Background: The escalating incidence of antimicrobial resistance in Gram-negative bacteria has elicited worldwide apprehension owing to its profound ramifications, such as the emergence of infections that cannot be effectively treated and may result in mortality. The poultry industry is of significant concern due to its substantial utilization of antimicrobials, including those regarded as last-resort therapies for complex multi-drug resistant Gram-negative infections. Methods: A comprehensive collection of ninety cloacal swabs was undertaken from a diverse range of thirty poultry farms located within the Nablus governorate during the period spanning from March to June in the year 2019. The swabs, specifically collected from broilers, were cultured on appropriate culture media to isolate Gram-negative bacteria under optimal conditions. The separated bacteria were subjected to various microbiological techniques, such as Gramme staining, oxidase testing, and API20E, to aid in their identification. The susceptibility of the isolates for 16 antibiotics was evaluated using the disc diffusion method, while colistin was assessed using the micro-broth dilution method. Additionally, molecular characterization was conducted using polymerase chain reaction (PCR) to identify the existence of MCR genes. Result: A total of 244 isolates of Gram-negative bacteria were collected and subjected to characterization, identification, and categorization into two groups: the Enterobacteriaceae group consisting of 170 isolates (69.7%) and the remaining 74 isolates (30.3%) categorized as others. The initial cohort consisted of 103 Escherichia coli (42.2%), 24 Proteus mirabilis (9.8%), and 19 Salmonella spp. (7.8%). The second group consisted of 62 Serratia spp. (25.4%), 1 Aeromonas hydrophila (0.4%), and 11 other unidentified species (4.51%). The study observed varying levels of antibiotic resistance among different antibiotics. The highest resistance rates were found for trimethoprim (100%), tetracycline (94.3%), trimethoprim-Sulfamethoxazole (90.6%), ciprofloxacin (86.1%), chloramphenicol (80.7%), ampicillin (75.4%), gentamicin (50.4%), fosfomycin (17.2%), cefuroxime (10.2%), cefotaxime (10.2%), ceftriaxone (7%), ceftazidime (2%), amikacin (1.2%), and meropenem (0.4%). Furthermore, the percentages of multidrug-resistant (MDR), extensively drug-resistant (XDR), and Extended-spectrum beta-lactamase (ESBL) isolates were 83.8%, 4.8%, and 18.6%, respectively, with corresponding proportions of 140/167, 8/167, and 31/167. Notably, a significant proportion of the total isolated bacteria, specifically 65.4%, exhibited resistance to colistin as determined through the micro-broth dilution method. In our study, 167 Enterobacteriaceae isolates were examined, of which 111 (66.5%) were resistant to colistin. Among these colistin-resistant isolates, 35 (31.5%) were found to possess variants of the MCR gene. The gene variant MCR-7 was the most prevalent among the isolates, with 12 occurrences. Subsequently, the presence of MCR-1 and MCR-8 was detected in 10 and 8 isolates, respectively. Conclusion: This study represents the inaugural report on colistin resistance in Gram-negative bacteria obtained from poultry farms in the West Bank region of Palestine. The results of our study serve as a catalyst for raising awareness about the emergence of colistin resistance in animal farms, particularly in poultry, and the significant implications this has for human health.

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