| Summary: | A cross-sectional study was used to identify and assess prevalence and phenotypic antimicrobial resistance (AMR) profiles of <i>Escherichia coli</i> and other enterobacteria isolated from healthy wildlife and livestock cohabiting at a 10,000 acres game ranch near Lusaka, Zambia. Purposive sampling was used to select wildlife and livestock based on similarities in behavior, grazing habits and close interactions with humans. Isolates (<i>n</i> = 66) from fecal samples collected between April and August 2018 (<i>n</i> = 84) were examined following modified protocols for bacteria isolation, biochemical identification, molecular detection, phylogenetic analysis, and antimicrobial susceptibility testing by disc diffusion method. Data were analyzed using R software, Genetyx ver.12 and Mega 6. Using Applied Profile Index 20E kit for biochemical identification, polymerase chain reaction assay and sequencing, sixty-six isolates were identified to species level, of which <i>Escherichia coli</i> (72.7%, 48/66), <i>E. fergusonii</i> (1.5%, 1/66), <i>Shigella sonnei</i> (22.7%, 14/66), <i>Sh. flexinerri</i> (1.5%, 1/66) and <i>Enterobacteriaceae bacterium</i> (1.5%, 1/66), and their relationships were illustrated in a phylogenetic tree. Phenotypic antimicrobial resistance or intermediate sensitivity expression to at least one antimicrobial agent was detected in 89.6% of the <i>E. coli</i>, and 73.3% of the <i>Shigella</i> isolates. The <i>E. coli</i> isolates exhibited the highest resistance rates to ampicillin (27%), ceftazidime (14.3%), cefotaxime (9.5%), and kanamycin (9.5%). Multidrug resistance (MDR) was detected in 18.8% of <i>E. coli</i> isolates while only 13.3% <i>Shigella</i> isolates showed MDR. The MDR was detected among isolates from impala and ostrich (wild animals in which no antimicrobial treatment was used), and in isolates from cattle, pigs, and goats (domesticated animals). This study indicates the possible transmission of drug-resistant microorganisms between animals cohabiting at the wildlife–livestock interface. It emphasizes the need for further investigation of the role of wildlife in the development and transmission of AMR, which is an issue of global concern.
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