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Abstract

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.

Funding
This study was supported by the:
  • Science Foundation Ireland (Award 08/RFP/GEN1225)
    • Principle Award Recipient: AoifeBoyd
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2022-05-10
2024-03-29
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References

  1. Nair GB, Ramamurthy T, Bhattacharya SK, Dutta B, Takeda Y et al. Global dissemination of Vibrio parahaemolyticus serotype O3:K6 and its serovariants. Clin Microbiol Rev 2007; 20:39–48 [View Article] [PubMed]
    [Google Scholar]
  2. O’Boyle N, Boyd A. Manipulation of intestinal epithelial cell function by the cell contact-dependent type III secretion systems of Vibrio parahaemolyticus. Front Cell Infect Microbiol 2014; 3:114 [View Article] [PubMed]
    [Google Scholar]
  3. Joseph SW, Colwell RR, Kaper JB. Vibrio parahaemolyticus and related halophilic Vibrios. Crit Rev Microbiol 1982; 10:77–124 [View Article] [PubMed]
    [Google Scholar]
  4. Chowdhury MAR, Yamanaka H, Miyoshi S, Shinoda S. Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region. FEMS Microbiol Lett 1990; 74:1–9 [View Article]
    [Google Scholar]
  5. Daniels NA, Shafaie A. A review of pathogenic Vibrio infections for clinicians. Infect Med 2000; 17:665–685
    [Google Scholar]
  6. Broberg CA, Calder TJ, Orth K. Vibrio parahaemolyticus cell biology and pathogenicity determinants. Microbes Infect 2011; 13:992–1001 [View Article] [PubMed]
    [Google Scholar]
  7. O’Boyle N, Houeix B, Kilcoyne M, Joshi L, Boyd A. The MSHA pilus of Vibrio parahaemolyticus has lectin functionality and enables TTSS-mediated pathogenicity. Int J Med Microbiol 2013; 303:563–573 [View Article] [PubMed]
    [Google Scholar]
  8. Makino K, Oshima K, Kurokawa K, Yokoyama K, Uda T et al. Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholerae. Lancet 2003; 361:743–749 [View Article] [PubMed]
    [Google Scholar]
  9. Hubbard TP, Chao MC, Abel S, Blondel CJ, Abel Zur Wiesch P et al. Genetic analysis of Vibrio parahaemolyticus intestinal colonization. Proc Natl Acad Sci U S A 2016; 113:6283–6288 [View Article] [PubMed]
    [Google Scholar]
  10. Morita Y, Kodama K, Shiota S, Mine T, Kataoka A et al. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob Agents Chemother 1998; 42:1778–1782 [View Article] [PubMed]
    [Google Scholar]
  11. Poirel L, Liard A, Rodriguez-Martinez J-M, Nordmann P. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J Antimicrob Chemother 2005; 56:1118–1121 [View Article] [PubMed]
    [Google Scholar]
  12. Saga T, Kaku M, Onodera Y, Yamachika S, Sato K et al. Vibrio parahaemolyticus chromosomal qnr homologue VPA0095: demonstration by transformation with a mutated gene of its potential to reduce quinolone susceptibility in Escherichia coli. Antimicrob Agents Chemother 2005; 49:2144–2145 [View Article] [PubMed]
    [Google Scholar]
  13. Davidsen T, Beck E, Ganapathy A, Montgomery R, Zafar N et al. The comprehensive microbial resource. Nucleic Acids Res 2010; 38:D340–5 [View Article] [PubMed]
    [Google Scholar]
  14. Yu NY, Wagner JR, Laird MR, Melli G, Rey S et al. PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010; 26:1608–1615 [View Article] [PubMed]
    [Google Scholar]
  15. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J et al. The Pfam protein families database. Nucleic Acids Res 2012; 40:D290–301 [View Article] [PubMed]
    [Google Scholar]
  16. Waterfield NR, Sanchez-Contreras M, Eleftherianos I, Dowling A, Yang G et al. Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts. Proc Natl Acad Sci U S A 2008; 105:15967–15972 [View Article] [PubMed]
    [Google Scholar]
  17. Mehmood MA, Gai Y, Zhuang Q, Wang F, Xiao X et al. Aeromonas caviae CB101 contains four chitinases encoded by a single gene chi1. Mol Biotechnol 2010; 44:213–220 [View Article] [PubMed]
    [Google Scholar]
  18. Battle SE, Meyer F, Rello J, Kung VL, Hauser AR. Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals. J Bacteriol 2008; 190:7130–7140 [View Article] [PubMed]
    [Google Scholar]
  19. Yi C, Chen J, Hu D, Song B. First report about the screening, characterization, and fosmid library construction of Xanthomonas oryzae pv. oryzae strain with resistance to Fubianezuofeng. Pestic Biochem Physiol 2020; 169:104645 [View Article] [PubMed]
    [Google Scholar]
  20. Charlop-Powers Z, Banik JJ, Owen JG, Craig JW, Brady SF. Selective enrichment of environmental DNA libraries for genes encoding nonribosomal peptides and polyketides by phosphopantetheine transferase-dependent complementation of siderophore biosynthesis. ACS Chem Biol 2013; 8:138–143 [View Article] [PubMed]
    [Google Scholar]
  21. Pang H, Zhang P, Duan C-J, Mo X-C, Tang J-L et al. Identification of cellulase genes from the metagenomes of compost soils and functional characterization of one novel endoglucanase. Curr Microbiol 2009; 58:404–408 [View Article] [PubMed]
    [Google Scholar]
  22. Cheng G, Hu Y, Yin Y, Yang X, Xiang C et al. Functional screening of antibiotic resistance genes from human gut microbiota reveals a novel gene fusion. FEMS Microbiol Lett 2012; 336:11–16 [View Article] [PubMed]
    [Google Scholar]
  23. Feng Y, Duan C-J, Pang H, Mo X-C, Wu C-F et al. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl Microbiol Biotechnol 2007; 75:319–328 [View Article] [PubMed]
    [Google Scholar]
  24. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY et al. Pfam: the protein families database. Nucleic Acids Res 2014; 42:D222–30 [View Article] [PubMed]
    [Google Scholar]
  25. Piddock LJV. Multidrug-resistance efflux pumps - not just for resistance. Nat Rev Microbiol 2006; 4:629–636 [View Article] [PubMed]
    [Google Scholar]
  26. Tran JH, Jacoby GA, Hooper DC. Interaction of the plasmid-encoded quinolone resistance protein Qnr with Escherichia coli DNA gyrase. Antimicrob Agents Chemother 2005; 49:118–125 [View Article] [PubMed]
    [Google Scholar]
  27. Shashikant CS, Carr JL, Bhargava J, Bentley KL, Ruddle FH. Recombinogenic targeting: a new approach to genomic analysis--a review. Gene 1998; 223:9–20 [View Article] [PubMed]
    [Google Scholar]
  28. Krachler AM, Ham H, Orth K. Outer membrane adhesion factor multivalent adhesion molecule 7 initiates host cell binding during infection by gram-negative pathogens. Proc Natl Acad Sci U S A 2011; 108:11614–11619 [View Article] [PubMed]
    [Google Scholar]
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