A Comparison Study on Antimicrobial Susceptibility of Campylobacter spp. Isolates from Faecal Samples of Domestic Animals and Poultry in India and Iran

MATERIALS AND METHODS

Isolation of Campylobacter from environmental samples: In all 246 faecal samples were collected from healthy domestic animals and poultry at different farms of India and Iran. Out of all, 126 samples were collected from buffalo, cow, ox, sheep, goat and poultry in Pune, India and 120 samples were collected from cow, horse and Poultry in Shiraz, Iran. The faecal samples were collected using sterile sticks and polyethylene bags and transferred to the laboratory within one hour of sampling. The samples were subjected for detection of Campylobacter immediately upon arrival in the laboratory. The method of Camplylobacter detection in this study was pre-treatment-Kapadnis Baseri (prêt- KB) method and medium was blood and antibiotic free Kapadnis Baseri (KB) medium (Baserisalehi et al., 2004).

To perform this method faecal samples were emulsified at 10% (w/v) in sterile phosphate- buffered saline (0.1 M, pH = 7) to give 10% suspension. The suspension was centrifuged at 8500 rpm for 10 min followed by holding them at room temperature. After 10-15 min, 0.1 mL supernatant from the tube was plated on the KB medium.

All suspected colonies grew on the KB medium were picked up and 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 (Atabay and Corry, 1997). These tests included H₂S by lead acetate strip, nitrate reduction, growth in 1% glycine and 3.5% NaCl, growth at temperatures 25, 37 and 42°C and resistance to nalidixic acid (30 μg) and cephalothin (30 μg). All thermophilic campylobacters were confirmed using hippurate hydrolysis, indoxyl acetate and urease tests.

Antibiotic susceptibility by disc diffusion method and E- test: Antimicrobial susceptibility of Campylobacter spp. isolates in this study was determined by disc diffusion method (Bauer et al., 1966) and E-test (Baker, 1992). For disc diffusion test, the antibiotic discs were chloramphenicol 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). The disc strengths and the zone size interpretation was in accordance with National Committee for Clinical Laboratory standards (NCCLS, 2002).

The antibiotic strips for E-test were tetracycline, erythromycin, gentamicin, ciprofloxacin, ampicillin and chloramphenicol obtained from AB Biodisk, Sweden.

To perform the disc diffusion test, each culture was grown in 5 mL of Muller-Hinton broth until the turbidity corresponded to 0.5 MacFarland standard tubes (1.5x10⁸ cells mL^-1). The suspension was spread inoculated using sterile cotton swab onto Muller-Hinton agar plate and various antibiotic discs were placed on it. After incubating the plates at 37°C under microaerophilic conditions for 48h the inhibition zones were recorded.

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 h 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.

RESULTS

Isolation and identification of Campylobacter spp.: Seventy and thirty seven Campylobacter spp. were isolated from faecal samples of domestic animal and poultry in India and Iran respectively. Out of seventy Campylobacter isolates in India 27 were belonged to C. jejuni, 18 to C. coli and 25 to C. lari and Out of thirty seven isolates in Iran 15 were belonged to C. jejuni, 10 to C. coli and 12 to C. lari species.

Antibiotic susceptibility of Campylobacter isolates: The results on antibiotic susceptibility of Campylobacter isolates from faecal samples of domestic animal and poultry by disc diffusion method indicated that all Campylobacter isolates were sensitive to ciprofloxacin whilst, different responses to the other antibiotics have been observed among the Campylobacter isolates from both of the countries. In addition, present finding showed that frequency of existence of antibiotic sensitive strains of Campylobacter in Iran was relatively high. For instance, all Campylobacter strains isolates in India were resistant to Cephalexin and Cefotaxime whereas, the sensitive strains of Campylobacter to these antibiotics were found among the isolates in Iran. Furthermore, the rate of existence of Ampicillin resistant strains of Campylobacter in India was relatively high (Table 1).

Minimal Inhibitory Concentration (MIC) of antibiotics against environmental isolates of Campylobacter from domestic animals and poultry in India and Iran by E-test: Minimal inhibitory concentrations of six important antibiotics against Campylobacter spp. isolates from domestic animals and poultry were determined by 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.

As shown in Table 2 and 3, varied ranges of MIC values were observed for different antibiotics due to varied responses of the Campylobacter isolates. The lowest MIC values against the Campylobacter isolates from both of the areas were found for ciprofloxacin (2 μg mL^-1) and highest MIC values were found for ampicillin and chloramphenicol with 256 μg mL^-1 in case of the isolates in India and 64 μg mL^-1 in case of the isolates in Iran. Furthermore, the range of MIC values for ciprofloxacin was narrow while, for the other antibiotics tested was wide. Besides, good correlation was found between sensitivity data of Campylobacter isolates by disc diffusion method and lowest MIC value obtained for ciprofloxacin in E-test.

Table 1:	Susceptibility of environmental campylobacters isolates from domestic animals and poultry in India and Iran by disc diffusion method
† Campylobacter isolates from domestic animals and poultry in India, ‡ Campylobacter isolates from domestic animals and poultry in Iran, *Ch, Chloramphenicol,. Ce, Cephalexin, Co, Co-trimoxazole,. Cf, Cefotaxime,. Am, Ampicillin,. Ci, Ciprofloxacin, Te, Tetracycline,. Er, Erythromycin, Ge, Gentamicin

Table 2:	Minimal inhibitory concentrations of antibiotics against environmental Campylobacter isolates from domestic animals and poultry in India by E-test
*27 isolates, † 18 isolates, ‡ 25 isolates were tested. Cumulative percentage of the MIC concentration at which 50% (MIC50) and 90% (MIC90) of the bacterial isolates were inhibited from growth

Table 3:	Minimal inhibitory concentrations of antibiotics against environmental Campylobacter isolates from domestic animals and poultry in Iran by E-test
*15 isolates, † 10 isolates, ‡ 12 isolates were tested. Cumulative percentage of the MIC concentration at which 50% (MIC50) and 90% (MIC90) of the bacterial isolates were inhibited from growth

DISCUSSION

The present study clearly demonstrated the significance of domestic animals and poultry as extensive reservoirs of campylobacters. Present finding illustrated that frequency of occurrence of Campylobacter was high in the both areas of investigation. In addition, presence of different species of Campylobacter suggested that the domestic animals and poultry harbour a variety of the pathogenic Campylobacter spp. Therefore, close contact of the people with infected animals and consumption of contaminated animal food products can be a cause of Campylobacter enteritis.

Although, pathogenic Campylobacter spp. were detected in both of the regions, frequency of occurrence of them in India was relatively high. Similar to present data Baserisalehi et al. (2007) expressed, high faecal carriage of campylobacters for domestic animals and poultry in India increased the risk of infections among the people who living and working in the farms of this area. They also opined that existence of campylobacters in the environment depended on weather status of the countries as well as diet of the animals. Hence, suitable environmental conditions and favourable diet might be considered as reasons for existence of campylobacters in India with high frequency compared to Iran.

On the other hand, present data showed that pathogenic Campylobacter isolates from domestic animals and poultry in both of the countries were sensitive to ciprofloxacin while, varied responses to the other antibiotics were found among the isolates. Furthermore, the results obtained from susceptibility of the isolates to the antimicrobial agents elucidated that frequency of occurrence of antibiotic sensitive Campylobacter isolates from domestic animals and poultry in Iran was relatively high. Although, parallel to present data isolation rate of antibiotic sensitive Campylobacter in developing countries was high (Isenbarger et al., 2002; Taylor and Courvalin, 1988), the rate of antibiotic resistant Campylobacter is increasing in developed countries (Ge et al., 2003). In general, due to high frequency of occurrence of ampicillin resistant Campylobacter spp. in India, 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. Similar to India, ciprofloxacin should be considered as a drug of choice for treatment of campylobacteriosis in Iran. In addition, the existence of antibiotic sensitive Campylobacter in Iran with high frequency increased possibility to select effective antibiotics for treatment of Campylobacter enteritis. Nevertheless based on foregoing evidence ciprofloxacin resistance is not yet a problem in these regions as it is in Styria, Austria (Feierl et al., 1999).