A Phenotypically Silent vanB2 Operon Carried on a Tn1549-Like Element in Clostridium difficile

Abstract

In the last decade, Clostridium difficile infection (CDI) has reached an epidemic state with increasing incidence and severity in both health care and community settings. Vancomycin is an important first-line therapy for CDI, and the emergence of resistance would have significant clinical consequences. In this study, we describe for the first time a vanB2 vancomycin resistance operon in C. difficile, isolated from an Australian veal calf at slaughter. The operon was carried on an ~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon linked with the emergence and global dissemination of vancomycin-resistant enterococci (VRE). Notably, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly as a result of an aberrant vanRB gene. As observed for other anaerobic species of the animal gut microbiota, C. difficile may be a reservoir of clinically important vancomycin resistance genes. IMPORTANCE In an era when the development of new antimicrobial drugs is slow, vancomycin remains the preferred antimicrobial therapy for Clostridium difficile infection (CDI), the most important health care-related infection in the world today. The emergence of resistance to vancomycin would have significant consequences in relation to treating patients with CDI. In this paper, we describe for the first time a complete set of vancomycin resistance genes in C. difficile. The genes were very similar to genes found in vancomycin-resistant enterococci (VRE) that were associated with the emergence and global dissemination of this organism. Fortunately, the C. difficile strain did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), possibly because of a small difference in one gene. However, this observation signals that we may be very close to seeing a fully vancomycin-resistant strain of C. difficile.

Keywords: Clostridium difficile infection; antimicrobial resistance; mobile genetic element; vanB.

FIG 1

Comparative genomic analysis of Tn1549-like element in C. difficile strain AI0499 and prototypical Tn1549 of Enterococcus faecalis (GenBank accession no. AF192329). Arrows indicate open reading frames (ORFs) and direction of transcription. Excisionase (xis) and integrase (int) genes are shown in yellow, and genes comprising the vanB2 operon (vanXB, vanB, vanHB, vanW, vanYB, vanSB, and vanRB) are shown in green, with the remaining ORFs shown in blue. The figure was prepared using Easyfig (minimum blast hit length of 100 bp and a maximum E value of 0.001) (10). Vertical blocks between sequences indicate regions of homology with Blast nucleotide identity shown on a colored scale ranging from 70% (light gray) to 100% (dark gray). Bm3R1 and ORFs KU558763_5, KU558763_6, KU558763_7, KU558763_10, and KU558763_25 are shown to be present in strain AI0499 but absent from the sequence with accession no. AF192329 with Bm3R1 and KU558763_7 interrupting vanRB (vanRB_1 and vanRB_2 fragments shown). Overall sizes of elements in strain AI0499 and the sequence with accession no. AF192329 are 42,375 bp and 33,805 bp, respectively.