Human infections caused by antibiotic-resistant bacteria such as E. coli have limited treatment options due to their recurrent transmission between animals, humans and environment. Shiga toxigenic E. coli (STEC), a virulent subtype of E. coli, is a food-borne pathogen that causes frequent outbreaks of bloody diarrhoea and haemolytic uraemic syndrome (CDC, 2023). STEC mediated illnesses are being reported globally from contaminated dairy products and other foods (Aslantaş et al., Reference Aslantaş, Yılmaz, Büyükaltay and Kocabağ2022). Antibiotic resistance in STEC has further complicated this issue as dissemination of these AMR food pathogens to the public poses a serious health risk (Sethulekshmi et al., Reference Sethulekshmi, Latha and Anu2018). Milk, a widely consumed dairy product can serve as a vehicle for the transmission of antibiotic resistant STEC to humans, but there are only a few studies that has reported the presence of multidrug resistant STEC in milk (Joseph et al., Reference Joseph, Sankarshanan and Kalyanikutty2022; Liu et al., Reference Liu, Meng, Dong, Zhang, Wang and Zheng2021). This study aimed to test whether or not the milk supplied by local Indian household retailers has high prevalence of ESBL resistant STEC strains.
Materials and methods
Isolation and identification of STEC isolates
Raw milk samples (n = 131) from household suppliers in Kerala were collected in sterile 100 ml screw-capped bottles and transported aseptically to the laboratory in an ice bucket. Isolation of STEC strains was carried out as per the protocol of Joseph et al. (Reference Joseph, Sankarshanan and Kalyanikutty2022). An enrichment using immune-magnetic bead (Dynabead®, Applied Biosystems, Norway), followed by selective isolation in cefixime tellurite sorbitol MacConkey medium (CT-SMAC: HiMedia, India) was carried out for the screening of E. coli O157. At the same time, E. coli non-O157 isolates were identified after enrichment in modified Escherichia coli broth containing 1.5 g/l bile salt and 20 mg/l novobiocin and further isolation in CT-SMAC medium (Joseph and Kalyanikutty, Reference Joseph and Kalyanikutty2021). Subsequently, latex agglutination tests were performed using E. coli O157 monoclonal antisera (HiMedia, India) and E. coli OK O Antisera (Statens Serum Institute, Denmark) to confirm the serotype.
Antibiotic susceptibility testing
The antibiotic sensitivity of the screened STEC isolates was assessed using amoxyclav (10 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), cefalexin (10 μg) and cefotaxime (10 μg) antibiotic discs, as per CLSI (2021). The MIC values for ESBL antibiotics: amoxicillin, amoxyclav, cefepime, cefotaxime, piperacillin, imipenem and meropenem were also determined using CLSI protocols. E. coli ATCC25922 and K. pneumoniae ATCC700603 were used as the controls.
Genotypic analysis
We used PCR with EmeraldAmp GT PCR master mix to examine the presence of β-lactam genes bla TEM, bla SHV, bla VIM, and bla CTX-M in STEC isolates. The primer sequences are listed in online Supplementary Table S1.
Multilocus sequence typing (MLST)
The MLST typing revealed multiple allelic variations in the housekeeping genes of the representative STEC strain (M under Bioproject PRJA1002082). These variations were then used to construct a phylogenetic tree using iTOL. Besides, Tajima's D test was employed to evaluate the evolutionary significance of the MLST sequences.
Results and discussion
STEC was identified in 41.2% of fresh raw milk samples (54 of 131 samples). The sample quantity from each location varied based on the size and access granted by the local farms that were visited. The prevalence of E. coli O157 and E. coli non O157 in the samples was 55.56 (n = 30) and 44.4% (n = 24) respectively.
The antibiotic resistance profiling showed that 31.48% (17) of the total 54 confirmed STEC pathotypes were phenotypically resistant to at least one β-lactam antibiotic. While all of the 17 isolates were resistant to penicillin G and ampicillin (Table 1), 23.5% (4) of the isolates exhibited resistance to all of the antibiotics tested. This is consistent with the studies conducted by Joseph et al. (Reference Joseph, Sankarshanan and Kalyanikutty2022) and Sethulekshmi et al. (Reference Sethulekshmi, Latha and Anu2018). The molecular analysis revealed the presence of β-lactam genes bla TEM in 94.1% (16), bla SHV in 47.05% (8), bla VIM in 58.8% (10) and bla CTX−M in 11.8% (2) of the isolates. The downregulation of gene bla CTX−M suggests that its role in the spread of STEC strains may not be as strong as the bla TEM, which is expressed at higher levels.
Table 1. Antibiotic resistance profile of STEC isolates from raw milk, Kerala
An in-silico analysis of the MLST sequences revealed a unique ST type (NovelST). Nine pathogenic STEC sequence types were considered for comparative analysis (online Supplementary Table S2). The housekeeping genes showed allelic variation with the highest frequency being at adk6. adk6 (prevalent in 60% (6) of the isolates), while mdh11 was present only in 10% (2: Fig. 1). This allelic variation may be attributed to the presence of a new sequence type (Novel ST) of STEC in milk. The evolutionary significance analysis using Tajima's D test (Tajima, Reference Tajima1989) that measured genetic diversity and natural selection showed that NovelST exhibits a high-frequency polymorphism under balancing selection (online Supplementary Table S3). As such, it can be considered a potential zoonotic pathogen posing a public health risk. This information on the variation of pathogenic STEC strain can contribute to develop effective strategies to increase the safety and quality of milk.
Figure 1. The MLST profile of STEC isolate from raw milk. Novel ST represents the ST type of the strain isolated from milk in this study. iTOL visualization of the neighbourhood joining tree shows an outer layer of related MLST genes, while the inner layer represents its corresponding bootstrap values.
In conclusion, the detection of ESBL-resistant STEC strains in samples collected from household farms in Kerala serves as a stark reminder of the potential risks associated with consuming raw milk. It also underscores the need for proper milk handling and processing to prevent transmission of antibiotic-resistant bacteria to the human population.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029924000268
