MATERIALS AND METHODS

Bacterial stains: A total of 12 S. Gallinarum isolated from various sources in different regions of India and 3 reference S. Gallinarum strains were procured from the National Salmonella Centre, Indian Veterinary Research Institute, Izatnagar (Table 1). Additional 5 Salmonella serotypes (7 isolates) viz., S. heidelberg, S. typhimurium, S. ayinde, S. essen and S. kastrup isolated from poultry tissue samples and eggs in Bareilly district (Taddele et al., 2011), Uttar Pradesh, India were also included in this study.

Maintenance of strains: S. Gallinarum strains obtained from the National Salmonella Centre, IVRI, Izatnagar were checked for their biochemical profiles and inoculated on nutrient agar slants and kept at 4°C. Sub-culturing was done periodically to test the purity, morphological and biochemical characteristics of the strains.

Antimicrobial resistance testing: All the 22 Salmonella isolates including the 03 standard reference Salmonella strains were subjected to drug sensitivity test using sixteen (16) commonly used antimicrobial agents {ampicillin 10 μg (A10), amoxicillin/clavulanic acid 30 μg (Ac30), gentamicin 10 μg (G10), kanamycin 30 μg (K30), enrofloxacin 5 μg (Ex5), ciprofloxacin 5 μg (Cf5), erythromycin 15 μg (Em15), ofloxacin 5 μg (Of5), tetracycline 30 μg (T30), colistin 10 μg (Cl10), cephalexin 30 μg (Cp30), nalidixic acid 30 μg (Na30), chloramphenicol 30 μg (C30), amoxicillin 10 μg (Aml10), neomycin (N30) and sulfa-trimethoprim 25 μg (SXT25)} by disc diffusion method as described by Miles and Amyes (1996) and the interpretation was made as per the zone size interpretation chart provided by the manufacturer of antimicrobial discs (Himedia, India). The antibiogram profiles were categorized into four groups based on sensitivity pattern and results were scored as highly resistant (0), less sensitive (1), moderately sensitive (2) and highly sensitive (3) and the scored data was analysed statistically.

Table 1:	Host and epidemiological sources of S. Gallinarum isolates

RESULTS

Morphological and biochemical characterization of Salmonella isolates: The Salmonella Gallinarum isolates (15) and the serotypes (5) isolated from poultry showed typical cultural, morphological and biochemical characteristics of the genus and species. Subculturing was done periodically to test the purity, morphological and biochemical properties of the strains. All the isolates produced H₂S in TSI, were non-lactose fermenters, non-motile, positive in MR test, catalase test and negative with oxidase test and urease test and showed gas production from glucose.

Antimicrobial resistance testing for S. Gallinarum isolates: All the 15 S. Gallinarum isolates including the 03 reference strains were 100% resistant to erythromycin and 13 of them were resistant to nalidixic acid where as the two strains, E-4627 and E-4661, were less sensitive to this antimicrobial. Five (33.3%) isolates showed 100% resistance to neomycin and 8 (53.3%) were less sensitive to the same antimicrobial. Fourteen (93.3%) isolates were 100% sensitive to amoxicillin/clavulanic acid and gentamicin whereas strains E-4668 and E-4684 were moderately sensitive to amoxicillin/clavulanic acid and gentamicin, respectively. More than 53% of the isolates were either 100% resistant or less sensitive to the commonly used antimicrobials, kanamycin and tetracycline. The antimicrobials ciprofloxacin, ofloxacin, colistin and sulfa-trimethoprim were 88.8% effective where as enrofloxacin and chloramphenicol showed 82.2% efficacy against the S. Gallinarum isolates tested including reference strains. The reference strain, E-402, showed maximum resistance against ampicillin, erythromycin, tetracycline, cephalexin, nalidixic acid and neomycin and it was less sensitive to kanamycin, enrofloxacin, chloramphenicol and amoxicillin (Table 2 and Fig. 1). The other two reference strains (E-75 and E-76) showed resistance to three antimicrobials viz., erythromycin, nalidixic acid and neomycin. The overall sensitivity of isolates including reference strain to the tested antimicrobials was in the range of 0-98% (Fig. 1).

Table 2:	Antimicrobial sensitivity pattern of S. Gallinarum isolates and reference strain
3Em15-erythromycin 15 μg, 4K30-kanamycin 30 μg, 5T30-tetracycline 30 μg, 6Na30-nalidixic acid 30 μg, 7N30 - neomycin, 8Cp30-cephalexin 30 μg, 9Aml10-amoxicillin 10 μg, 10A10-ampicillin 10 μg, 11Ac30-amoxicillin/clavulanic acid 30 μg, 12G10-gentamicin 10 μg, 13Ex5-enrofloxacin 5 μg, 14Cf5-ciprofloxacin 5 μg, 15Of5-ofloxacin 5 μg, 16Cl10-colistin 10 μg, 17C30-chloramphenicol 30 μg and 18SXT25-sulfa-trimethoprim 25 μg

Table 3:	Antimicrobial sensitivity pattern of Salmonella serotypes isolated during the present study

Antimicrobial resistance testing for 5 Salmonella serotypes: Among the five Salmonella serotypes (S. heidelberg, S. typhimurium, S. ayinde, S. essen and S. kastrup) tested S. typhimurium showed resistance to 6 antimicrobials viz., kanamycin, enrofloxacin, erythromycin, tetracycline, ofloxacin and neomycin and it was highly sensitive only to ciprofloxacin. S. kastrup was also resistant to kanamycin, erythromycin, tetracycline, nalidixic acid and neomycin. The remaining isolates were resistant to at least one antimicrobial and were sensitive to at least 5 antimicrobials (Table 3 and Fig. 2). Four antimicrobials (gentamicin, ciprofloxacin, cephalexin and sulfa-trimethoprim) were the most effective antimicrobials showing about 90.5% efficacies (Fig. 2).

Fig. 1:	Stacked column showing the sensitivity of S. Gallinarum isolates and reference strains to different antimicrobials tested

Fig. 2:	Stacked column showing the sensitivity of different Salmonella serotypes to different antimicrobials tested

DISCUSSION

The clinical management of salmonellosis in man and animals is mainly based on antimicrobials and fluid therapy which is quite expensive; and indiscriminate use of antimicrobials may lead to emergence of multiple drug resistant strains which are threat to human and animal population. The emergence of multidrug resistance among Salmonella spp. is an increasing concern. Salmonella strains of avian origin are also often resistant to variety of antimicrobials approved for poultry including tetracycline, oxytetracycline, penicillin, aminoglycosides, sulfisoxazole and fluoroquinolones (Kabir, 2010; Oliveira et al., 2006; Parveen et al., 2007). Multiple drug resistance against 6-8 drugs has been reported in several Salmonella serotypes of human and animal origin in India by several investigators (Mandal et al., 2006; Prakash et al., 2005; Singh et al., 2007). Emergences of multiple drug resistant Salmonella strains have often been alleged to be responsible for frequently occurring outbreaks and hyperendemicity of salmonellosis in India. The present study was undertaken to test the antimicrobial sensitivity pattern of S. Gallinarum isolates and other 5 Salmonella serotypes.

In the present study, all S. Gallinarum isolates including reference strains were found to be 100% resistant to erythromycin and 13 (86.7%) of them were resistant to nalidixic acid. The resistance pattern for erythromycin was in accordance with previous findings of Khan et al. (2005) in Bangladesh; whereas the resistance to nalidixic acid is in close agreement with the prevalence rate of 92-96% reported from India (Lakshmi et al., 2006) and 96% from Bhutan (Dahal, 2007). High prevalence of nalidixic acid resistance among poultry isolates (89%) was also reported from France (Cailhol et al., 2005). Sensitivity to amoxicillin/clavulanic acid and gentamicin has been observed in 93.3% of the Gallinarum isolates which was in close harmony with previous observations (Dahal, 2007; Gaedirelwe and Sebunya, 2008; Lee et al., 2003). The Salmonella isolates in India from 1996-99 and 2001 were reported to be 100% chloramphenicol sensitive and sensitivity as high as 79% was reported in 2000 (Mandal et al., 2004). The present study also showed chloramphenicol sensitivity of around 82%. This re-emergence of chloramphenicol sensitivity could be attributed to the limited use of the antimicrobial during the last decade in India (Khan and Shukla, 2004; Achla et al., 2005; Mohanty et al., 2006). However, a report by Khan et al. (2005) from Bangladesh showed less sensitivity of Salmonella isolates to chloramphenicol.

On an average the antimicrobial pattern for ciprofloxacin, ofloxacin, tetracycline, colistin and sulfa-trimethoprim observed against S. Gallinarum isolates in this study was in close agreement with earlier reports of Lee et al. (2003) in Korea on the same organism but the pattern for ampicillin, enrofloxacin and kanamycin did not correlate exactly with their results, which may be due to molecular heterogeneity of the strains and use of different branded antimicrobials. In addition, a particular antimicrobial is effective at the time of introduction to the market but later it may become ineffective due to inadvertent use for long duration in sub optimal doses. A resistance to trimethoprim-sulfamethoxazole among poultry isolates has been reported from India (Prakash et al., 2005), Mexico (Zaidi et al., 2006), Senegal (Bada-Alambedji et al., 2006) and USA (Zhao et al., 2006). However, sensitivity to sulfa-trimethoprim was around 89% in this study.

Among the other Salmonella isolates 5/7 (71.4%) were resistant and 2/7 (28.6%) of them were less sensitive to erythromycin. Resistance to nalidixic acid was observed in 3/7 (43%) of these isolates and the remaining 4/7 (57%) were less sensitive to this antimicrobial. Reports from Bangladesh (Khan et al., 2005) and France (Cailhol et al., 2005) have showed the same observation with regard to the resistance pattern to these antimicrobials.

Out of a total of 22 isolates tested for different antimicrobials 4/22 (18.2%) were resistant to one antimicrobial and the remaining 81.8% were resistant to at least two or more antimicrobials. The maximum resistance was observed in S. Gallinarum reference strain E-402 and S. typhimurium, which were resistant to 6 antimicrobials followed by S. kastrup, which showed resistance to 5 antimicrobials. The other S. Gallinarum reference strains, E-75 and E-76, were resistant to three antimicrobials. S. typhimurium was sensitive only to ciprofloxacin. Antimicrobial resistance has been reported to be more common in S. typhimurium than the other serovars in India (Prakash et al., 2005).

Advances to prevent and control salmonellosis in the food animal industry by various means such as improved biosecurity, vaccination, use of competitive exclusion products and the introduction of novel immuno-potentiators with limited success has necessitated the use of antimicrobial chemotherapy in the treatment and control of salmonellosis (Zhao et al., 2007). The occurrence and proliferation of antibiotic-resistant Salmonella in environmental samples, poultry and other animals and humans may be due to the use of medicated feeds, the practice of dipping hatching eggs in solutions containing antimicrobial agents, routine inoculation of day-old poults with antibiotics and treatment of other animals and humans with antibiotics (Kabir, 2010). The widespread use of antimicrobials for therapeutic purposes in food animals and as growth promoters in animal feeds have been implicated in promoting emergence and spread of antimicrobial resistance among salmonellae (Humphrey, 2001; White et al., 2001), through mutation and acquisition of resistance encoding genes (Fluit, 2005). The circumstances in India may be aggravated due to easy accessibility of antimicrobials at cheaper price and their extensive use in poultry production (Prakash et al., 2005; Singh et al., 2007). The other major contributing factor is widespread availability of different brands and uncontrolled use of these antimicrobials.

CONCLUSION

Present study showed for the emergence and widespread prevalence of multidrug resistant Salmonella species in poultry. Therefore, measures need to be focussed to avoid indiscriminate use of antibiotics in food animals and quality control of antibiotics should be initialized to prevent uncontrolled use of antibiotics. Surveillance, identification and antibiotic sensitivity of the prevalent Salmonella serotypes in the country would help devise suitable prevention and control programme for this important poultry pathogen having food-borne zoonosis.

ACKNOWLEDGMENTS

Material and financial support provided by the division of Bacteriology and Mycology, Indian Veterinary Research Institute (IVRI), Izatnagar, Bareilly (U.P.), India is acknowledged by the authors. The national Salmonella centre, IVRI, Izatnagar, Bareilly (U.P.), India is also highly acknowledged for providing the Salmonella Gallinarum isolates including reference starins. The authors are also thankful to Prof. S.K. Khar for his contribution in editing this manuscript.