PCR run comprised of preheat at 94°c for 10 minutes followed by 36 cycles of denaturation at 94°C for 1 minute, annealing 58°C for 45 seconds, extension at 72°C for 2 minutes with final extension at 72°C for 10 minutes.
Amplified products were run on to horizontal gel electrophoresis in 1.5% agarose (Bethesda Research Laboratories) in 1X TBE buffer at room temperature at 100 volt (50 mA) for 30 to 35 minutes. Five µl amplified DNA mixed with tracking dye was then loaded into an individual well of the gel (5 mm thick). One hundred bp DNA molecular size marker was loaded into well at the middle or at two sides of the gel for comparing with the base pair of identified band. DNA bands were detected by staining with ethidium bromide (0.5 µl/ml) for 30 minutes at room temperature and then destained with distilled water for 15 minutes. Photographs were taken using digital camera with UV transilluminator (Gel Doc, Major science, Taiwan).

Antimicrobial Susceptibility Test
All the identified Campylobacter spp. was tested for antimicrobial susceptibility performed by Kirby Bauer disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute [23]. The antimicrobial disks were used according to the standard antibiotic panel for specific sample and isolated organisms. Antibiotic disks were obtained from commercial sources (Oxoid, UK). The isolated organisms were tested against ampicillin (10 µg), tetracycline (30 µg), cotrimoxazole (25 µg), nalidixic acid (30 µg), tetracycline (30 µg), cephalothin (30 µg), erythromycin (15 µg), and ciprofloxacin (5 µg). Antibiotic sensitivity testing of identified C. jejuni and C. coli was performed in blood agar media at 42°C for 24 hours.

RESULTS AND DISCUSSION
Campylobacters are one of the most frequent causes of foodborne gastroenteritis in developing as well as developed countries [12]. It is noteworthy that C. jejuni and C. coli are the two main species isolated from the stool of diarrheal children [25].
In the present study, among 200 patients, 27 (13.50%) were positive for Campylobacter spp. which is mentioned in Table  respectively from diarrheal children. These are in accordance with the present study [4,5,26]. Various types of Campylobacter spp. were isolated from diarrheic stool samples such as 13% in Thailand, [27] 17.7% in Algeria, [28] 18% in Tanzania, [29] 18% in Pakistan, [7] 12.5% in the US [30] and 18.6% in Peru [31] which support the present study. The difference in frequency of Campylobacter spp. in different parts of the world is probably due to varying standards of living conditions, water supply and feeding habits as the infection occurs through water and food. The frequency of socioeconomic status of Campylobacter spp. positive cases has been stated in Fig. 2. In both developed and developing countries poultry is an important source of Campylobacteriosis. Strains isolated from human and chickens were matched both phenotypically and genotypically, confirming that chickens are an important source of human Campylobacteriosis [32].
In the present study, 5 (2.5%) patients had both Campylobacter and DEC. Campylobacter is isolated frequently with another enteric pathogen in patients with diarrhea in developing countries. In some cases half or more patients with Campylobacter enteritis also had other enteric pathogens [5].
In the present study, among 27 Campylobacter spp., 13 (48.15%) were from 6 months to 12 months of age group and 6 (22.22%) from 13 to 24 months of age group and 8 (29.63%) were above 24 months. This difference in Campylobacter infection in different age groups is not statistically significant (p>0.05). In Bangladesh, Haq and Rahman reported that Campylobacter spp. infection was 38.8% in children of ≤ 12 months of age and 15.9% in children of >12 months of age [33]. These findings agree with the findings of the present study. Ali et al. and Feizabadi et al. reported peak incidence of C. jejuni infection in <24 months of age group, which is also similar to the present study [7,17]. In this study, 8 (4%) cases were from above 24 months of age group. Similarly, Haque et al. reported that 5.8% C. jejuni were isolated from 2 years to 5 years of diarrhea patients [34].
In the present study, among 27 Campylobacter positive cases, 16 (59.26%) were male, 11 (40.74%) were female and ratio of male and female was 1.5:1, which has mentioned in Table  1. In chi-square test, χ2 = 0.414, df=2, p=3.84 and p>0.05, statistically not significant. In Bangladesh, Huda and Alam et al. reported male and female ratio of Campylobacter spp. were 1.4: 1 and 1.8: 1 respectively which are in accordance with the present study [26,35]. Ali et al. reported the ratio between male and female was 1.7:1 in Rawalpindi which correlate with the present study [7]. Tardy et al. showed male and female ratio of 1:1.2 in France which was not in agreement with the present study [24]. There is a preponderance of males among Campylobacter infected persons but the reason of this type of sex distribution remains unknown [36,12].
In this study, among 27 isolated Campylobacter spp, 24 (88.89%) were C. jejuni and 3 (11.11%) were C. coli. The distribution of Campylobacter spp. has depicted in Fig. 1 [37]. Similar study was performed by Linton et al. in UK and reported that 80% were C. jejuni and 10% were C. coli and rest 10% comprised of mixed infection [38]. Feizabadi et al that 85.8% were C. jejuni and 14.2% were from Iran [17]. The colonies with typical morphology were further identified using gram stain, catalase test, oxidase test, susceptibility to nalidixic acid and cephalothin.
In this study Present study showed that among 27 Campylobacter spp., 24 were positive by hippurate hydrolysis test and all 24 were also positive by polymerized chain reaction by using species specific primer of C. jejuni. samples were negative by hippurate hydrolysis test and identified as C. coli and they were also negative by PCR using C. jejuni specific ( Table 2).
C. jejuni and C. coli are closely related by phylogenetic and by genetic criteria [39] so identification of Campylobacter at species level is difficult. Although the hippurate hydrolysis test is widely used to differentiate C. jejuni species of Campylobacter [40]. This test is not entirely reliable because C. jejuni negative strains have been isolated leads to mis identification. So, for reliable and absolute identification, molecular tests due to their relative ease, low cost and potential application in large-scale screening programs, by means of automated technologies, appear to be attractive candidates [43]. Serotyping alone may be insufficient, cross reactivity may occur and expensive to characterize C. jejuni coli but may be useful in conjunction with other typing schemes such as PCR [44]. In present study, primer of flaA gene specific for was used for identification of the desired strain (Fig. 3).
Most cases of Campylobacteriosis do not require antimicrobial treatment since they are clinically mild and self-limiting in nature, although antimicrobial therapy is required for serious enteritis and systemic infections. Macrolides and  [17]. The colonies with typical morphology were further identified using gram stain, catalase test, oxidase test, susceptibility to In this study Present study showed that among , 24 were positive by hippurate hydrolysis test and all 24 were also positive by polymerized chain reaction by using C. jejuni. Three purate hydrolysis and they were also specific primer are closely related by phylogenetic and by genetic criteria [39] so at species level is difficult. Although the hippurate hydrolysis test is C. jejuni from other [40]. This test is not C. jejuni hippuratenegative strains have been isolated [41,42] which identification. So, for reliable and absolute identification, molecular tests due to their relative ease, low cost and potential scale screening programs, by means of automated technologies, appear to active candidates [43]. Serotyping alone may be insufficient, cross reactivity may occur C. jejuni and C. but may be useful in conjunction with other typing schemes such as PCR [44]. In present e specific for C. jejuni was used for identification of the desired strain do not require antimicrobial treatment since they are clinically limiting in nature, although antimicrobial therapy is required for serious enteritis and systemic infections. Macrolides and fluoroquinolones are considered as drugs of choice for the treatment of enteric infections and intravenous aminoglycoside for those cases present with systemic manifestations [45,46]. There are some previous reports that have shown a marked increase in resistance to quinolones in both developed and develop countries [47,48]. In the present study, the sensitivity of C. jejuni was 91.67% to tetracycline, 75% to erythromycin, 70.83% to ampicillin, 58.33% to ciprofloxacin 50% to nalidixic acid. Twenty four (100%) C. jejuni were resistant to cephalothin and 91.67% to co ( Table 3).  11.11%

C. jejuni C. coli
; Article no. BMRJ.19818 fluoroquinolones are considered as drugs of for the treatment of enteric infections and intravenous aminoglycoside for those cases present with systemic manifestations [45,46]. There are some previous reports that have shown a marked increase in resistance to quinolones in both developed and developing countries [47,48]. In the present study, the was 91.67% to tetracycline, 75% to erythromycin, 70.83% to ampicillin, 58.33% to ciprofloxacin 50% to nalidixic acid.
were resistant to 1.67% to co-trimoxazole    [17]. In Thailand and Nigeria the resistance to ciprofloxacin increased from 0% to 84% (1991)(1992)(1993)(1994)(1995)(1996) as a result of inappropriate use of quinolones. Resistance to quinolones is contributed to the indiscriminate use of drug with the initiation of diarrhea [37].
In the present study, all C. coli. were sensitive to tetracycline, 66.66% to nalidixic acid, 33.33% to both ciprofloxacin and erythromycin. All (100%) C. coli were resistant to ampicillin, cephalothin and co-trimoxazole. Increasing antimicrobial resistance among Campylobacter was noted which may be due to frequent use of these antimicrobials, self-medication, inappropriate and incomplete dose schedule in developing countries [49].

CONCLUSION
In tropical developing countries, Campylobacter infections are hyper endemic among young children. Asymptomatic infections occur commonly in both children and adults, whereas, in developed countries, asymptomatic Campylobacter infections are unusual. Nevertheless, in both developed and developing countries, Campylobacter remains one of the most common bacterial causes of diarrhea. Routine use of antibiotic prophylaxis to prevent Campylobacter infections is not recommended. There is much possibility to develop critical complications of the carriers. So, regular diagnosis of Campylobacter Spp. should be practiced in clinical laboratories. For precise and meticulous detection PCR should be incorporated routinely in diagnostic laboratory.