Antibiotic resistance profiles in broiler production:
A total of 173 E. coli isolates were successfully recovered from the collected samples, with varying distributions across the different stages of the production pyramid. Specifically, 55 isolates were obtained from the 0th day, while 52 and 66 isolates were retrieved from the 20th and 40th days of the production pyramid, respectively. Among the tested antimicrobial agents, E. coli isolates displayed notable resistance to Cefixime (100%) and Amoxicillin (83.3%). In contrast, maximum sensitivity was observed for Gentamicin (83.3%), Chloramphenicol (83.3%), and Doxycycline (83.3%). Tetracycline and Co-trimoxazole showed mixed responses, with a 50% resistance rate and 50% sensitivity rate, respectively. These findings underscore the intricate spectrum of antimicrobial responses exhibited by E. coli isolates, highlighting the diverse challenges and opportunities for effective treatment strategies (Figure: 1A).
ESBL gene prevalence and potential transmission factor:
Among the collected E. coli isolates, a substantial portion, accounting for 44.5% (77/173) of the total, were found to carry the ESBL-producing gene. Specifically, 34.68% (60/173) of the isolates tested positive for the blaCTXM gene through PCR amplification, yielding a characteristic amplified product size of 540 bp. Additionally, 23.1% (40/173) of the isolates exhibited positivity for the blaSHV gene, as evidenced by characteristic amplified products measuring 792 bp. However, another finding was the co-occurrence of both CTX-M and SHV genes in 13.29% (23/173) of the isolates. While assessing the prevalence of ESBL-producing E. coli at different time points within the broiler production cycle, our analysis unveiled intriguing trends. In the initial day-old sampling, which included cloacal, feed, and water samples, we observed a prevalence of ESBL-producing E. coli at 50.9% (28/55). However, on the 20th day of production, there was a notable decline in prevalence, accounting for 26.9% (14/52) of the isolates. A significant shift occurred on the 40th day of production when the prevalence surged to 53.03% (35/66) of the isolates (see Figure: 1B). Our investigation has unveiled the dynamic nature of ESBL-producing E. coli prevalence within the broiler production pyramid. Notably, a discernible decline in prevalence occurred from the day-old sampling to the 20th day, followed by a marked resurgence from the 20th day to the 40th day of the production cycle. This initial rise in prevalence raises the possibility of vertical transmission of microbiota from parent to offspring [12,13]. However, contemporary research underscores the intricate web of transmission dynamics in broiler production, suggesting that alternative modes such as fomite transmission or horizontal gene transfer mediated by other bacterial species might exert a more significant influence on the dissemination of resistance traits [18]. One plausible explanation for the observed fluctuations in ESBL-E. coli prevalence in our study is the potential role of pond water as a source of drinking water for the birds within the context of a small-scale farm setting. While our study did not directly confirm this hypothesis, it is worth noting that pond water often carries a heightened risk of contamination with human faecal matter due to its multifunctional use, including bathing and personal hygiene practices, particularly after defecation, among local villagers [19]. Also, in poultry farming practices, drinkers are typically suspended from the roof, their height adjusted to match the average tail height of the flock, ensuring that birds do not need to stoop or bend excessively to access water. However, it is noteworthy that all our observations (Supplementary Figure: 1) indicate that the drinkers in this study were placed on the floor rather than being suspended, potentially increasing the likelihood of water contamination. Furthermore, irregular water change practices in the drinkers on the farm may have contributed to the accumulation of ESBL-E. coli, exacerbated by contamination from bird droppings. This situation underscores the need for further investigations and interventions to elucidate and mitigate the role of pond water and suboptimal drinking water practices in the dissemination of antibiotic resistance in broiler production systems.
Variants of ESBL and their dynamics:
Upon investigating the occurrence of ESBL on a variant level we have encountered comparatively similar trends. At the initiation of the production pyramid, specifically on the day-old chick stage, the prevalence of blaCTX-M was observed at 32.14% (9/28) in cloacal swabs, with corresponding prevalence rates of 6.6% (1/15) and 25% (3/12) in water and feed samples, respectively. As the production cycle progressed to the 20th day, prevalence rates of blaCTX-M shifted to 30.7% (8/26), 21.05% (4/19), and 28.5% (2/7) in cloacal swabs, water, and feed samples of poultry, respectively. Intriguingly, by the 40th day of the production pyramid, a noticeable decline was observed in prevalence, reaching 17.14% (6/35) in cloacal swabs, while prevalence surged significantly to 89.2% (25/28) in water samples and 66.6% (2/3) in feed samples (Figure: 1C). Shifting focus to the prevalence of blaSHV, similar trends unfolded. At the outset of the production pyramid, the prevalence of blaSHV was 28.5% (8/28) in cloacal swabs and 66.6% (10/15) in water samples, while feed samples displayed an absence of the SHV gene. As the production cycle progressed to the 20th day, the prevalence of blaSHV witnessed a decline, standing at 3.84% (1/26) in cloacal swabs, with an absence observed in both water and feed samples. On the 40th day, a further decrease was noted, with a prevalence rate of 2.8% (1/35) in cloacal swabs, contrasting with a heightened prevalence of 67.8% (19/28) in water samples and 33.3% (1/3) in feed samples (Figure: 1D).
These findings underscore the dynamic nature of ESBL gene prevalence throughout the broiler production pyramid, emphasizing the potential influence of fomites/management practices and environmental factors that contribute to these fluctuations. This warrants meticulous consideration in the context of antimicrobial resistance control strategies to ensure food safety. Moreover, these observed variations shed light on potential shifts in transmission routes and factors influencing gene dissemination within the intricate web of poultry production. To delve in to a deeper understanding of ESBL gene dynamics, a subset of 9 bacterial isolates was selected for nucleotide sequencing. Within this subset, 6 isolates harbored the blaCTX-M gene, while 3 showcased the presence of the blaSHV gene. Among the 6 isolates bearing blaCTX-M, 4 emanated from water samples, 1 emerged from feed samples, and 1 was gleaned from cloacal swabs. The entirety of the blaSHV gene-bearing isolates hailed exclusively from water sources, revealing a unique habitat preference for gene dissemination. After the sequencing endeavour, the generated nucleotide sequences were deposited into the esteemed DNA Data Bank of Japan (DDBJ) https://ddbj.nig.ac.jp/submission , solidifying their accessibility for future research endeavours. The results of the nucleotide sequencing endeavour for blaCTX-M gene unveiled a repertoire of genetic signatures, unveiling sequences such as CTX-M-15-w81(1) (Accession number- LC738775), CTX-M-15-w81 (Accession number- LC738776), CTX-M-15-w-44 (Accession number- LC738777), CTX-M-15-w-44 (Accession number- LC735992). The nucleotide sequencing of the blaSHV gene provided insight into sequences encompassing SHV-1 (Accession number- LC738898), SHV-27 (Accession number- LC738864), and SHV-45 (Accession number- LC738863). To emphasize the potential for ESBL transmission through the food chain, we constructed a phylogenetic tree (Figure: 2A) incorporating these ESBL gene variants isolated from our study, clinical samples [20], and other poultry bird samples [1,5]. The phylogenetic analysis reveals a partial clonal relationship among the ESBL sequences, suggesting a shared genetic ancestry. This finding reinforces the notion that ESBL variants identified in poultry production systems may have parallels in clinical settings, further supporting the potential bidirectional transmission between birds and humans.