Reduced Minimum Inhibitory Concentration of Antibiotics Associated with DT 104 Phage Type of Salmonella Enterica Serovar Typhimurium

| There has been worldwide emergence of a multi-drug resistant S. typhimurium phage type DT104. It is reported to be resistant to five antibiotics viz., ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline (ACSSuT) and a major health concern for human and animals alike. A study was undertaken to illuminate the MIC levels of five antibiotics among Indian isolates of S. typhimurium. Examination of 100 strains of S. typhimurium revealed that all the strains were sensitive to chloremphenicol and streptomycin and 95% of tested strains were sensitive to ampicilline. However, sulfisoxazole and tetracycline expressed resistance of 93% and 57%, respectively. The MIC values of all the antibiotics determined in this study were much below the break point except for one isolate, which showedMICvalue of >240 μg/ml for sulfisoxazole. The study provides valuable information on the MIC value of S. typhimurium and it appears that this organism circulating in India have not yet acquired resistance against most of these antibiotics. Judicious use of these antibiotics in human and veterinary practice and regular monitoring is required to keep a check on the situation.

The increasing concern is the emergence of multidrug resistant phenotypes among Salmonella serotypes, in particular S. typhimurium (Mirza and Wamola, 1989;Singh et al., 2011).Antibiotic resistance in many bacteria including zoonotic salmonellas has been attributed to the injudicious use of antibiotics in food-producing animals with onward transmission to humans through the food chain (Threlfall, 2002;Tiwari and Dhama, 2014).
S. typhimurium definitive phage type DT104 resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT) was first isolated in the 1960s and later emerged in the 1990s as major pathogen (de Jong et al., 2012;Sahu et al., 2013).About 15% of isolates have been found to exhibit decreased susceptibility to ciprofloxacin (Threlfall, 2002).MDR DT104 has caused numerous outbreaks throughout the world (Threlfall et al., 2005).This strain of S. typhimurium is recognized to be particularly virulent in animal and human hosts and has been isolated increasingly from humans and animals in the United Kingdom and several other European countries and more recently, in the United States and Canada (Poppe et al., 1998).Humans may acquire the infection from foods of animal origin contaminated with the infective organism.
There is no report/very little information available on the DT 104 phage type and MIC levels of antibiotics to which it is resistant.Therefore, the present study was undertaken to detrmine the MIC levels of five antibiotis to which S. Typhimurium D 104 are resistant.
A total of 100 strains of Salmonella typhimurium were taken from the repository of the National Salmonella Center (Veterinary), Indian Veterinary Research Institute, Izatnagar, India.All the strains were tested for their purity, morphological and biochemical characteristics (Barrow, 1993;Edwards and Ewing, 1972) and serotypically by slide agglutination test using polyvalent antisera and tube agglutination test using somatic and flagellar group specific and factor antisera available at National Salmonella Centre (Veterinary), I.V.R.I., Izatnagar.
All the strains were subjected to testing for minimum inhibitory concentration (Table 1) by HiComb MIC test (Himedia, Mumbai).Briefly, young broth culture of each isolate was swabed on Mueller Hinton agar plates three times, turning the plate at 60 o angle between each streaking to get the homogenous growth of culture.The inoculum was allowed to dry for 5-15 min HiComb MIC strip was applied to the agar surface with the MIC scale facing upwards.Plates were incubated at 37°C and examined after 24hrs.The zone of inhibition was in the form of ellipse.MIC value was determined as the value at which the zones convene the comb like projections of the strips and not the handle.If the MIC value interpreted from part A of the strip was not same for part B, in that case MIC was interpreted as lower of the two.One hundred strains of S. typhimurium were initially subjected to testing for 5 individual antibiotics.
Results revealed that all the strains were sensitive to chloromphenicol and streptomycin and 95% of strains were sensitive to ampcillin.However, resistance was observed with sulfisoxazole by 93% strains and with tetracyclin by 43% strains.The high degree of chloromphenicol and streptomycin susceptibility to S. enterica isolates has also been reported from many other parts of India (Mandal et al., 2004;Singh et al., 2010;Choudhary et al., 2013).This may be due to less use of these drugs in human and animal practice.However, sensitivity to ampiciln appears to be new phenomenon.Recently, Choudhary et al. ( 2013) observed 90% Salmonella Typhi and Paratyphi strains to be sensitive to ampicillin, which may be due to its reduced use in clinical practice.One of the tests prescribed by CLSI for determining minimum inhibitory concentration is E test.However, its cost and limited availability in India may restrict its use.HiComb MIC test (Himedia) is considered to be an alternative to the E test (Harish et al., 2008).In this study, we used HiComb MIC test for determining the minimum inhibitory concentration of selected antibiotics (ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracyclin) against which S. typhimurium isolates were found to be sensitive.
The observations were in agreement to the results of antibiotic sensitivity test.The MIC values of all the antibiotics determined in this study were much below the break point except for one isolate which showed-

Table 1 :
Antibiotics used for MIC determination S.

Table 2 :
Result of minimum inhibitory concentration of individual S. typhimurium strain (R=Resistance)