Antimicrobial Resistance Pattern of Salmonella Species Recovered from Livestock and Poultry

Among the food-borne pathogens, cases associated with non-typhoidal salmonellosis (NTS) are predominant. Several environmental reservoirs of NTS exist and these organisms are present in the intestinal tract of domesticated and wild animals. The immediate risk of transmission to humans is through contaminated food and water. Animal origin foods like contaminated raw poultry meat are considered an important source of Salmonella spp. Most of the studies on Salmonella spp. have targeted poultry and poultry meat however, Salmonella spp. were also detected in other foods (Makwana et al., 2015; Prabhakar et al., 2020). In India, Salmonella enterica subspecies enterica has been detected in the feces of cattle and buffalo suffering from chronic diarrhoea (Hassan et al., 2020).
       
Salmonella species are responsible for a wide range of food- and water-borne diseases. Fecal shedding of Salmonella by humans and animals increases the risk of environmental contamination. It can enter the agricultural environment and contaminate the plants and surface water. The emergence of antimicrobial resistance (AMR) in foodborne pathogens including Salmonella species is a matter of concern. Resistance to extended-spectrum cephalosporins and fluoroquinolones has been reported in Salmonella enterica (Yan et al., 2005). Monitoring of AMR traits is necessary to understand the trends and magnitude of food borne pathogens like Salmonella spp. Present study was aimed to determine the prevalence of resistant Salmonella in the fecal samples of livestock and poultry and raw foods like milk, meat, fish and water.
 

Sampling and bacterial culturing
 
A total of 620 samples (Table 1) were collected from different locations from August to December 2020. The work was carried out in the department of VPH, KNP College of Veterinary Science, Shirwal, Maharashtra. Fecal samples of livestock and poultry were directly collected using rectal/cloaca swabs. Fecal droppings of wild pigeons were collected from their roosting places without touching or handling them. Water samples were collected from poultry farms and duck ponds. Raw food samples viz. chevon, mutton, milk and fish were collected from the local market. Isolation of Salmonella spp. was done as per the standard protocols. All the fecal swabs were pre-enriched with 10 ml buffered peptone water and incubated at 37°C for 18-24 h. Later, 1 ml of pre-enriched culture was inoculated with 9 ml tetrathionate broth and after incubation at 37°C for 24 h, a loopful of culture was streaked on Xylose Lysine Desoxycholate (XLD) and Brilliant Green agar (BGA). All the plates were incubated at 37°C for 24 h and presumptive colonies were further subcultured and processed for identification of Salmonella species through Gram’s staining, catalase, oxidase, IMViC test and H₂S production.


 
Extraction of DNA and PCR conditions
 
Genomic DNA from pure bacterial colonies was extracted by boiling and snap chilling method. All the PCR conditions were performed in 25 µl volume containing 12.5 µl 2 x PCR master mix, 1 µl each forward and reverse primers, DNA and nuclease free water to make final volume. Detection of invA gene for invasiveness was done by PCR as per the method described previously (Rahn et al., 1992). Primers used for amplification of invA gene (284 bp) were F- GTG AAA TTA TCG CCA CGT TCG GGC AA and R-TCA TCG CAC CGT CAA AGG AAC C. In this PCR 3 µl DNA and 7.5 µl nuclease-free water was used. PCR conditions were set as initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 52°C for 45 s and extension at 72°C for 60 s. The final extension was carried out at 72°C for 7 min. PCR products were run in 1.5% agarose gel stained with ethidium bromide.
       
PCR for the detection of quinolone-resistant genes viz., QnrA, QnrB and QnrS PCR was performed as per the method of Cattoir et al. (2007). Annealing temperatures were 60, 56, 57 degrees Celsius, respectively for QnrA, QnrB and QnrS genes. Other PCR conditions were initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing as above for 30 s and extension at 72°C for 1 min and a final extension of 72°C for 10 min. The expected amplicon size for QnrA, QnrB and QnrS was 550, 264 and 428 base pairs, respectively.
       
Attempts were also made to detect mobile colistin-resistant genes (mcr1-mcr5) by the multiplex PCR developed by Lescat et al. (2018). Reactions were set at denaturation at 94°C for 4 min; followed by 30 cycles of 94°C for 5 s, 59°C for 20 s and a single, final, elongation step at 72°C for 5 min. Gel electrophoresis was carried out in 2.5% agarose gel stained with ethidium bromide. Detection of major beta-lactam encoding genes viz., bla_SHV, bla_TEM, bla_CTX-M and bla_OXA was also performed by multiplex PCR as per the method described earlier (Fang et al., 2008). The cycling conditions were set as initial denaturation (95°C/15 min-1 cycle) followed by 30 cycles of denaturation (94°C /30 sec), annealing (62°C/90 sec) and extension (72°C /60 sec). The final extension was set at 72°C for 10 min and at 4°C. Five microliters amplified product was further separated by electrophoresis in 1.5% agarose gel dissolved in 0.5x TBE stained by ethidium bromide.
 
Antibiogram
 
Antimicrobial resistance and sensitivity patterns of Salmonella species were studied by Kirby Bauer disk diffusion method results were interpreted using criteria suggested by the Clinical and Laboratory Standards Institute (2017). A total of 15 antimicrobial agents were used (Table 2). ESBL production was evaluated using double-disk synergy test (DDST). The procedure involved two steps, initial screening followed ESBL confirmation. Cefotaxime (30 µg) and ceftazidime (30 µg) antimicrobial discs were used for initial screening followed by ESBL confirmation using cefotaxime/clavulanic acid (30/10 µg) and ceftazidime/clavulanic acid (30/10 µg) discs. Isolates showing the difference in a zone of inhibition of ≥5 mm of cephalosporin discs and cephalosporin plus clavulanic acid containing disc were considered as potential ESBL producers. Detection of carbapenemase (KPC), Metallo-beta-lactamase (MBL), ampC and colistin resistance was also assessed by epsilon test using EzyMIC strips (HiMedia) and results were interpreted accordingly.


 
 

Prevalence
 
During this investigation, 39 Salmonella spp. were recovered from 620 samples with a prevalence rate of 6.29%. Fecal prevalence of Salmonella species was observed more in the broiler (20%) and layers (16%) followed by pigs (8.75%). Similarly, Salmonella species were detected in raw chicken (6%) and fish (8%) samples. Present findings affirm that healthy food animals and poultry birds harbor Salmonella species in their intestine. Foods of animal origin especially chicken and chicken products have been studied extensively for the detection of Salmonella spp. Some researchers also highlighted its presence in other foods like mutton, chevon, milk and fish (Makwana et al., 2015; Singh et al., 2018; Prabhakar et al., 2020). An outbreak of Salmonellosis was also detected in the poultry farms in India (Rajagopal and Mini, 2013), indicating that food animals and poultry are the predominant reservoirs of NTS.
       
As observed in the present study, Salmonella spp. were detected in raw chicken meat (9%) and chevon (7%) samples collected from different districts of Chhattisgarh by Naik et al. (2015).  From a food safety point, all the foods intended for human consumption either in the raw or processed form must be negative for Salmonella species. Thus, its presence in some of the animal-origin raw foods warrants the application of good hygienic practices at retail meat shops and vendors selling fish.
 
Invasiveness
 
Salmonella species isolated during this investigation were virulent strains as they have exhibited the presence of invA gene (Fig 1) which encodes for a protein on the inner membrane that is necessary for the invasion of epithelial cells. The presence of invA gene in Salmonella spp. is indicative of its virulence and generally virulent strains express the same along with other genes like fimA, spvR and spvC gene. The presence of invA has been reported by several researchers across the globe in Salmonella spp. isolated from foods and present findings are in agreement with previous studies (Mthembu et al., 2019).


 
Antibiogram
 
Salmonella species under study were mostly sensitive to monobactam and cephalosporin groups of antimicrobials. They have shown a high degree of sensitivity to aztreonam, cefepime, cefoxitin, ceftazidime, cefpodoxime and piperacillin-tazobactam. Over 71.79% of isolates were also sensitive to ciprofloxacin which belongs to the fluoroquinolone group. However, isolates showed resistance to ampicillin (53.84%), amoxyclav (33.33%), colistin (66.66%), tetracycline (58.57%) and sulfafurazole (23.07%). Mostly pig and poultry origin isolates shown resistance to more than two groups of antimicrobials indicating their multidrug resistance status. A total of 11 (28.20%) isolates were multidrug-resistant and MAR index varies from 0.2 to 0.6 which is suggestive of high-risk source. A study from Ethiopia also recorded a very high prevalence (86%) of multidrug-resistant Salmonella species isolated from caeca of slaughtered broiler birds (Afsaw Ali  et al., 2020). In India, some of the Salmonella strains resistant to amoxicillin and cephalosporins were isolated from ready-to-eat street foods (Anukampa et al., 2017). Another study from India also recorded moderate resistance in Salmonella Newport isolated from poultry and animal origin foods. However, a high degree of sensitivity was observed against ciprofloxacin, chloramphenicol and cefuroxime (Kumar et al., 2016). Isolates of our study were mostly sensitive to cephalosporins but they have shown resistance to colistin, tetracycline and certain penicillin group of antimicrobials. About 17.94% (7/39) isolates of this study were ESBL producing strains (Table 3). ESBL producing and colistin-resistant Salmonella spp. was detected in pigs and pork in Thailand recently (Lay et al., 2021). We have also screened all the Salmonella isolates for carbapenem, MBL and ampC production by epsilon test but all the isolates were negative.  


 
Colistin resistance
 
We could not detect mobile colistin-resistant mcr genes in Salmonella spp. Detection of mcr genes is rare in the foodborne Salmonella spp. and there are no reports from India. Globally, researchers have detected mcr genes in Salmonella enterica (Sia et al., 2020). Polymyxins, including polymyxin B and E (colistins), is regarded as the last-resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative organisms, especially carbapenem-resistant Enterobacteriaceae. We have phenotypically detected colistin resistance using the epsilon test (Fig 2) and disk diffusion method. Phenotypically, about 66.66% Salmonella spp. were resistant to colistin.


 
Quinolone resistance
 
Out of 39 isolates, 3 (7.69%) showed the presence of QnrS gene and all were resistant to ciprofloxacin which is a second-generation quinolone (Fig 3). They were isolated from poultry. Fluoroquinolones are the most widely used antibiotics for treating salmonellosis in both humans and animals because of their broad spectrum in antimicrobial activity. The Qnr genes provide a low resistance level to quinolones in the Enterobacteriaceae. The rate of detection of Qnr genes is relatively low among Salmonella species, however, risk of spread of plasmid-mediated Qnr genes through foods is speculated. Quinolone resistant genes were detected in Salmonella species isolated from raw meat products from China (Zhou et al., 2019).


 
Beta-lactam genes
 
All the ESBL positive strains were studied by multiplex PCR for the presence of genes encoding for beta-lactamases. One isolate each from cattle and broiler feces was positive for bla_TEM gene (Fig 4). In contrast to our findings, high prevalence of bla_TEM and bla_CTXM was recorded in the Salmonella spp. isolated from retail meat samples by Zhou et al. (2019). The worldwide incidence of resistance in Salmonella spp. isolated from the poultry and other foods of animal origin are varying, including ESBL productivity and rate of detection of AMR genes.  However, in the context of AMR and One Health, epidemiological investigations need to be conducted to examine the local situation of prevalence and antimicrobial resistance in priority food-borne pathogens like Salmonella species.

Present study highlighted the importance of detecting Salmonella species in fecal samples and raw foods of animal origin obtained from the retail market. Detection of virulent, ESBL positive and multidrug-resistant strains in food samples and feces of livestock and poultry poses public health, food safety and environmental risk and therefore AMR traits and spread of animal origin NTS should be critically monitored.
 

The authors are thankful to the Hon’ble Vice-Chancellor, Maharashtra Animal and Fishery Sciences University, Nagpur for providing the MAFSU Research Grant (MAFSU/365/19) for the completion of this work.