Short Communication

 

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

 

Mudasir Ahmad1, Rajesh Kumar Agarwal2, Jinu Mary John3, Joy Lalmuanpuia Kataria1, Blessa Sailo4

1Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India; 2National Salmonella Centre (Vet), Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India; 3College of Veterinary and Animal Sciences, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut; 4ICAR Research Complex for NEH region, Manipur Center, Lamphelpat, Imphal, India.

Editor | Kuldeep Dhama, Indian Veterinary Research Institute, Uttar Pradesh, India.

Received | November 01, 2014; Revised | November 13, 2014; Accepted | November 14, 2014; Published | November 21, 2014

*Correspondence | Rajesh Kumar Agarwal, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India; Email: grace_bly@yahoo.com

Citation | Ahmad M, Agarwal RK, John JM, Kataria JL, Sailo B (2014). Reduced minimum inhibitory concentration of antibiotics associated with DT 104 phage type of Salmonella enterica Serovar typhimurium. Adv. Anim. Vet. Sci. 2 (10): 592-598.

DOI | http://dx.doi.org/10.14737/journal.aavs/2014/2.10.592.598

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2014 Ahmad et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

Salmonellosis is one of the important diseases affecting human and animal health worldwide. Many serovars of Salmonella have been identified to cause infection both in humans and animals (Agarwal et al., 1999; Crump and Mintz, 2010; Singh et al., 2013). Among different serovars, S. typhimurium is most commonly associated with enteric infection in man and animals (de Jong et al., 2012). S. typhimurium has a diverse host range, which include humans, cattle, pig, sheep, horse, rodent and birds (Townsend et al., 2001; Chaudhuri et al., 2013). S. typhimurium and other serovars can cause a more invasive infection in immunocompromised patients such as those with AIDS, certain cancers and under chemotherapy, which can be life threatening (Hachfi et al., 2009). It is also recognized as the most common saviour of Salmonella causing cardiovascular, bone and joint infections (Kalpana et al., 1998).

 

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 S. typhimurium strains were initially examined for their antimicrobial drugs susceptibility/ resistance pattern in triplicate by disc diffusion method (CLSI, 2005) on Mueller Hinton agar (Himedia, Mumbai, India) using 5 different antimicrobial agents viz., streptomycin 300 µg,, ampicillin 10 µg, tetracycline 30 µg, sulfisoxazole 300 µg and chloramphenicol 30 µg.

 

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 60o 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.

 

Table 1: Antibiotics used for MIC determination

S. No.	Name of antibiotic	Range (µg)
1	Ampicillin (A)	A: 256 - 0.1 B: 4 - 0.001
2	Chloramphenicol ©	A: 240 - 0.01 B: 8 - 0.001
3	Streptomycin (S)	A: 240 - 0.01 B: 30 - 0.001
4	Sulfisoxazole (Sf)	A: 240 - 0.001 B: 30 - 0.001
5	Tetracycline (T)	A: 240 - 0.01 B: 5 - 0.01

 

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.

 

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

S. No.	S. typhimurium isolates	MIC (µg/ml)
		Ampicillin	Chloramphenicol	Streptomycin	Sulfisoxazole	Tetracyclin
1	E-4767	0.512	0.1	1.0	R	R
2	E-2924	0.512	0.1	1.0	R	R
3	E-421	0.256	0.1	0.1	R	R
4	E-2919	0.512	0.1	1.0	R	R
5	E-4638	0.256	0.1	1.0	>240	0.1
6	E-828	0.512	0.1	1.0	R	R
7	E-2563	0.512	0.1	0.1	R	R
8	E-4231	0.512	0.1	1.0	R	0.1
9	E-2928	0.128	0.1	1.0	R	R
10	E-773	0.256	0.1	0.01	R	0.1
11	E-2416	0.512	0.1	1.0	R	0.1
12	E-5215	R	0.1	0.1	R	0.1
13	E-2700	0.512	0.1	0.01	R	R
14	E-2393	0.512	0.1	0.1	R	R
15	E-557	0.016	0.5	1.0	R	R
16	E-5257	0.256	0.1	0.01	R	R
17	E-4256	0.256	0.1	0.01	3	0.01
18	E-4806	0.512	0.1	1.0	R	0.01
19	E-1159	0.256	0.1	1.0	R	R
20	E-4885	0.512	0.1	1.0	R	0.01
21	E-427	1.024	0.1	1.0	R	R
22	E-2443	0.256	0.1	0.01	R	R
23	E-2382	R	0.1	0.01	R	R
24	E-4483	0.512	0.1	1.0	R	R
25	E-4808	0.512	0.1	1.0	R	R
26	E-5268	0.512	0.1	1.0	R	R
27	E-4807	0.256	0.1	1.0	R	R
28	E-4836	0.512	0.1	1.0	R	R
29	E-2381	R	1.0	3.0	R	R
30	E-4810	0.256	0.1	1.0	R	0.01
31	E-4851	0.064	0.1	1.0	R	0.01
32	E-420	0.256	0.01	0.01	R	R
33	E-872	0.256	0.1	1.0	R	R
34	E-5220	0.256	0.1	1.0	R	0.01
35	E-5256	0.256	0.1	1.0	R	R
36	E-5266	0.128	0.01	0.001	R	R
37	E-2398	0.256	0.1	0.001	R	R
38	E-1114	R	0.1	0.001	R	R
39	E-5202	0.256	0.1	0.001	R	R
40	E-425	0.032	0.1	0.001	R	R
41	E-2391	0.512	0.1	0.1	R	R
42	E-4769	0.512	0.1	0.1	R	0.1
43	E-4891	0.512	0.1	0.1	R	0.01
44	E-2614	0.256	0.1	0.001	R	R
45	E-5262	0.256	0.1	0.01	R	0.01
46	E-2392	0.512	0.1	1.0	R	0.1
47	E-2959	0.512	0.01	0.01	R	R
48	E-5227	0.512	0.1	1.0	R	R
49	E-4857	0.512	0.1	1.0	R	0.1
50	E-2377	0.128	0.1	1.0	3.0	0.01
51	E-3139	0.512	0.01	0.01	3.0	0.1
52	E-2919	0.064	0.01	0.01	R	R
53	E-5255	0.512	0.01	0.01	R	0.1
54	E-4862	0.256	0.01	1.0	R	0.1
55	E-5264	0.256	0.01	1.0	R	R
56	E-608	0.512	0.1	1.0	R	R
57	E-4858	0.512	0.01	1.0	R	0.1
58	E-4242	0.512	0.1	1.0	R	R
59	E-4630	0.512	0.1	1.0	R	R
60	E-2689	1.024	0.1	0.1	R	R
61	E-4658	0.512	0.1	1.0	R	R
62	E-5270	0.256	0.1	0.001	R	R
63	E-4863	0.064	0.1	1.0	R	0.01
64	E-660	0.256	0.5	0.01	R	R
65	E-5226	0.064	0.1	1.0	R	R
66	E-4856	0.256	0.1	0.001	R	0.1
67	E-4803	0.512	0.1	1.0	R	0.1
68	E-5265	0.256	0.1	1.0	R	R
69	E-4775	0.256	0.1	1.0	R	0.01
70	E-2622	0.512	0.1	1.0	R	R
71	E-4809	1.024	0.1	1.0	R	0.1
72	E-4811	0.256	0.1	0.001	R	0.1
73	E-2443	0.128	0.1	0.001	R	R
74	E-2950	0.256	0.1	0.001	R	0.1
75	E-4854	0.128	0.1	0.01	R	0.01
76	E-2920	0.128	0.1	0.01	R	0.01
77	E-2597	0.256	0.1	0.01	R	R
78	E-2387	0.256	0.1	0.01	R	0.1
79	E-5235	0.256	0.1	1.0	R	R
80	E-2693	0.256	0.1	0.01	R	R
81	E-5158	0.128	0.1	1.0	R	0.1
82	E-5254	0.256	0.1	0.01	R	R
83	E-4227	0.256	0.1	0.01	R	R
84	E-5269	0.256	0.1	0.01	R	0.01
85	E-958	0.064	0.1	0.01	3.0	0.01
86	E-3135	0.256	0.1	0.01	3.0	0.1
87	E-4757	0.512	0.1	0.01	R	0.1
88	E-462	0.128	0.1	0.01	R	R
89	E-1115	0.256	0.1	0.01	R	R
90	E-556	0.256	0.1	1.0	R	R
91	E-381	0.064	2.0	1.0	R	R
92	E-711	0.256	0.1	1.0	R	R
93	E-3126	0.064	0.1	1.0	R	0.1
94	157B	0.256	0.1	1.0	R	0.01
95	E-2595	0.128	0.1	1.0	5.0	0.01
96	E-759	0.256	0.1	1.0	R	0.1
97	E-970	0.512	0.1	1.0	3.0	0.1
98	E-4629	0.256	0.1	1.0	R	0.1
99	E-774	0.512	0.1	1.0	R	1.0
100	E-3140	R	0.5	1.0	R	R
Total resistant		5	0	0	93	57

 

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 results are presented in table 2, MICs of ampicillin sensitive strains ranged between 0.016 μg/ml to 1.024 μg/ml. MIC level of 0.256 μg/ml was recorded in 40 isolates, 0.512 μg/ml in 34 isolates, 0.128 μg/ml in 9 isolates, 0.064 μg/ml in 7 isolates, 1.024 μg/ml in 3 isolates, 0.032 and 0.016 μg/ml in 1 isolate each.

 

MICs of chloramphenicol sensitive strains ranged between 0.01 μg/ml to 2 μg/ml. MIC level of 0.1 μg/ml was recorded in 86 isolates, 0.01 μg/ml in 9, 0.5 μg/ml in 3, 1 and 2 μg/ml in 1 isolate each.

 

MICs of streptomycin sensitive strains ranged between 0.001 μg/ml to 3 μg/ml. MIC level of 1 μg/ml was recorded in 54 isolates, 0.01 μg/ml in 26, 0.001 μg/ml in 11, 0.1 μg/ml in 8 and 3 μg/ml in one isolate. MICs of sulfisoxazole sensitive strains ranged between 3.0 μg/ml to >240 μg/ml. MIC level of 3 μg/ml was recorded in 6 isolates, 5 μg/ml in 1, greater than 240 μg/ml in 1 isolate. MICs of tetracyclin sensitive strains was between 0.01μg/ml to 1.0 μg/ml. MIC level of 0.1 μg/ml was recorded in 24 isolates, 0.01 μg/ml in 18, 1 μg/ml in 1 isolate. The MIC results revealed that all the isolates were within the prescribed concentrations for sensitivity for the antibiotics tested viz., ampicillin (=8 μg/ml), chloramphenicol (=8 μg/ml), streptomycin (=8 μg/ml), sulfisoxazole (=256 μg/ml) and tetracyclin (=4 μg/ml). The observations were in agreement to the results of antibiotic sensitivity test performed in our experiment.

 

The determined minimum inhibitory concentration of the tested antibiotics (ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracyclin) in our study, were much below to the findings of De Jong et al. (2009) who observed MIC value of >128 μg/ml for ampicillin, chloramphenicol, streptomycin, tetracyclin and 1024 μg/ml for sulfisoxazole. It appears that Indian isolates have not yet acquired resistance to these antibiotics. Highest MIC observed in our study for ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracyclin were 1.024 μg/ml, 2 μg/ml, 3 μg/ml, >240 μg/ml and 1.0 μg/ml, respectively. The MIC results revealed that all the isolates were within the prescribed concentrations for sensitivity for the antibiotics tested viz., ampicillin (=8 μg/ml), chloramphenicol (=8 μg/ml), streptomycin (=8 μg/ml), sulfisoxazole (=256 μg/ml) and tetracyclin (=4 μg/ml). 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- MIC value of >240 μg/ml for sulfisoxazole. The reason for higher breakpoint for this isolate is not clear.

 

Overall, it appears that S. typhimurium isolates circulating in India have not yet acquired resistance against these antibiotics, which may be due to less use of these antibiotics in human and veterinary practice.

 

 

Acknowledgements

 

The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar (U.P) for providing all the necessary facilities to carry out the above research work.

 

 

Conflict of Interest

 

There is no conflict of interest.

 

 

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