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Indian Journal of Medical Microbiology, Vol. 23, No. 1, January-March, 2005, pp. 48-51 Brief Communication Antimicrobial susceptibility of thermophilic Campylo-bacter spp. isolated from environmental samples Baserisalehi M, Al-Mahdi AY, Kapadnis BP Department of Microbiology, University of Pune, Pune-411 007, Maharashtra Code Number: mb05011 ABSTRACT Environmental samples were subjected to determine frequency of occurrence of pathogenic campylobacters in the environment. The antimicrobial susceptibility of the isolates was tested to evaluate the level of antibiotic sensitive campylobacters in the environment of investigation. In all, 70 Campylobacter isolates were obtained from water and domestic animal faeces samples using Kapadnis-Baseri device and antimicrobial susceptibility of them was determined by disc diffusion test and E- test. The results indicated that all the isolates of Campylobacter were sensitive to ciprofloxacin and resistant to cefotaxime, cephalexin and ampicillin. Lowest MIC values were observed for ciprofloxacin and gentamicin (2µg/mL) and highest MIC values for ampicillin and chloramphinicol (256µg/mL). In general, pathogenic Campylobacter spp. were prevalent in large numbers in the environment, however, they were sensitive to ciprofloxacin.Key words: Antimicrobial susceptibility, Campylobacter spp. Campylobacter is the most common cause of bacterial acute gastroenteritis in human beings. The natural habitat of most of the Campylobacter species is the intestine of birds and other warm-blooded animals, including seagulls and several other wild birds. Campylobacter may enter the environment, including water and food through the faeces of animals, birds, or infected humans. These organisms are unable to grow but may survive in the environment for several weeks at temperatures around 4°C.[1] The genus Campylobacter comprises 14 species, out of which, C. jejuni, C. coli and C. lari are responsible for cases of gastroenteritis. Antimicrobial chemotherapy in case of patients with acute Campylobacter enteritis involves treatment with erythromycin, tetracyclines and fluoroquinolones.[2] However, there are reports on development of resistance in Campylobacter species to erythromycin, tetracyclines and fluoroquinolones from developed[3],[4] and developing countries.[5] For example, the increased fluoroquinolone-resistant campylobacters in Thailand, from 0-84% during 1990-1995 questions continued use of flouroquinolones for treatment of patients suffering from Campylobacter enteritis. In addition, there is report of increased fluoroquinolone-resistant strains of Campylobacter jejuni/ coli from Austria.[5] Therefore, based on foregoing evidence, and because, investigations on bacteriological, pathological, clinical and epidemiological aspects of campylobacters in India are relatively recent, the present study was undertaken to determine frequency of occurrence of pathogenic campylobacters in the environment and to determine antimicrobial susceptibility patterns of the isolates. MATERIALS AND METHODS Isolation of Campylobacter from environmental samples One hundred and twenty six faecal samples collected from healthy domestic animals (buffalo, cow, ox, sheep, goat) from different farms in Pune city, India, were collected using sterile sticks and polyethylene bags and transferred to the laboratory within one hour of sampling. The samples were subjected to detection of Campylobacter immediately upon arrival in the laboratory. In addition, 86 water samples were collected from different sites along the rivers, Mula and Pavana, in Pune city, India. The sampling sites were selected based on extensive use of water by the people living at the bank of rivers. The water samples were collected in 500 mL sterile bottles and transported to the laboratory at ambient temperature, and stored at 4°C. They were analyzed within two hours. Faecal and water samples were collected for 12 months during 2002-2003. Campylobacter strains were isolated using Kapadnis-Baseri device (KB device) on nonselective nutrient agar.[6] All suspected colonies were confirmed by typical morphology, darting motility, Gram staining, oxidase and catalase tests. The isolates exhibiting characteristics of Campylobacter were subjected to standard Campylobacter phenotypic identification tests.[7] These tests included H2S by lead acetate strip, nitrate reduction, growth in 1% glycine and 3.5% NaCl, growth at temperatures 25°C, 37°C and 42°C and resistance to nalidixic acid (30µ g) and cephalothin (30µg). All thermophilic campylobacters were confirmed using hippurate hydrolysis, indoxyl acetate and urease tests. Serological test for confirmation of C. jejuni isolates was performed on the basis of heat - stable (HS) antigen.[8] Antiserum was obtained from MAST ASSURE, Manchester Public Health Laboratory, UK and used according to manufacturer′s instructions. Antibiotic susceptibility by disc diffusion method and E- test The antimicrobial susceptibility pattern of the isolates under study was determined by disc diffusion method[9] and E-test.[10] For disc diffusion test, the antibiotic discs included: chloramphenicol 30 µg, norfloxacin 10 µg, kanamycin 30 µg, co-trimoxazole 25 µg, cefotaxime 30 µg, ampicillin 10 µg, ciprofloxacin 5 µg, tetracycline 30 µg, erythromycin 15 µg, gentamicin 10 µg and cephalexin 30 µg (Hi Media, Mumbai). To perform the E-test three different antibiotic E-test strips were applied on each plate. The plates were incubated at 37°C for 48 hours under microaerophilic conditions and inhibitory concentration of each antibiotic was read at the point where the elliptical zone of inhibition intersected the E-test strip. The antibiotic strips used for E-test were tetracycline, erythromycin, gentamicin, ciprofloxacin, ampicillin and chloramphenicol and were obtained from AB Biodisk, Sweden. RESULTS Isolation and identification of Campylobacter spp. Seventy isolates of thermophilic Campylobacter were isolated from water and animal faeces. Out of these, 27 belonged to C. jejuni, 18 to C. coli and 25 to C. lari species. Among C. jejuni isolates, 21 were isolated from the faeces of domestic animals and six of them from river water. Among C. coli isolates, 10 were isolated from animal faeces and eight from water samples. Out of 25 C. lari isolates, 5 and 20 were from animal faeces and water samples respectively. All of the C. jejuni isolates exhibited positive reaction in the serological test. Hence, heat stable antigens existed in all of the C. jejuni isolates. Antibiotic susceptibility of Campylobacter isolates The results on antibiotic susceptibility of Campylobacter from domestic animal faeces and river water, by disc diffusion method indicated that all isolates of Campylobacter were sensitive to ciprofloxacin. In addition, all isolates of C. coli were sensitive to tetracycline. All Campylobacter isolates were resistant to cefotaxime, cephalexin and ampicillin. Similarly, all C. lari isolates were resistant to co-trimoxazole. Among C. jejuni isolates, 74 (70%) of them were sensitive to gentamicin and kanamycin while 59 (55%) of them were sensitive to erythromycin and norfloxacin respectively. Less than 50% of the C. jejuni isolates were sensitive to chloramphenicol, tetracycline and co-trimoxazole and less than 50% isolates of C. coli were sensitive to rest of the antibiotics except co-trimoxazole and chloramphenicol. The number of C. coli isolates sensitive to antibiotics was relatively less than that of C. jejuni. Besides, less than 50% of C. lari isolates were sensitive to chloramphenicol, gentamicin, norfloxacin, kanamycin and erythromycin except tetracycline. In general, all of the Campylobacter isolates [Table - 1] were sensitive to ciprofloxacin and resistant to cefotaxime, cephalexin and ampicillin. Minimal inhibitory concentrations of six important antibiotics against 70 Campylobacter isolates were determined using E-test. Swarming of some Campylobacter isolates coupled with hazy growth at the edge of the inhibition zone affected precise reading of the E-test results. Minimal inhibitory concentration (MIC) of antibiotics against environmental isolates of Campylobacter by E-test As shown in [Table - 2], varied ranges of MIC values were observed for different antibiotics due to varied responses of Campylobacter spp. The lowest MIC values were observed for ciprofloxacin and gentamicin (2µg/mL) and highest MIC values were observed for ampicillin and chloramphenicol (256 µg/mL). The range of MIC values of ciprofloxacin (2-4 µg/mL), ampicillin (128-256 µg/mL), erythromycin (8-32 µg/mL) and chloramphenicol (16-256 µg/mL) against different Campylobacter isolates were similar. While the range of MIC values of tetracycline and gentamicin varied with the Campylobcater isolates. Lowest MIC value of tetracycline was against C. coli, and C. lari and highest against C. jejuni and C. lari isolates. The range of MIC values for tetracycline against C. lari was relatively wide. Lowest MIC value of gentamicin was against C. jejuni and highest against rest of the species of Campylobacter. The range of MIC values for ciprofloxacin was narrow while for the other antibiotics it was relatively wide. Our observations indicate that all Campylobacter isolates showed similar response to ciprofloxacin while the response isolates was variable to rest of the antibiotics. Good correlation was found between sensitivity data of Campylobacter isolates by disc diffusion method and lowest MIC value obtained for ciprofloxacin in E-test. Besides, lowest MIC value of tetracycline (4µg/mL) for C. coli correlated with that by disc diffusion method. The antibiotic resistance data for all Campylobacter by disc diffusion method correlated with that by E-test. DISCUSSION The present study demonstrated the significance of domestic animals and river water as reservoirs of Campylobacter spp. In addition, the presence of different species of Campylobcater suggested that domestic animals and river water could harbour a variety of thermophilic Campylobacter spp. C. jejuni was isolated with high frequency from domestic animal faeces while C. lari was isolated with high frequency from river water. Frequency of occurrence of C. coli was approximately equal from both the sources. Thermophilic Campylobacter spp. such as C. jejuni may enter the environment including water and food through contamination by domestic animal faeces and humans could be infected due to consumption of such contaminated water and food. Therefore, Campylobacter may be easily transmitted to human and animals from these environmental sources and/or by close contact. Two water-borne outbreaks of C. jejuni affecting thousands of people in central Norway[11] were reported and correlated with contamination of drinking water by stool of birds. On the other hand, campylobacteriosis is considered as a zoonotic disease. Therefore, close contact with infected animals and consumption of contaminated animal food products can be a cause of enteritis due to Campylobacter.[12] Our observations indicated that pathogenic Campylobacter isolates were sensitive to ciprofloxacin. But 40.8% of C. jejuni (MIC 32μg/mL), 66.6% of C. coli (MIC 32μg/mL) and 56% of C. lari (MIC 64μg/mL) isolates were found to be resistant to erythromycin. These results are contrary to reports by Isenbarger et al., who reported that most of the Campylobacter isolates in Vietnam and Thailand were sensitive to erythromycin.[4] On the other hand, most of the Campylobacter isolates were resistant to ampicillin, which was earlier correlated with β-lactamase in resistant strains of Campylobacter spp.[3] It is possible that production of β-lactamase by Campylobacter isolates in this study is responsible for high frequency of ampicillin resistant campylobacters. Our data also indicated that 67% and 24% of C. jejuni and C. lari isolates, respectively from environmental samples, were resistant to tetracycline, which was earlier demonstrated to be plasmid-mediated[2]. Hence, there is possibility of gene transfer among campylobacters and other bacteria in the environment. Therefore, high levels of tetracycline resistance in Campylobacter environmental isolates was not surprising. Gentamicin has been recommended as a drug of choice against Campylobacter enteritis.[13] However, our data illustrated that the gentamicin with MIC values in the range, 2 - 64 μg/mL for C. jejuni and 4 - 8 μg/mL for C. coli and C. lari, could not be a drug of choice. In general, due to high frequency of occurrence of ampicillin resistant Campylobacter spp. in this area, the ampicillin could not be a drug of choice for treatment of campylobacteriosis. Tetracycline and gentamicin are recommended as alternative treatment, while ciprofloxacin would be a drug of choice for treatment of campylobacteriosis in this geographical area. Therefore, we concluded that in this geographical region, ciprofloxacin resistance is not yet a problem as it is in Styria, Austria.[5] In conclusion, thermophilic Campylobacter spp. are ubiquitous in our region. Therefore, people working in farms and coming in contact with domestic animals should take necessary precautions. REFERENCES
Copyright 2005 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb05011t2.jpg] [mb05011t1.jpg] |
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