Abstract
<title>Abstract</title> <p> Objective <italic>Vibrio cholerae</italic> remains a major cause of cholera outbreaks in Ethiopia, yet information on the distribution of virulence-associated genes among circulating strains is limited. This study aimed to detect major virulence genes in culture-confirmed <italic>V. cholerae</italic> isolates recovered from cholera outbreak sites in Ethiopia using multiplex polymerase chain reaction. Result description: A total of 125 culture-confirmed <italic>V. cholerae</italic> isolates recovered from fecal samples collected during cholera outbreaks in Ethiopia were analyzed. Following sub-culture and genomic DNA extraction, multiplex PCR assays were performed to detect nine virulence-associated genes ( <italic>ompW, tcpA, rfbO1, zot, toxR, rtxC, ace, hlyA</italic> , and <italic>ompU</italic> ). All nine target genes were identified among the isolates, although their frequencies varied. The most frequently detected gene was <italic>hlyA</italic> (84.0%, 105/125), followed by <italic>ompW</italic> (80.8%, 101/125), <italic>rfbO1</italic> (76.8%, 96/125), <italic>tcpA</italic> (75.2%, 94/125), <italic>zot</italic> (72.8%, 91/125), <italic>ompU</italic> and <italic>toxR</italic> (71.2%, 89/125 each), <italic>ace</italic> (69.6%, 87/125), and <italic>rtxC</italic> (68.8%, 86/125). Similar distributions of the virulence genes were observed among isolates obtained from outbreak sites in the Amhara, Oromia, and Addis Ababa regions. The detection of multiple virulence-associated genes highlights the pathogenic potential of outbreak-associated <italic>V. cholerae</italic> strains and provides baseline molecular data for future epidemiological and genomic investigations in Ethiopia. </p>