Detection of ESBL and ACBL-producing Salmonella enterica in Cattle from Different Districts of South Bengal

Livestock is a source of subsidiary income for many families in India especially the resource-poor who maintain few heads of animals. Cows and buffaloes if in milk will provide regular income to the livestock farmers through the sale of milk (BAHS, 2014).  Thus, the animal husbandry sector plays an important role in a country’s national economy and socio-economic development. Out of the 535.78 million livestock populations in India, cattle represent over 35.97% (192.49 million) of the animals, the 2nd highest in the World. In West Bengal, out of a total 37.4 million livestock population, cattle represent over 50.80 per cent (19 million) of the animals [Annual Report 2018-19, DAHD].
       
Salmonellosis in cattle is one of the main zoonotic diseases worldwide. Salmonella is a genus of gram-negative, facultative anaerobic bacteria that belong to the family of Enterobacteriaceae. Salmonella enterica is an important public health pathogen causing foodborne zoonoses. Salmonella infections cause gastrointestinal symptoms predominantly with generalist serotypes and are often characterized by high morbidity but low mortality (Hoelzer et al., 2011). Salmonella spp. can produce ESBLs and can show resistance to extended-spectrum cephalosporins due to the action of the plasmid AmpC gene (Rodriguez  et al., 2009).
       
Salmonella organisms carried zoonotic importance. It is the most common food-borne pathogenic bacteria in humans as well as in animals (Cummings et al., 2010). Foodborne infections are the most common form (Scallan et al., 2011; Cummings et al., 2012) of Salmonellosis caused by undercooked foods and unpasteurized dairy products. Thus, the drug-resistant Salmonella spp. can be a matter of great concern in starting a foodborne zoonosis.        

In this context, the present study was aimed at the prevalence study of salmonellosis in cattle, identification of the pathogen and detailed characterization, detection of antibiotic resistance (ESBL) properties in the positive strains and in-vitro drug resistance study of ESBL-producing Salmonella spp isolates.

A total of 170 faecal samples (135 non-descript and 35 crossbreed Jersey cows) were collected from Indigenous or Deshi and Crossbred Jersey cattle during the period January to July 2022. All these animals were reared on free-range systems with no history of disease outbreaks. All the samples were collected on a random basis from these apparently healthy cattle from Purba Barddhaman, Birbhum and North 24 Parganas districts of West Bengal (Table 1). The faecal swab samples were aseptically collected directly from animals and were kept in sterile containers with peptone water (HiMedia, India) under ice cover for transport.


       
All the swabs were enriched into Selenite F broth (HiMedia, India) at 37^oC for 24 hours and were streaked into Xylose Lysine Deoxycholate (XLD) Agar (HiMedia, India) and Salmonella Shigella agar (SSA, HiMedia, India) separately and incubated at 37^oC, for approx. 16-18hrs. The tentatively positive colonies were preserved on separate nutrient agar (HiMedia, India) slants for subsequent biochemical characterization (Quinn et al., 1994). The tentatively positive isolates were checked morphologically by Gram’s method of staining and biochemically by different tests as per methods described by Quinn et al., (1994) with some modifications.
       
Molecular confirmation of the Salmonella spp. isolates was done by PCR. Bacterial DNAs were extracted as per standard methods (Wani et al., 2004). PCR detection of the 16S rRNA gene as described by Pradhap et al., (2011) was followed for confirmation of the isolates. The primer sequences and predicted length of the PCR products are listed in Table 2.


       
Phenotypic detection of ESBL and ACBL production in Salmonella enterica isolates was done by disc diffusion method (Bauer et al., 1966) following standard protocols (CLSI, 2021; Tan et al., 2009).
       
Detection of the major beta-lactamases-producing genes in all Salmonella enterica strains were attempted by PCR as per Bert et al., (2002); Colom et al., (2003); Weill et al., (2004) and Feria et al., (2002). All the PCR primer details and PCR conditions are listed in Table 2. The PCR products were visualized by gel doc system (UVP, UK) after electrophoresis in 1.5% (w/v) agarose (SRL, India) gel containing ethidium bromide (0.5 μg/ml) (SRL, India).
       
In-vitro antibiotic sensitivity test of ESBL-producing Salmonella enterica isolates against 12 different antibiotics (Table 3) by the disc diffusion method as per Bauer et al., (1966) and CLSI (2021).

A total of 170 faecal samples screened from cattle from different agroclimatic zones [Table 1] (119 samples from the Gangetic alluvial zone and the rest 51 samples from the Red and Undulating Laterite zone), yielded 35 Salmonella spp. isolates with typical characters (Quinn et al., 1994). Scientists like Singh et al., (2018), Manishimwe et al., (2021), Ranjbar et al., (2020) and López-Martín  et al. (2016) also reported similar positivity of salmonellosis from bovine faecal samples in their studies.
       
All the Salmonella spp. isolates showed standard results in all the biochemical tests such as catalase (+ve), oxidase (-ve), indole (-ve), methyl red (+ve), Voges-Proskauer (-ve), citrate utilization tests (+ve) and urease (-ve) [Quinn et al., 1994; Samanta et al., 2014]. Similarly, Ranjbar et al., (2020) and Adel et al., (2021) showed similar typical results.
       
Molecular conformation of the Salmonella spp. isolates showed the presence of the 16S rRNA gene (1428 bp) [Fig 1]  specific for this genus in all 35 samples, thus confirmed as Salmonella enterica. Ranjbar et al., (2020) and Doosti et al., (2017) also confirmed Salmonella spp. by detection of the 16S rRNA gene in their studies.


       
The prevalence of Salmonellosis in bovine faecal samples was quite significantly high (20.6%) which almost, matches the 23.2% positivity of Salmonella spp. from beef calve samples by Obaidat (2020) and 18.1% positivity of Salmonella spp., was reported by El-Seedy et al., (2016) from the diarrheal calf samples. A prevalence of 3-9.6% Salmonella spp. from diarrhoeal cattle was observed by Murugkar et al., (2005); Sudhanthirakodi (2016). and Sharma and Joshi (2020) from different states of India.
       
The prevalence of Salmonella enterica strains among the faecal samples showed a higher positivity in Cross Breed jersey cows (88.57%), younger cattle (up to 2 years of age) [24.53%], male cattle (42.86%) and from the Gangetic Alluvial zone samples (25.21%). These were also reported by Nuzhat et al., (2020), Olaogun et al., (2016), Khan et al., (2021), Ibrahim et al., (2023) and Banerjee et al., (2019).
       
Eighteen (51.42%) and 29 (82.86%) Salmonella spp. isolates were found to be positive phenotypic ESBL and ACBL production respectively (Drieux et al., 2008). Adel et al., (2021) showed 42% and Salvia et al., (2022) revealed 72% ESBL and ACBL positivity phenotypically in earlier studies which almost match with these results. 
       
Molecular detection of ESBL genes revealed that 26(74.29%) Salmonella spp. isolates were positive for at least one of the blaCTX-M (Fig 2) and blaSHV (Fig 3) genes. No blaTEM gene was detected in this study. Again 32 (91.43%) isolates showed the presence of the blaAmpC gene (Fig 4) which was the highest among all genes (Worku et al., 2022) (Table 1). Only 8 isolates showed the presence of both genes. Similarly, Adel et al., (2021) observed the prevalence of the blaCTX-M (32.4%) and the blaSHV-12 (14.7%) genes in Salmonella enterica isolates which almost matches the current findings. Ahmed et al., (2014), and Banerjee et al., (2019) also reported 52-70% ACBL positivity in Salmonella spp. isolates in their studies.






       
In vitro antibiogram of the ESBL-producing Salmonella spp. isolates (n=26) showed high-level resistance to most of the antibiotics like cefotaxime, enrofloxacin, ampicillin/cloxacillin, doxycycline etc. (Akinyemi et al., 2017, Kolhe et al., 2022) whereas few antibiotics like azithromycin, chloramphenicol, HL gentamicin, etc. were comparatively sensitive to them (Table 3). Scientists like Khan et al., (2021), Adzitey et al., (2020) and Ahmed et al., (2014) reported similar findings in their studies and thus confirm the present report. The antibiotic resistance pattern observed in this report partially matches the antibiotic usage patterns on those animals as per the data collected from those areas. The antimicrobial resistance is quite an alarming phenomenon now worldwide due to the human-animal interaction and transferrable nature of antimicrobial resistance.

The authors thank the University Authorities and the Faculties of the Department of VMEJ, VCC and VMC, F/VAS, West Bengal University of Animal and Fishery Sciences, West Bengal, India for providing the necessary funds, research facilities and support for this study. They are also thankful to the farm owners, dairy farmers and Field Vets of the districts under study in West Bengal, India for their technical and field support.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors. The authors are responsible for the accuracy and completeness of the information provided but do not accept any liability for any direct or indirect losses resulting from using this content.
 
Informed consent
 
This study does not involve an animal experiment, so the permission of the University Animal Ethics Committee is also not required.

The authors declare that there are no conflicts of interest among themselves regarding the publication of this article.