AUTHOR=Isidro Joana , Menezes Juliana , Serrano Mónica , Borges Vítor , Paixão Pedro , Mimoso Margarida , Martins Filomena , Toscano Cristina , Santos Andrea , Henriques Adriano O. , Oleastro Mónica 

TITLE=Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of Resistance

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02994

DOI=10.3389/fmicb.2018.02994

ISSN=1664-302X

ABSTRACT=<p><bold>Background:</bold><italic>Clostridium difficile</italic> infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of <italic>C. difficile</italic>.</p><p><bold>Methods:</bold> A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The <italic>C. difficile</italic> isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest<sup>®</sup>strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS).</p><p><bold>Results:</bold> Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were <italic>tcdA</italic><sup>-</sup>/<italic>tcdB</italic><sup>+</sup> and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in <italic>gyrA</italic>, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in <italic>rpoB</italic> for rifampicin resistance, the <italic>tetM</italic> gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, <italic>aadE</italic> and <italic>aac(6′)-aph(2″)</italic>. Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes <italic>ermG</italic>, <italic>mefA</italic>/<italic>msrD</italic> and <italic>vat</italic>, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance.</p><p><bold>Conclusion:</bold> We describe an epidemic, <italic>tcdA</italic><sup>-</sup>/<italic>tcdB</italic><sup>+</sup>, multidrug resistant clone of <italic>C. difficile</italic> from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of <italic>C. difficile</italic>.</p>