Emerging of Multiple Drug Resistance in Salmonella typhimurium Serovars Isolated from Indian Ganga River

Semmannan Kalaiyarasu^1*, Mumtesh Kumar Saxena², Radhe Shyam Gupta³

*Corresponding author:[email protected]

ARTICLE CITATION: Kalaiyarasu S, Saxena MK, and Gupta RS (2013). Emerging of multiple drug resistance in Salmonella typhimurium serovars isolated from Indian Ganga River. Adv. Anim. Vet. Sci. 1 (1S): 1 – 3
Received: 201 3–08–17, Revised: 2013–08–20, Accepted: 2013–08–21
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ABSTRACT

Salmonella is an important pathogen responsible for food borne illness and other enteric diseases in human and animals. Development of drug resistance in these microbes leads to failure of therapeutic attempts and posing the public health risk. Present study was undertaken to observe the drug resistanance profile of Salmonella typhimurium isolated from different locations of Indian Ganga river. A total of 44 Salmonella isolates including 24 S. typhimurium serovars were subjected to antibiogram test by disc diffusion method. The plasmid was isolated and compared with drug resistance pattern to describe its role in resistance mediation. All S. typhimurium serovars were resistant to ampicillin (100%) followed by tetracycline (91.6%), cephatoxime (75%) and nitrofurantoin (66.6%). A few isolates (6.8%) showed resistant to even ciprofloxacin. Gentamicin was the only antibiotic that showed 100% sensitivity to all isolates. Though these isolates showed resistance to most of the commonly used antibiotics, plasmid mediation of drug resistance was not observed. Our study indicates the presence of pathogenic Salmonella serovars in Indian water body and their increasing percentage of multiple drung resistance which is not solely mediated by plasmids. ll

Some of the newly –emerging resistant bacteria are transmitted to human population mainly via food and water. The best–known examples are the food borne pathogens like Salmonella and Campylobacter and commensal Enterococcus. Some Salmonella serovars are more pathogenic and common bacterial causes of food–borne diarrhoea complaint worldwide (Becker et al., 2013). Though the infection by these organisms can be treated easily using commonly available antibiotics, development of drug resistant strains become common phenomena now a day due to indiscriminate use of antibiotics at sub–therapeutic concentrations and as feed supplements and growth promoters in animal husbandry practices. These practices are often associated with the development of resistance to multiple numbers of antibiotics. Multiple drug–resistant (MDR) strains of Salmonella are now encountered frequently and the rate of multidrug–resistance development has increased considerably in recent years (Mijovic et al., 2012). Emerging of multiple drug resistancy among several Salmonella serovars including typhimurium, ^newport and other serovars noticed wordwide (Barraw et al., 1988; Zhao et al, 2003; Li et al., 2012; Hannan et al., 2013; Van Kessel et al., 2013 Even worse, some variants of Salmonella has developed multiple drug–resistances as an integral part of the genetic material of the organism, therefore likely to retain their drug–resistant genes even when antimicrobial drugs are no longer used. The emergence of MDR Salmonella strains with resistance to fluoroquinolones and third–generation cephalosporins (Bhattacharya et al., 2011) is a serious issue, which results in severe limitation of the possibilities for effective treatment of human and animal infections. Hence, an attempt has been made to understand drug sensitivity pattern of these Salmonella isolates and the role of plasmids in resistance mediation among the isolates of Indian Ganga river.

Salmonella serovars (isolated from Indian Ganga river water at different places and serotyped at National Salmonella Centre, Indian Veterinary Research Institute, Izatnagar, India) were obtained from Animal Biotechnology Centre, College of Veterinary and Animal Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India. Stock cultures were revived by streaking on MaConkey Lactose Agar (MLA) and Brilliant Green Agar (BGA) and observed the colony characteristics specific to Salmonella genus. Biochemical tests were performed to confirm the Salmonella using HiSalmonellaTM Identification kit (Himedia, Mumbai, India). In total, 44 isolates of Salmonella were used in the study which includes S. typhimurium (24) , ^{. abuja, S. lagos, S. pontypridd, S. oritamerin, S. chinkual, S. Goldenberg} and ^{S. zwickau} ( isolated from different locations of ganga river bank including Haridwar, Hastinaupur, Gurmukteshwar and Narora ). Out of 44 isolates characterized, 24 isolates of S. typhimurium tested for its antibiotic sensitivity due to its common occurance as food borne pathogen.

Antibiotic sensitivity test was performed according to Kirby–Bauer method (Bauer et al., 1966) Sterile Muller Hinton Agar plates were prepared and incubated for overnight at 37°C for testing its sterility. Two mL of overnight grown culture was spread over the surface of the agar plate and allowed to absorb as described. Antibiotic discs (Himedia, Mumbai, India) used were as follows; Ciprofloxacin (5µg), Ofloxacin (5 µg), Norfloxacin (10 µg), Nitrofurantoin (300 µg), Cotrimaxazole (25 µg), Cephotaxime (30 µg), Cephalexin (30 µg), Chloramphenicol (30 µg), Nalidixic acid (30 µg), Oxytetracycline (30 µg), Ampicillin (10 µg), Gentamicin (10 µg), Streptomycin (10 µg), and Tetracycline (30 µg).The antibiotic discs were placed properly at equal distance with the help of sterile forceps. Plates were incubated for 24 hours at 37°C and the zone of inhibition for each disc was measured in millimeter (mm) and compared with respective standard minimum inhibitory concentration (MIC) to interpret the results of test culture as resistant (R) or sensitive (S).

From the overnight grown culture, plasmid isolation was carried out as per the manufacturer protocol (QIAGEN, USA). Briefly, 50 mL of overnight grown culture was pelleted by centrifugation and 4 mL of buffer P1 was added and mixed. Then 4 mL of buffer P2 was added and mixed by inverting and kept at room temperature for 5 minutes followed by addition of 4 mL of P3.. Lysate was transferred to the QIAfilter (provided with kit) cartridge and kept at room temperature for 10 minutes. Meanwhile QIAGEN–tip 100 was equilibrated with 10 mL of buffer QBT and the content of catridge was plunged in to the tip. After complete emptying, plasmid DNA was eluted with 5 mL of buffer QF and precipitated by adding 3.5 mL of isopropanol. Then it was dried and resuspended in 50 µL of TE buffer (pHp 8.0). The size of the plasmids was determined by gel electrophoresis (1% agarose) run along the ruler plasmid (E.coli, MTCCV517)

Among these 24 isolates of S. typhimurium, G–20 and G–39 showed resistance to all except Chloramphenicol and Gentamicin, Norfloxacin and Gentamicin respectively. Isolate G–19 showed sensitivity to only 4 antibiotics (Nx, Cp, S, G). Remaining all the isolates showed resistance to more than two antibiotics. A total of 15 types of drug resistance pattern were observed in S. typhimurium (indicated in Table 1. Gentamicin was the only antibiotic found to be sensitive to all the isolates followed by Norfloxacin which was resistant to isolate G–20 only. S. typhimurium showed 100% resistance to ampicillin, 91.6% to tetracycline, 75% to cephotaxime, 70.8% to Oxytetracycline, 66.6% to nitrofurantoin and 33.3% to streptomycin.

Among 24 Salmonella typhimurium isolates, 17 isolates shown the presence of 30MDa size plasmid. Two isolates namely G–23 and G–36 had shown presence of four (16 MDa, 8.6 MDa, 4.2 MDa and 3.2 MDa) and two (16MDa and 3.9 MDa) small plasmids respectively apart from large plasmids. Remaining isolates did not show presence of any plasmids. Presence of large as well as small plasmids of isolates compared with respective drug resistance pattern. Presence of neither large nor small plasmid positively correlated with its drug resistant pattern.

Presence of typhoidal and non–typhoidal Salmonella with multiple drug resistance from the river water indicate the risk of people living on the river bank. Multiple drug resistance against more than 6–8 antibiotics (Cf, Of, Nx, Nf, Co, Ce, Cp, C, O, A, G, S and T) in S. typhimurium has been reported earlier (Saxena et al., 2004; Chiu et al., 2006 and Li et al., 2012) and it is mediated by large size plasmids (Barrow et al., 1988; Diaz et al., 2006; Chain et al., 2012). Present study shows 100 % resistance to ampicillin which is in agreement with previous studies in India (Tankhiwale et al., 2003). Though previous report says most of the Salmonella strains were sensitive to chloramphenicol and ciprofloxacin (Chande et al., 2002) here serious issue is that three isolates (6.8%) showed resistance to even ciprofloxacin. Gentamicin was the only antibiotic to which all the isolates (100%) were sensitive followed by norfloxacin (97.72%), chloramphenicol and ofloxacin (93.18%). Recent drug resistance study in India showed ciprofloxacin resistance in Salmonella isolates less than 2 percent and chloramphenical resistance upto 9% (Bhattacharya et al., 2011). The results of our study indicate the increasing of ciprofloxacin resistance in Salmonella isolates but more or less same to chloramphenicol. The acquisition of quinolone resistance is mainly due to chromosomal mutations, although a plasmid mediating quinolones resistance described (Kawakara et al., 1988). Resistance to fluoroquinolones is acquired in stepwise fashion with the introduction of chromosomal mutations that alter the target proteins either DNA gyrase as well as topoisomerase IV, or it may lessen intracellular drug accumulation by active drug efflux. Recent days, plasmid mediated low level of quinolone resistance observed in Klebsiella pneumonia (Li et al., 2012) and E.coli (mirmomeni et al., 2007). Interestingly in our observations, the isolates showing resistance to ciprofloxacin (G–20 and G–38) also having large sized plasmid but mediation of resistance by these plasmids has to be proved. Our study showed the maximum sensitivity to gentamicin (100 %) and maximum resistance to ampicillin followed by nitrofurantoin where as in earlier reports maximum resistance was observed against nitrofurazone while gentamicin was the most effective antibiotic and reported 83.33 % sensitivity to gentamicin and chloramphenicol (Zhao et al., 2003).

When comparing the plasmid profile with the multiple drug resistance no correlation could be made because resistance to most of the common antibiotics has been observed in both the isolates, i.e., harboring the plasmids and isolates without plasmids. For example 100 % resistance was observed to ampicillin in all isolates of S. typhimurium. Similar pattern was observed in case of tetracycline also as all the isolates without plasmid were not 100% sensitive to this drug and all with plasmid did not show 100% resistance. MDR in these isolates not solely depend on the presence of plasmids of neither large size nor small size but other factors like chromosomal mutations, physiological adaptations also need to be considered to characterize the MDR completely. Plasmid mediated ciprofloxacin resistance in two isolates has to be investigated further in more number of isolates before coming to a conclusion. Regarding therapeutic point of view, though the use of quinolones is a common practice and resistant to these drugs is keep on increasing and now the time to rethink about alternate antibiotics apart from highly sensitive gentamicin. It is necessary to take some precautions like restricted antibiotic use in the treatment of human beings as well as animal husbandry practices to control increasing frontline antibiotic resistance. From these observations, we can conclude that ciprofloxacin resistance in Salmonella is emerging and resistance to most commonly used antibiotics is increasing in Indian continent.

AKNOWLEDGEMENTS

Department of Science andTechnology, India is highly acknowledged for providing funds for study.

REFERENCES

Barraw PA and Lovell MA (1988). The association between a large molecular mass plasmid and virulence in a strain of Salmonella Pullorum. J Gen Microbiol. 134: 2307–2316.

Bauer AW, Kirby WMM, Sherris JC and Turk M (1966). Antibiotic susceptibility testing by a standard single disc method. Am J Clin Pathol. 45: 493–496.
PMid:5325707

Becker SL, Vogt J, Knopp S, Panning M, Warhurst DC, Polman k, Marti H, Miiller LV, Yansouni CP, Jacobs J, Bottien E, SAcko M, Rijal S, Meyanti F, Miles MA, Boelart M, Lutumba P, Lieshout LV, Goren EKN, Chappuis F and Utzinger (2013). Persistent digestive disorders in the tropics: causative infectious pathogens and reference diagnostic tests. Fems Infect Dis. 13:1-21.

Bhattacharya SS, Das U and Choudhury BK (2011). Occurrence and antibiogram of Salmonella Typhi and S.Paratyphi A isolated from Rourkela, Orissa. Indian J Med Res. 133:431–433.
PMid:21537098 PMCid:PMC3103178

Chain AK and Hall RM (2012). Evolution of a multiple antibiotic resistance region in IncHI1 plasmids: reshaping resistance regions in situ. J Antimicrob Chemother. 67: 2848–53.
http://dx.doi.org/10.1093/jac/dks317
PMid:22888274

Chande C, Shrikande S, Kapale S, Agrawal S and Fule RP (2002). Change in antimicrobial resistance pattern of Salmonella Typhi in central India. Ind J Med Res. 115: 248–250.
PMid:12440196

Chiu CH, Su LH, Chu CH, Wang MH, Yeh CM, Weill FX and Chu C (2006). Detection of multidrug–resistant Salmonella enterica serovar Typhimurium phage types DT102, DT104, and U302 by multiplex PCR. J Clin Microbiol. 44: 2354–2358.
http://dx.doi.org/10.1128/JCM.00171-06
PMid:16825349 PMCid:PMC1489530

Diaz MA, Cooper RK, Cloeckaert A and Siebeling RJ (2006). Plasmid mediated high-level gentamicin resistance among enteric bacteria isolated from pet turtles in Louisiana. Appl. Environ. Microbiol. 72: 306-312.
http://dx.doi.org/10.1128/AEM.72.1.306-312.2006
PMid:16391058 PMCid:PMC1352233

Hannan A, Asghar S, Naeem T, Ikram UM, Ahmed I, Aneela S and Hussain S (2013). Antibacterial effect of mango (Mangifera indica Linn.) leaf extract against antibiotic sensitive and multi-drug resistant Salmonella typhi. Pak J Pharm Sci. 26 : 715-9.
PMid:23811447

Kawakara K, Haraguchi Y, Tsuchimoto M, Terakado N and Danbara H (1988). Evidence of correlation between 50kb plasmid of Salmonella Cholera Suis and its virulence. Pakistan J Biol Sci. 4: 155-163.

Li L, Cheng CK, Cheung MK, Law PT, Ling JM, Kam KM, Cheung WM and Kwan HS (2012). Draft genome sequence of Salmonella enterica serovar Typhimurium ST1660/06, a multidrug-resistant clinical strain isolated from a diarrheic patient. J Bacteriol. 194:6319-20.
http://dx.doi.org/10.1128/JB.00363-12
http://dx.doi.org/10.1128/JB.01593-12
PMid:23105062 PMCid:PMC3486384

Mijovic G, Andric B, Terzic D, Lopicic M, Dupanovic, B (2012). Antibiotic susceptibility of Salmonella spp.: a comparison of two surveys with a 5 years interval. Jn of IMAB. 1: 216-219.

Mirmomeni MH, Colagar AH and Ghazaey S (2007). Molecular study of Salmonella Enteritidis in poultry sample by PCR, plasmid curing, antibiotic resistance and protein pattern analysis. Pakistan J Biol Sci. 10: 1562-1570.
http://dx.doi.org/10.3923/pjbs.2007.1562.1570
PMid:19086500

Saxena MK, Singh VP, Saxena A, Lakhchaura BD and Sharma B (2004). Detection of virulence in Indian isolates of Salmonella by Polymerase Chain Reaction. Ind J Biotechnol. 3: 37–40.

Tankhiwale SS, Agrawal G and Jalgoankar SV (2003). A preliminary report on current antibiogram of Salmonella enteric serotype Typhi in Nagpur. Indian J Med Microbiol. 21: 292.
PMid:17643050

Van Keesel JS, Sonnier J, Zhao S and Karns JS (2013). Antimicrobial resistance of Salmonella enterica isolates from bulk tank milk and milk filters in the United States. J food Prot. 76: 18–25.
http://dx.doi.org/10.4315/0362-028X.JFP-12-263
PMid:23317852

Zhao S, Qaiyumi S, Friedman S, Singh R, Foley SL and White DG, et al. (2003). Characterization of Salmonella enterica Serotype Newport Isolated from Humans and Food Animals. J Clin Microbiol.41: 5366–5371.
http://dx.doi.org/10.1128/JCM.41.12.5366-5371.2003
PMid:14662912 PMCid:PMC309039