Knight, D.R.

Riley, T.V.

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         Intrinsic and acquired antimicrobial resistance repertoire of Australian isolates of Clostridium difficile identified on whole genome sequencing   Daniel R. Knight1 and Thomas V. Riley1,2,*  1 Microbiology & Immunology, School of Pathology and Laboratory Medicine, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands 6009, WA 2 * Department of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands 6009, WA  OBJECTIVES: A 2013 report from the CDC ranks Clostridium difficile as the most important antimicrobial resistant threat to public health in the USA (1). In this study, whole genome sequencing (WGS) was used to investigate intrinsic and acquired antimicrobial resistance in C. difficile in Australia.  METHODS: A total of 171 C. difficile strains isolated from humans (n=104) and livestock (n=67) in the last decade was studied. WGS was performed using standard illumina protocols (2). Acquired antimicrobial resistance genes were identified using SRST2 (3). To identify intrinsic resistance, and to characterise the genetic elements underlying any acquired resistance, annotated de novo assemblies were interrogated against a custom Tn/recombinase sequence library with manual curation (4-5). Minimum inhibitory concentrations were determined using the CLSI agar dilution methodology and CLSI/EUCAST breakpoints (6-8).  RESULTS: Fluoroquinolone resistance was observed in 8.2% of isolates (n=14, all human sequence type (ST) 11) and conferred by mutations in gyrA and gyrB. Forty-six isolates (26.9%) showed a MLSB phenotype, 29 of which (human ST11 and porcine STs 2,13 and 49) harboured ermB genes carried on Tn6194-like elements. The remaining 17 isolates with a MLSB phenotype were negative for ermB/Tn6194. Tetracycline resistance (tetR) was observed in 29.2% of isolates (n=50). tetM was found in 47 of 50 tetR isolates, carried on Tn916-like (human ST11) and Tn5397-like (porcine STs 2,13 and 49) elements. Of the tetM positive strains, 48.0% (n=24) harboured the efflux gene tet40 from Strep. suis, whilst 14.0% (n=7) carried tetW from TnB1230 of the ruminent anaerobe Butyrivibrio flavescens. Despite C. difficile being inherently resistant to aminoglycosides, 23.0% (n=40) of isolates carried an aminoglycoside-streptothricin resistance cassette (ant6-Ia,aph3-III,Sat4A). All isolates harboured gene clusters for bacitracin and tellurium resistance and carried the blaR gene conferring intrinsic resistance to penicillins and nearly all cephalosporins. Notably, all pig isolates carried the cryptic cme gene which confers multidrug resistance in E. faecalis. All isolates were susceptible to vancomycin, metronidazole, rifaximin, fidaxomicin, meropenem, Augmentin and pipericillin-tazobactam in vitro.  CONCLUSIONS: WGS demonstrated that C. difficile posseses a diverse repetoire of intrinsic and acquired mechanisms of resistance to clinically relevant antimicrobials, some of which are expressed phenotypically.  REFERENCES  1) Centers for Disease Control and Prevention. 2013. Antibiotic resistance threats in the United States, 2013. http://www.cdc.gov/drugresistance/threat-report-2013/ 2) Eyre DW, Tracey L, Elliott B et al. Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012. Euro Surveill. 2015. 20:21059 3) Inouye M, Dashnow H, Raven L et al. SRST2: Rapid genomic surveillance for public health and hospital microbiology labs. Genome Med. 2013. 6:90 4) Bankevich A, Nurk S, Antipov D et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. 2012. J. Comput. Biol. 19:455-477 5) Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014. 30:2068-2069 6) CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard-Seventh Edition. Clinical and Laboratory Standards Institute. M11-A7:Wayne PA. 2013 7) CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. M100-S23:Wayne PA. 2013 8) EUCAST. Clinical Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0. London, United Kingdom. http://www.eucast.org/ Accessed 18th November 2015. Proffered Paper 2.1 View publication stats

Intrinsic and acquired antimicrobial resistance repertoire of Australian isolates of Clostridium difficile identified on whole genome sequencing

https://researchrepository.murdoch.edu.au/id/eprint/58590/1/whole%20genome%20sequencing.pdf

Intrinsic and acquired antimicrobial resistance repertoire of Australian isolates of Clostridium difficile identified on whole genome sequencing

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