1887

Abstract

The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria are exposed to anaerobic conditions within the gastrointestinal tract, yet little is known about how oxygen exposure influences AMR. The facultative anaerobe was chosen as a model to address this knowledge gap. We obtained isolates from 66 cholera patients, sequenced their genomes, and grew them under anaerobic and aerobic conditions with and without three clinically relevant antibiotics (ciprofloxacin, azithromycin, doxycycline). For ciprofloxacin and azithromycin, the minimum inhibitory concentration (MIC) increased under anaerobic conditions compared to aerobic conditions. Using standard resistance breakpoints, the odds of classifying isolates as resistant increased over 10 times for ciprofloxacin and 100 times for azithromycin under anaerobic conditions compared to aerobic conditions. For doxycycline, nearly all isolates were sensitive under both conditions. Using genome-wide association studies, we found associations between genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations. These AMR phenotypes were more heritable, and the AMR-associated genetic elements were more often discovered, under anaerobic conditions. These AMR-associated genetic elements are promising targets for future mechanistic research. Our findings provide a rationale to determine whether increased MICs under anaerobic conditions are associated with therapeutic failures and/or microbial escape in cholera patients. If so, there may be a need to determine new AMR breakpoints for anaerobic conditions.

Funding
This study was supported by the:
  • Génome Québec
    • Principle Award Recipient: B.Jesse Shapiro
  • Genome Canada
    • Principle Award Recipient: B.Jesse Shapiro
  • National Institutes of Health
    • Principle Award Recipient: EricJ Nelson
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000905
2022-12-05
2024-06-17
Loading full text...

Full text loading...

/deliver/fulltext/mgen/8/12/mgen000905.html?itemId=/content/journal/mgen/10.1099/mgen.0.000905&mimeType=html&fmt=ahah

References

  1. CLSI Performance Standards for Antimicrobial Susceptibility Testing, 27th edn Wayne, PA: Clinical and Laboratory Standards Institute; 2017
    [Google Scholar]
  2. Bueno E, Sit B, Waldor MK, Cava F. Genetic dissection of the fermentative and respiratory contributions supporting Vibrio cholerae hypoxic growth. J Bacteriol 2020; 202:e00243-20 [View Article]
    [Google Scholar]
  3. Bueno E, Pinedo V, Cava F. Adaptation of Vibrio cholerae to hypoxic environments. Front Microbiol 2020; 11:739 [View Article]
    [Google Scholar]
  4. Xu Q, Dziejman M, Mekalanos JJ. Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro. Proc Natl Acad Sci USA 2003; 100:1286–1291 [View Article]
    [Google Scholar]
  5. Mandlik A, Livny J, Robins WP, Ritchie JM, Mekalanos JJ et al. RNA-Seq-based monitoring of infection-linked changes in Vibrio cholerae gene expression. Cell Host Microbe 2011; 10:165–174 [View Article]
    [Google Scholar]
  6. Beaber JW, Hochhut B, Waldor MK. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 2004; 427:72–74 [View Article]
    [Google Scholar]
  7. Narendrakumar L, Gupta SS, Johnson JB, Ramamurthy T, Thomas S. Molecular adaptations and antibiotic resistance in Vibrio cholerae: a communal challenge. Microb Drug Resist 2019; 25:1012–1022 [View Article]
    [Google Scholar]
  8. Das B, Verma J, Kumar P, Ghosh A, Ramamurthy T. Antibiotic resistance in Vibrio cholerae: understanding the ecology of resistance genes and mechanisms. Vaccine 2020; 38 (Suppl. 1):A83–A92 [View Article]
    [Google Scholar]
  9. Bueno E, Sit B, Waldor MK, Cava F. Anaerobic nitrate reduction divergently governs population expansion of the enteropathogen Vibrio cholerae. Nat Microbiol 2018; 3:1346–1353 [View Article]
    [Google Scholar]
  10. Nelson EJ, Nelson DS, Salam MA, Sack DA. Antibiotics for both moderate and severe cholera. N Engl J Med 2011; 364:5–7 [View Article]
    [Google Scholar]
  11. WHO The Treatment of Diarrhoea – a Manual for Physicians and Other Senior Health Workers, 4th rev Geneva: World Health Organization; 2005
    [Google Scholar]
  12. Leibovici-Weissman Y, Neuberger A, Bitterman R, Sinclair D, Salam MA et al. Antimicrobial drugs for treating cholera. Cochrane Database Syst Rev 2014; 2014:CD008625 [View Article]
    [Google Scholar]
  13. Khan AI, Mack JA, Salimuzzaman M, Zion MI, Sujon H et al. Electronic decision support and diarrhoeal disease guideline adherence (mHDM): a cluster randomized controlled trial. Lancet Digit Health 2020; 2:e250–258 [View Article]
    [Google Scholar]
  14. Biswas D, Hossin R, Rahman M, Bardosh KL, Watt MH et al. An ethnographic exploration of diarrheal disease management in public hospitals in Bangladesh: from problems to solutions. Soc Sci Med 2020; 260:113185 [View Article]
    [Google Scholar]
  15. Ingle DJ, Levine MM, Kotloff KL, Holt KE, Robins-Browne RM. Dynamics of antimicrobial resistance in intestinal Escherichia coli from children in community settings in South Asia and sub-Saharan Africa. Nat Microbiol 2018; 3:1063–1073 [View Article]
    [Google Scholar]
  16. Towner KJ, Pearson NJ, Mhalu FS, O’Grady F. Resistance to antimicrobial agents of Vibrio cholerae E1 Tor strains isolated during the fourth cholera epidemic in the United Republic of Tanzania. Bull World Health Organ 1980; 58:747–751
    [Google Scholar]
  17. Burrus V, Marrero J, Waldor MK. The current ICE age: biology and evolution of SXT-related integrating conjugative elements. Plasmid 2006; 55:173–183 [View Article]
    [Google Scholar]
  18. Hooper DC, Jacoby GA. Topoisomerase inhibitors: fluoroquinolone mechanisms of action and resistance. Cold Spring Harb Perspect Med 2016; 6:a025320 [View Article]
    [Google Scholar]
  19. Garriss G, Waldor MK, Burrus V. Mobile antibiotic resistance encoding elements promote their own diversity. PLoS Genet 2009; 5:e1000775 [View Article]
    [Google Scholar]
  20. Fonseca EL, Dos Santos Freitas F, Vieira VV, Vicente ACP. New qnr gene cassettes associated with superintegron repeats in Vibrio cholerae O1. Emerg Infect Dis 2008; 14:1129–1131 [View Article]
    [Google Scholar]
  21. Fyfe C, Grossman TH, Kerstein K, Sutcliffe J. Resistance to macrolide antibiotics in public health pathogens. Cold Spring Harb Perspect Med 2016; 6:a025395 [View Article]
    [Google Scholar]
  22. Grossman TH. Tetracycline antibiotics and resistance. Cold Spring Harb Perspect Med 2016; 6:a025387 [View Article]
    [Google Scholar]
  23. Zhu Z, Surujon D, Ortiz-Marquez JC, Huo W, Isberg RR et al. Entropy of a bacterial stress response is a generalizable predictor for fitness and antibiotic sensitivity. Nat Commun 2020; 11:4365 [View Article]
    [Google Scholar]
  24. Wood S, Zhu K, Surujon D, Rosconi F, Ortiz-Marquez JC et al. A pangenomic perspective on the emergence, maintenance, and predictability of antibiotic resistance. In Tettelin H, Medini D. eds The Pangenome: Diversity, Dynamics and Evolution of Genomes Cham: Springer; 2020 pp 169–202
    [Google Scholar]
  25. Warrier I, Ram-Mohan N, Zhu Z, Hazery A, Echlin H et al. The transcriptional landscape of Streptococcus pneumoniae TIGR4 reveals a complex operon architecture and abundant riboregulation critical for growth and virulence. PLoS Pathog 2018; 14:e1007461 [View Article]
    [Google Scholar]
  26. Jensen PA, Zhu Z, van Opijnen T. Antibiotics disrupt coordination between transcriptional and phenotypic stress responses in pathogenic bacteria. Cell Rep 2017; 20:1705–1716 [View Article]
    [Google Scholar]
  27. van Opijnen T, Dedrick S, Bento J. Strain dependent genetic networks for antibiotic-sensitivity in a bacterial pathogen with a large pan-genome. PLoS Pathog 2016; 12:e1005869 [View Article]
    [Google Scholar]
  28. Cain AK, Barquist L, Goodman AL, Paulsen IT, Parkhill J et al. A decade of advances in transposon-insertion sequencing. Nat Rev Genet 2020; 21:526–540 [View Article]
    [Google Scholar]
  29. Dörr T, Delgado F, Umans BD, Gerding MA, Davis BM et al. A transposon screen identifies genetic determinants of Vibrio cholerae resistance to high-molecular-weight antibiotics. Antimicrob Agents Chemother 2016; 60:4757–4763 [View Article]
    [Google Scholar]
  30. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007; 130:797–810 [View Article]
    [Google Scholar]
  31. Dwyer DJ, Belenky PA, Yang JH, MacDonald IC, Martell JD et al. Antibiotics induce redox-related physiological alterations as part of their lethality. Proc Natl Acad Sci USA 2014; 111:E2100–E2109 [View Article]
    [Google Scholar]
  32. Staerck C, Gastebois A, Vandeputte P, Calenda A, Larcher G et al. Microbial antioxidant defense enzymes. Microb Pathog 2017; 110:56–65 [View Article]
    [Google Scholar]
  33. Smirnova G, Muzyka N, Oktyabrsky O. Transmembrane glutathione cycling in growing Escherichia coli cells. Microbiol Res 2012; 167:166–172 [View Article]
    [Google Scholar]
  34. Bryan LE, Kwan S. Mechanisms of aminoglycoside resistance of anaerobic bacteria and facultative bacteria grown anaerobically. J Antimicrob Chemother 1981; 8 (Suppl. D):1–8 [View Article]
    [Google Scholar]
  35. Bryan LE. General mechanisms of resistance to antibiotics. J Antimicrob Chemother 1988; 22 (Suppl. A):1–15 [View Article]
    [Google Scholar]
  36. Bryant RE, Fox K, Oh G, Morthland VH. Beta-lactam enhancement of aminoglycoside activity under conditions of reduced pH and oxygen tension that may exist in infected tissues. J Infect Dis 1992; 165:676–682 [View Article]
    [Google Scholar]
  37. Dwyer DJ, Kohanski MA, Hayete B, Collins JJ. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol 2007; 3:91 [View Article]
    [Google Scholar]
  38. Hong Y, Li Q, Gao Q, Xie J, Huang H et al. Reactive oxygen species play a dominant role in all pathways of rapid quinolone-mediated killing. J Antimicrob Chemother 2020; 75:576–585 [View Article]
    [Google Scholar]
  39. Luan G, Hong Y, Drlica K, Zhao X. Suppression of reactive oxygen species accumulation accounts for paradoxical bacterial survival at high quinolone concentration. Antimicrob Agents Chemother 2018; 62:e01622-17 [View Article]
    [Google Scholar]
  40. Zhao X, Drlica K. Reactive oxygen species and the bacterial response to lethal stress. Curr Opin Microbiol 2014; 21:1–6 [View Article]
    [Google Scholar]
  41. Van Alst AJ, Demey LM, DiRita VJ. Vibrio cholerae requires oxidative respiration through the bd-I and cbb(3) oxidases for intestinal proliferation. PLoS Pathog 2022; 18:e1010102 [View Article]
    [Google Scholar]
  42. Van Alst AJ, DiRita VJ. Aerobic metabolism in Vibrio cholerae is required for population expansion during infection. mBio 2020; 11:e01989-20 [View Article]
    [Google Scholar]
  43. Nelson EJ, Chowdhury A, Harris JB, Begum YA, Chowdhury F et al. Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea. Proc Natl Acad Sci USA 2007; 104:19091–19096 [View Article]
    [Google Scholar]
  44. Sprouffske K, Wagner A. Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics 2016; 17:172 [View Article]
    [Google Scholar]
  45. CLSI Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, M45, 3rd edn Wayne, PA: Clinical and Laboratory Standards Institute; 2017
    [Google Scholar]
  46. Drezen E, Rizk G, Chikhi R, Deltel C, Lemaitre C et al. GATB: genome assembly & analysis tool box. Bioinformatics 2014; 30:2959–2961 [View Article]
    [Google Scholar]
  47. Lees JA, Galardini M, Bentley SD, Weiser JN, Corander J. pyseer: a comprehensive tool for microbial pangenome-wide association studies. Bioinformatics 2018; 34:4310–4312 [View Article]
    [Google Scholar]
  48. Cook DE, Andersen EC. VCF-kit: assorted utilities for the variant call format. Bioinformatics 2017; 33:1581–1582 [View Article]
    [Google Scholar]
  49. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014; 30:1312–1313 [View Article]
    [Google Scholar]
  50. Saber MM, Shapiro BJ. Benchmarking bacterial genome-wide association study methods using simulated genomes and phenotypes. Microb Genom 2020; 6:000337 [View Article]
    [Google Scholar]
  51. Alexandrova L, Haque F, Rodriguez P, Marrazzo AC, Grembi JA et al. Identification of widespread antibiotic exposure in patients with cholera correlates with clinically relevant microbiota changes. J Infect Dis 2019; 220:1655–1666 [View Article]
    [Google Scholar]
  52. Leclercq SO, Wang C, Zhu Y, Wu H, Du X et al. Diversity of the tetracycline mobilome within a Chinese pig manure sample. Appl Environ Microbiol 2016; 82:6454–6462 [View Article]
    [Google Scholar]
  53. Grossman TH, Starosta AL, Fyfe C, O’Brien W, Rothstein DM et al. Target- and resistance-based mechanistic studies with TP-434, a novel fluorocycline antibiotic. Antimicrob Agents Chemother 2012; 56:2559–2564 [View Article]
    [Google Scholar]
  54. Noguchi N, Emura A, Matsuyama H, O’Hara K, Sasatsu M et al. Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2’-phosphotransferase I in Escherichia coli. Antimicrob Agents Chemother 1995; 39:2359–2363 [View Article]
    [Google Scholar]
  55. Chesneau O, Tsvetkova K, Courvalin P. Resistance phenotypes conferred by macrolide phosphotransferases. FEMS Microbiol Lett 2007; 269:317–322 [View Article]
    [Google Scholar]
  56. Humphries RM, Ambler J, Mitchell SL, Castanheira M, Dingle T et al. CLSI methods development and standardization working group best practices for evaluation of antimicrobial susceptibility tests. J Clin Microbiol 2018; 56:e01934-17 [View Article]
    [Google Scholar]
  57. Humphries RM, Abbott AN, Hindler JA. Understanding and addressing CLSI breakpoint revisions: a primer for clinical laboratories. J Clin Microbiol 2019; 57:e00203-19 [View Article]
    [Google Scholar]
  58. Ellington MJ, Ekelund O, Aarestrup FM, Canton R, Doumith M et al. The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST subcommittee. Clin Microbiol Infect 2017; 23:2–22 [View Article]
    [Google Scholar]
  59. Stokes JM, Lopatkin AJ, Lobritz MA, Collins JJ. Bacterial metabolism and antibiotic efficacy. Cell Metab 2019; 30:251–259 [View Article]
    [Google Scholar]
  60. Wang X, Zhao X, Malik M, Drlica K. Contribution of reactive oxygen species to pathways of quinolone-mediated bacterial cell death. J Antimicrob Chemother 2010; 65:520–524 [View Article]
    [Google Scholar]
  61. Kan B, Habibi H, Schmid M, Liang W, Wang R et al. Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions. Proteomics 2004; 4:3061–3067 [View Article]
    [Google Scholar]
  62. Mathur J, Waldor MK. The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides. Infect Immun 2004; 72:3577–3583 [View Article]
    [Google Scholar]
  63. Li CC, Crawford JA, DiRita VJ, Kaper JB. Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae. Mol Microbiol 2000; 35:189–203 [View Article]
    [Google Scholar]
  64. Buckley AM, Webber MA, Cooles S, Randall LP, La Ragione RM et al. The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis. Cell Microbiol 2006; 8:847–856 [View Article]
    [Google Scholar]
  65. Taylor DL, Bina XR, Bina JE. Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus. PLoS One 2012; 7:e38208 [View Article]
    [Google Scholar]
  66. Bina XR, Philippart JA, Bina JE. Effect of the efflux inhibitors 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide on antimicrobial susceptibility and virulence factor production in Vibrio cholerae. J Antimicrob Chemother 2009; 63:103–108 [View Article]
    [Google Scholar]
  67. Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 2018; 359:eaar4120 [View Article]
    [Google Scholar]
  68. Baddam R, Sarker N, Ahmed D, Mazumder R, Abdullah A et al. Genome dynamics of Vibrio cholerae isolates linked to seasonal outbreaks of cholera in Dhaka, Bangladesh. mBio 2020; 11:e03339-19 [View Article]
    [Google Scholar]
  69. Murphy SG, Johnson BA, Ledoux CM, Dörr T. Vibrio cholerae’s mysterious seventh pandemic island (VSP-II) encodes novel Zur-regulated zinc starvation genes involved in chemotaxis and autoaggregation. PLoS Genet 2021; 17:e1009624
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000905
Loading
/content/journal/mgen/10.1099/mgen.0.000905
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error