VanG-Type Vancomycin-Resistant Enterococcus faecalis Strains Isolated in Canada

Enterococcus faecalis G1-0247 (vancomycin MIC, 16 μg/ml) was found to harbor a vanG operon 99% identical to the vanG operon in E. faecalis BM4518. E. faecalis N03-0233 (vancomycin MIC, 16 μg/ml) was found to harbor a novel vanG operon, vanG2, on an element in a different chromosomal location than the vanG-harboring elements in G1-0247 and BM4518

Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate.

BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described

BLAST atlas of NAP1 genomes.

<p>BLASTn was used to compare NAP1 genomes listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053757#pone-0053757-t001" target="_blank">Table 1</a>. The rings, listed from inner to outer tracks are as follows: Orange peaks represent GC skew while the pink peaks represent GC content. The innermost black track is the R20291 reference genome. Purple tracks are the historical, pre-epidemic strains, CD196 and BI-1. The blue rings consist of Canadian NAP1 genomes, QCD32g58, QCD-97b34, QCD-37×79 and QCD-66c26. The green is the susceptible CD26A54_S while the red is the resistant subpopulation, CD26A54_R. The outermost ring contains the sequences for the two transposable elements, Tn<i>6104</i> and Tn<i>6106</i> to confirm their absence in all genomes except R20291.</p

Summary of genetic variation within coding regions relative to the reference R20291 genome unique to either CD26A54_R or CD26A54_S.

1<p>Wet lab confirmation of variant by sequenced PCR products using target specific primers.</p>2<p>Fold coverage of Illumina sequences at this position.</p>3<p>Frequency of the variant, presented as a percentage for comparison.</p

Growth of <i>C. difficile</i> in BHI broth.

<p>CD26A54_R, the metronidazole resistant strain (□) demonstrated aberrant growth compared to VLOO13 (□) at all time points between 5 and 24 hours (<i>p</i><0.05*). However, the CD26A54_S strain (□) only had a significantly greater cell density than CD26A54_R during late-log and early stationary time points (7–11 hours, <i>p</i><0.05**). There was no significant difference observed between VLOO13 and CD26A54_S throughout the growth curve.</p

Antibiotic susceptibility results of <i>C. difficile</i> strains by Etest®.

<p>N/A = not applicable as a quality control strain for this specific antibiotic.</p

Scanning electron microscopy of <i>C. difficile</i>.

<p>SEM images of VLOO13 (A), CD26A54_S (B) and CD26A54_R (C). (D) Histograms of calculated bacterial length are presented; they demonstrate the cell length variation that exists across the three <i>C. difficile</i> specimens that were imaged by SEM. The insets with arrows in panels (B, C) highlight the septum which partially forms between adjacent cells. Cell separation appears to be impaired resulting in the longer phenotype.</p

Variants within coding regions common to both CD26A54_R and CD26A54_S with reference to R20291.

1<p>Wet lab confirmation of variant by sequenced PCR products using target specific primers.</p>2<p>Fold coverage of Illumina sequences at this position.</p>3<p>Frequency of the variant, presented as a percentage for comparison.</p