To assess the prevalence of AMR enterococci in healthy finisher livestock and poultry, and their products in Bangladesh, we screened 230 samples from livestock and poultry at farms and 122 meat samples at retail markets. Our findings indicate that Enterococcus spp. are relatively common in poultry and livestock animals in Bangladesh, though the overall prevalence of E. faecium was lower than E. faecalis. Nonetheless, poultry has been demonstrated as the leading source of Enterococcus spp. in several studies conducted elsewhere (Maasjost et al. 2015; Stȩpień-Pyśniak et al. 2016). The prevalence of E. faecalis was significantly higher in poultry farms than at other livestock farms, but retail livestock meat yielded a higher prevalence of E. faecalis than poultry meat. This is likely to be indicative of post-slaughter contamination as well as the absence of, or lack of enforcement of, food safety regulations during the slaughtering process and meat product handling in Bangladesh.
Poultry and livestock are essential to food security in Bangladesh, the potential dissemination of resistant Enterococcus spp. in the food production continuum and strengthening of food safety regulations needs to be addressed. In the context of One Health and to further inform food safety interventions in Bangladesh, it is also important to identify the major reservoirs and their dissemination downstream of the production continuum, which was the intent of our study. Our data indicates that more than half of the samples were contaminated with Enterococcus spp. and the most predominant species was E. faecalis, the third most commonly identified pathogen in hospitals associated with increased mortality (Coque 2008). These findings are consistent with several preceding studies (Ngbede et al. 2017; Poeta et al. 2006; Yoshimura et al. 2000). There is no extensive data available on AMR amongst enterococci in livestock and poultry in Bangladesh because a national AMR surveillance program has not yet been established. However, one study showed high prevalence of enterococci among chickens (Banik et al. 2018) consistent with our study. Two relevant studies in involving human cases have implicated enterococci (Akram Hossain 2016; Suchi et al. 2018).
High prevalence of VNSE (44%; n=51) has been observed in this study which is higher than some other studies (Cosentino et al. 2010; Maasjost et al. 2015; Stȩpień-Pyśniak et al. 2016). Nonetheless, most alarming finding was the co-occurrence of linezolid resistance and VNSE, where most linezolid (90%) resistant isolates were VNSE, which could drive a therapeutic crisis for treating infection caused by VNSE (Bialvaei et al. 2017). In addition, this study detected low susceptibility of isolates to antimicrobials that are concurrently being used for both human infection and animal production in Bangladesh. In our study, the frequency of resistance to tetracyclines and macrolides were relatively high whereas comparatively low resistance were observed to DNA synthesis inhibitors including nitrofurantoin (10%) and ciprofloxacin (31%), which are consistent with similar studies conducted elsewhere (Cosentino et al. 2010; Maasjost et al. 2015; Stȩpień-Pyśniak et al. 2016).
MDR prevalence was high in farm and in fresh meat sold at retail markets (i.e. products originating from the same geographical locations where they were raised) and most of the VNSE isolates were also MDR (92%, 47/51). Remarkably, isolates that were possible-XDR were mostly recovered from poultry farms, this also raises the potential public health concern of exposure to and consumption of products from this species. These findings may be reflective of the indiscriminate use of antimicrobials in poultry in Bangladesh, (Ahmed et al. 2019) notably, the high use of CIAs (Imam et al. 2020) and thus support the need for surveillance of AMR and monitoring of AMU to inform changes in usage policy and to better understand AMR and AMU relationships. The comparative pairwise antibiotic resistance matrix reveals that the combination between chloramphenicol or ciprofloxacin with nitrofuratoin, linezolid or vancomycin may produce better efficiency against multidrug resistant isolates as combined resistance level to those antibiotics is very low compared to the other antibiotics (Figure 2). These findings warrant further research.
The presence of vanA gene in our study, primarily responsible for vancomycin resistance (Torres et al. 2018) further highlight the widespread dissemination of these resistant strains in animal populations. However, there was a discrepancy in detection of vancomycin resistance gene in our study; it was detected in both resistance and intermediate phenotypes, which might be showing that resistance is not dependent on gene presence alone rather depends on the gene expression level. We also found the presence of vanC1 and vanC2/3 gene among E. faecalis which was previously thought to be species specific to E. gallinarum and E. casseliflavus, respectively (Clark et al. 1998). The vanC gene cluster can be located on plasmids (as well as the chromosome), and can be transferred to other enterococci such as E. faecium and E. faecalis, (Moura et al. 2013; Sun et al. 2014) however the location of the vanC gene in the isolates from this study was not determined and may warrant further study in the future. We observed that most genotypic vancomycin-resistant enterococci (VRE) isolates were phenotypically non-resistant to vancomycin indicating vancomycin-variable enterococci (VVE), as described previously (Downing et al. 2015; Thaker et al. 2015). This could be a result of the mutation in van gene cassette including vanSR or vanHAXY gene cluster or any other novel mutations.(Hong et al. 2008) This particular finding has impact in clinical settings, where the misidentification in enterococcal infections may result to challenges in the development of an efficacious treatment regimen. The preceding studies found the relevance of VVE with only vanA gene, but in this study, we also found the association of vanB, vanC1 and vanC2/3 with VVE which would require further investigation. We also found some vancomycin resistant isolates without having any major VRE gene which may be due to the other type of resistance mechanism and beyond the scope of our current study. However, this discrepancy in vancomycin resistance pattern may evolves as a new therapeutic challenge for clinical setting. An immediate improvisation in diagnostic technique for VVE and continuous surveillance for VVE is utmost necessary as some other studies suggested the same (Downing et al. 2015; Szakacs et al. 2014).
Moreover, these VRE gene have been associated with vancomycin resistance and can easily be transferred to another susceptible isolates since there is a probability to be clustered in a mobile genetic elements like plasmid or transposons (Torres et al. 2018) and investigation of this in the future would be informative. This transfer could occur within livestock farms and their environment or throughout the food production chain from farm to fork as evidenced by the detection of MDR E. faecalis and E. faecium from diverse food animal species such as poultry, cattle, and goat and meat products/meat-derived products (Hammerum et al. 2010; Hoelzer et al. 2017).
The high prevalence of virulence indicators like gelatinase, sex pheromones, and aggregation factors were found among the MDR isolates. The presence of these factors in MDR enterococci can contribute to the colonization or formation of bacterial biofilm-like vegetations among immuno-compromised patients through urinary tract or blood and subsequently turned into untreatable urinary tract infection (UTI) or endocarditis respectively (Sharifi et al. 2013).