Validation of a random Vibrio parahaemolyticus genomic library by selection of quinolone resistance in a heterologous host

• Published in: Microbiology (Society for General Microbiology) Vol. 168; no. 5
• Main Authors: O'Boyle, Nicky, Boyd, Aoife
• Format: Journal Article
• Language: English
• Published: England 01.05.2022
• Subjects: Vibrio parahaemolyticus; qnr; genomic library; norfloxacin; resistance
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.001189

is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared genomic DNA and introduced this into HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by but not the heterologous host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with HB101 carrying an empty vector. The common locus contained within resistant clones included , a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.