|Year : 2014 | Volume
| Issue : 3 | Page : 240-246
Antibiotic susceptibility pattern and genotyping of campylobacter species isolated from children suffering from gastroenteritis
RM Abd El-Baky, M Sakhy, GFM Gad
Department of Microbiology, Faculty of Pharmacy, Minia University, Minia, Egypt
|Date of Submission||03-Apr-2013|
|Date of Acceptance||01-Sep-2013|
|Date of Web Publication||10-Jul-2014|
R M Abd El-Baky
Department of Microbiology, Faculty of Pharmacy, Minia University, Minia
Source of Support: None, Conflict of Interest: None
Purpose: To study the prevalence and the antimicrobial resistance of campylobacter species isolated from children suffering from gastroenteritis . Materials and Methods: A total of 125 stool samples were collected from children with gastroenteritis. The identification of isolates was performed with conventional methods as well as with molecular methods based on 16SrRNA species-specific gene amplification by PCR and product analysis. Resistance pattern of the isolated strains was determined using agar dilution method. Results: Conventional methods including sodium hippurate hydrolysis revealed that 12 (9.6%) samples were positive for Campylobacter species. Ten out of 12 Campylobacter spp. were identified as Campylobacter jejuni and 2 as Campylobacter coli but PCR assay revealed that five samples only were positive for Campylobacter and all were C. jejuni. Antimicrobial susceptibility to 10 antimicrobials was performed and all isolates (five isolates of C. jejuni) were susceptible to chloramphenicol, gentamicin and amikacin but all were resistant to ceftriaxone. Conclusion: PCR assay method allows reliable detection of C. jejuni. C. jejuni was the most prevalent Campylobacter species. Gentamicin, amikacin and chloramphenicol were the most effective antibiotic.
Keywords: Antibiotic resistance, campylobacter coli, campylobacter jejuni, gasteroenteritis, PCR
|How to cite this article:|
Abd El-Baky R M, Sakhy M, Gad G. Antibiotic susceptibility pattern and genotyping of campylobacter species isolated from children suffering from gastroenteritis. Indian J Med Microbiol 2014;32:240-6
|How to cite this URL:|
Abd El-Baky R M, Sakhy M, Gad G. Antibiotic susceptibility pattern and genotyping of campylobacter species isolated from children suffering from gastroenteritis. Indian J Med Microbiol [serial online] 2014 [cited 2020 Oct 19];32:240-6. Available from: https://www.ijmm.org/text.asp?2014/32/3/240/136550
| ~ Introduction|| |
Campylobacter is the most frequently reported cause of acute inflammatory gastroenteritis in developed countries and major cause of intestinal disease in children less than 2 years of age in these areas. A collective name for infections caused by members of these bacteria is called campylobacteriosis.  Sporadic human cases have been associated with consumption of under-cooked poultry meat, while larger outbreaks are associated with raw milk. , Contaminated drinking water has been the cause of sporadic cases, as well as of larger outbreaks. 
Campylobacteriosis is a self-limited disease, and antimicrobial therapy is not generally indicated in most cases. However, treatment can decrease the duration and the severity of illness if it is initiated early. Antibiotics have a role in reducing the symptoms, shortening the span of illness and controlling the transmission in the community.  In developing countries due to inappropriate detection method, a number of cases might have been undetected.  The fastidious growth requirements, complex taxonomy and unreliable biochemical tests, represent significant challenges in the identification of Campylobacter spp.  Furthermore, Campylobacter coli and Campylobacter jejuni are closely related by phylogenetic and genetic criteria,  so differentiation between Campylobacter species is difficult.  Differentiation between C. jejuni and C. coli is important in the treatment of human campylobacteriosis. So, development of simple methods for detection and reliable differentiation of the thermophilic Campylobacter species are necessary. 
Since 1977, membrane filtration  modified selective media  and molecular methods  have discovered other species that are rarely implicated as causes of disease. These species, namely Campylobacter hyointestinalis,  Campylobacter upsaliensis and Campylobacter fetus are inhibited by the high amount of cefoperazone contained in the selective medium. , Because most laboratories routinely use selective culture, these these less common are being missed. Moreover, biochemical identification common to the species level is limited and unreliable for Campylobacters; hence they are identified only to the genus level. The combination of not being able to detect the unusual species by the current method, and not identifying to the species level the common species that are isolated, has contributed to a limited understanding of the epidemiology of campylobacter gastroenteritis.
Recently, molecular methods have been developed that have the potential to replace classical bacteriological methods. Most molecular methods are based on the polymerase chain reaction (PCR) which is fast, simple and reliable. It allows not only detection of viable but also non-cultivable forms of Campylobacter., However, the high number of Campylobacter cases limits the routine use of genotype-based identification methods.
Antimicrobial resistance of Campylobacter spp. to fluoroquinolones, which are generally used for the empiric treatment of bacterial gastroenteritis, has increased during the past two decades, mainly as a result of the approval of this group of antimicrobials for the use in food-producing animals. , Macrolide-resistant Campylobacter spp. isolated from food, animals and humans have also been reported. ,, Resistances in Campylobacter spp. are most probably caused by the broad use of these antibiotics in veterinary medicine.  The antibiotic susceptibility patterns of Campylobacter seem to vary widely from country to country and from place to place.  So, the objective of this preliminary study was to determinate the prevalence of Campylobacter spp. (especially, C. jejuni and C. coli) in children suffering from gasteroentrities using conventional methods and PCR and to determine their antibiotic-resistance pattern.
| ~ Materials and Methods|| |
Collection of samples
This study was carried out prospectively at Suzan Mubarak and Onclostoma Hospitals between 30 October and 30 December 2011. Subjects were patients between the ages of 0 and 15 years, who presented with foul-smelling diarrhoea with or without mucous, fever and abdominal pain at the paediatrics units of these hospitals. One hundred and twenty-five fresh stool specimens were collected (50 samples from children <5 years and 75 from those of 5 - 15-years old). Most of the faecal specimens collected exhibited one or more of the following appearance (watery, bloody and/or mucoid) and some of them containing fecal leucocytes "pus cells". Swabs were immediately inoculated into 10-mL transport Campylobacter selective enrichment broth (Preston broth, Oxoid) and refrigerated at 5°C for a maximum of 2 h after sampling and before dispatch to the laboratory.
Conventional Culture method (using selective agar)
The samples in Preston broth were incubated for 48 h at 42°C. Following enrichment culture, samples were inoculated onto surface-dried plates of Campylobacter agar Charcoal Cefoperazone Deoxycholate Agar (CCDA). CCDA (LAB M laboratories, UK) was used as a selective agar for Campylobacter isolation. , The plates were incubated in jars with microaerophilic atmosphere generated by gas packs (LAB M laboratories) at 42°C for 48 h. The suspected colonies, showing typical morphology and positive for oxidase, H 2 S production in triple sugar iron agar, glucose utilisation and catalase reaction were microscopically examined after Gram stain and typical isolates were primarily identified through sodium hippurate hydrolysis.  All isolates were stored in brain heart infusion broth with 50% glycerol at -80°C for any further investigation.
Screening PCR assay
One mL aliquots of enrichment broths were centrifuged 13,000 × g, for 3 minutes. The pellets (from enrichment broths) and the colonies obtained from bacterial cultures on selective media plates were collected and suspended in sterile deionised distilled water and heated in a boiling water bath for 10 min. The samples were cooled immediately on ice for 5-10 min and centrifuged at 13000 × g for 5 min. The supernatants were used as DNA templates for PCR. 
Two sets of primers were used for PCR detection of campylobacter and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay, all samples were tested by PCR assay to identify campylobacter positive samples and if there are any non-cultivable strains based on their 16S rDNA gene sequences. PCR was performed in a DNA thermal cycler (UNO II Thermocycler; Biometra GmbH, Gottingen, Germany). The sequence of the forward primer, termed CCCJ609F, was 59- AAT CTA ATG GCT TAA CCA TTA-39, and the sequence of the reverse primer, termed CCCJ1442R, was 59-GTA ACT AGT TTA GTA TTC CGG-39. The primer pair was employed in a PCR with an annealing temperature of 58°C, generating an amplicon of 854 bp from all tested strains of both C. jejuni subspecies and C. coli. The second PCR assay, based on the hippuricase gene sequence, identifies strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The sequence of the forward primer termed HIP400F, was 59-GAA GAG GGT TTG GGT GGT G-39. The reverse primer termed HIP1134R, was 59-AGC TAG CTT CGC ATA ATA ACT TG- 39. The predicted product size was 735 bp. By using a PCR cycle with an annealing temperature of 66°C, an amplicon of this size was generated from tested strains of C. jejuni. The PCR product (5-μl aliquot) was separated by electrophoresis in 1% agarose gel at 100v for 40 min in Tris-acetate buffer visualised by ethidium bromide staining illuminated by UV transilluminator. 
Campylobacter susceptibility testing
Antimicrobial susceptibility testing was performed using agar dilution method according to the clinical and laboratory standard institute (CLSI) 2011.  The antibiotics used were ciprofloxacin, gentamicin, amikacin, chloramphenicol, ampicillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, ceftriaxone, cefoperazone and cefipime (all were obtained from Bayer, Milan, Italy). Preparation of stock solutions of the tested antibiotics was performed according to manufacturer's instructions. The MIC was defined as the lowest concentration of an antimicrobial agent that completely inhibited visible growth on agar surface. Since the Clinical Laboratory Standards Institute (CLSI) recommendations do not include specific breakpoints for defining resistance in Campylobacter spp. the criteria used in our study were those for Enterobacteriaceae.
| ~ Results|| |
In conventional culture method, 12 out of 125 samples were identified as Campylobacter genus. In differentiation tests, 10 samples were identified as C. jejuni and two samples as C. coli. By the use of PCR, to detect if there are any other forms of non cultivable camplyobacter, it was found that 5 samples only generated PCR products with the length of 854-bp specific for campylobacter spp. [Figure 1]. PCR positive samples were also positive CCDA cultures. It also revealed that all five samples positive for Campylobacter spp. are all C. jejuni as they were positive for 735bp gene (specific for C. jejuni) [Figure 2]. All samples positive for campylobacter were isolated from children <5 years [Table 1].
|Figure 1: Detection of Campylobacter genus in human stool samples by PCR assay amplifying 854 bp segment of 16S rRNA gene, specific for Campylobacter|
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|Table 1: Typing of Campylobacter spp. by conventional methods and result confirmation using PCR |
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|Figure 2: Detection of Campylobacter jejuni in human stool samples by PCR assay amplifying 735 bp segment of hip gene, specific for C. jejuni|
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[Table 2] shows that all isolates were susceptible to gentamicin, amikacin and chloramphenicol while all were resistant to ceftriaxone, 4 were resistant to ampicillin, 3 to ampicillin/sulbactam and cefoperazone, 2 to amoxicillin/clavulanic and one to ciprofloxacin and cefipime.
|Table 2: Antimicrobial resistance rates of Campylobacter spp. isolated from children suffering from gastroenteritis |
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| ~ Discussion|| |
Our study is the first report ever published from our country referring to the isolation, identification and antimicrobial resistance of Campylobacter spp. from children suffering from gastroenteritis. Assays for Campylobacter spp. have shown variation in sensitivity and specificity probably due to the high heterogeneity of the species,  implicating that a polyphasic strategy should be used to identify C. jejuni and C. coli. There was a remarkable discrepancy in the identification of Campylobacter strains between biotyping by the conventional methods and the molecular methods. In our study as well as in previous reports, Polymerase chain reaction and the detection of 16Sr RNA gene are excellent and more rapid methods for identification and differentiation between C. jejuni and C. coli as discrimination between the closely related species, C. jejuni and C. coli, is only based on the hippurate hydrolysis test,  but this phenotypic distinction is not always accurate because other amino acids or peptides which are transported from the culture media or produced during the incubation can give false-positive results. Also positive results from hippurate hydrolysis tests are based on the observation of the deep purple color. Pale purple colour is considered as negative. The judgement is based on qualitative criteria which are not reliable and may lead to misinterpretation. , Also, there are some species that are inhibited by the high amount of cefoperazone contained in the selective media, Due to these limitations, PCR is an alternative. In our study, the first set of primers was specific for Campylobacter genus and the other one was specific for C. jejuni. The sets of primers have also been used by Cloak and Fratamico  and Nayak et al., Our results revealed huge differences between conventional and molecular methods. As out of 125 samples, 12 (9.6%) were positive by conventional culture method but only 5 samples (4%) were detected as Campylobacter genus by PCR. These results are in agreement with the findings of Nayak et al.,  who reported that 67% of total Campylobacter isolates gave false results with conventional culture method. PCR assay revealed also that all campylobacter isolates were identified as C. jejuni that agrees with the findings of Eygor et al.,  and Sαenz et al., 
In the present study, the isolation rate of positive samples of Campylobacter spp. in the examined samples and that was proved by PCR was 4% from the total samples which was lower than that reported by Samuel et al.,  (8.2%), Mitikie et al.,  (10.5%), Kafetzis et al.,  who isolated Campylobacter spp. from 9% of 294 stool samples of hospitalised children and Aboderin et al.,  who reported a higher percentage (19.1%). On the other hand, our results agree with that reported by Maraki et al.,  who isolated Campylobacter spp. with a rate of 4.2% from 7090 human stool samples. These differences could be due to the difference in geographical location and study period where Campylobacter is hyperendemic in developing countries owing to poor sanitation and close contact with animals in home. All Campylobacter-positive samples (5/125) were seen in children below 5 years of age which agree with results obtained by Mitikie et al.,  and Taylor  who reported that Campylobacter infections are endemic in developing countries with peak isolation rate occurring in children less than 3-years old.
When either culture method detected a campylobacter to the genus level but PCR gave a negative result, this indicated that a pathogenic campylobacter species was unlikely to have been isolated. In these cases, the presence of commensal species as C concisus C rectus,  C hominis and the arcobacter species is possible. 
Since the Clinical Laboratory Standards Institute (CLSI) recommendations do not include specific breakpoints for defining resistance in campylobacter species, the criteria used in our study for the tested antibiotics were those of the CLSI for Enterobacteriaceae as described before by Englen et al.,  and by Luber et al.,  pending specific CLSI recommendations. So that, the breakpoints of enterobacteriaceae interpreted in CLSI 2011 were used in our study.
Antimicrobial treatment can shorten the duration and severity of illness if initiated early in the course of infection and antibiotic susceptibility studies are very necessary because variation occurs from country to country and from place to place. Regarding the antimicrobial susceptibility, there was a remarkably high resistance rate to ceftriaxone (100%), ampicillin (80%), cefoperazone and ampicillin/sulbactam (60%) amoxicillin/clavulanic (40%) but low resistance was shown against ciprofloxacin and cefipime (20%). High resistance rate may be either due to the potential use of these antimicrobials in open market and private pharmacies without prescription.  On the other hand, all isolates were susceptible to chloramphenicol, gentamicin and amikacin. This may be due to the fact that responded these antibiotics are prescribed less frequently in treating diarrheal cases. Many studies agree with our results. ,, As they showed that all tested isolates were susceptible to gentamicin and chloramphenicol but showed higher resistance to ciprofloxacin than reported in our study. Prats et al.,  reported low rates of resistance to Ciprofloxacin shown by C. jejuni isolated from humans which has increased from 3.5% in 1992 - 1993 to 12.7% in 1995-1997. Also, low rates for resistance (9%) to Ciprofloxacin were recorded by Wardak et al.,  On the other hand, Adekunle et al.,  showed that 66% of isolates of C. jejuni were resistant to ampicillin and all isolates were sensitive to gentamicin which is close to our results but they showed that all isolates were ciprofloxacin sensitive.
The rate of resistance to these drugs in developing countries is higher compared to that of developed countries. This is due to the use of these drugs for infections other than gastroenteritis and self medication which are often the main causes of increased resistance in developing countries.  Antibiotics which have poor in-vitro activity against C. jejuni include Cefoperazone and Ceftriaxone, may be incorporated into selective media for isolating C. jejuni from mixed flora such as that found in feces.
| ~ Conclusions|| |
The isolation, identification, and antimicrobial resistance of Campylobacter spp. from children less than 15 years old has been investigated and reported for the first time in our country. Our results showed a low prevalence of Campylobacter spp. in the geographical region included in this study with the predominance of C. jejuni in children younger than 5 years of age. There was a remarkably high resistance rate of C. jejuni isolates to cefoperazone, ampicillin, ampicillin/sulbactam and amoxicillin/clavulanic acid. The resistance rate of Campylobacter species increased through time which may be due to inappropriate usage of the commonly available drugs. Therefore, it is needed to update information about the resistance pattern of Campylobacter species in different areas. Also, we found that the application of PCR techniques is a short-time method for Campylobacter screening.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]