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

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.


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: antimicrobial resistance, Clostridium difficile infection, mobile genetic element, vanB S ince its first description as the causative agent of pseudomembranous colitis in 1978, Clostridium difficile has emerged as a major enteropathogen of humans and a significant burden to global health care systems (1). Vancomycin has been a first-line therapy for C. difficile infection (CDI) for almost 30 years, retaining good activity against C. difficile, including strains belonging to epidemic lineages and those with increased resistance to metronidazole (2). Despite sporadic reports of reduced susceptibility to vancomycin (MIC, Ն4 mg/liter; CLSI susceptibility breakpoint, Յ2 mg/liter), to date no underlying mechanisms have been identified (2). Sequencing of C. difficile genomes revealed the widespread presence of a vancomycin resistance operon (vanG Cd ) (3). Although it is often referred to as cryptic (phenotypically silent), transcriptional and biochemical studies showed that vanG Cd was functional, conferring a modest increase in MIC in C. difficile (from 1 mg/liter to 2 mg/liter) (4). Here, we present the first description of a cryptic vanB2 operon in C. difficile, carried on an~42-kb element showing significant homology and synteny to Tn1549, a conjugative transposon (CTn) linked with the emergence and global dissemination of vancomycin-resistant enterococci.
C. difficile strain AI0499 was recovered from the carcass of a calf (aged Ͻ7 days) in Victoria, Australia, in April 2013, identified as C. difficile by morphological and phenotypic traits as previously described (5), and confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. By PCR ribotyping, strain AI0499 was identified as ribotype (RT) 033 and thus negative for genes encoding large clostridial toxins A and B (tcdA Ϫ tcdB Ϫ ) but positive for genes encoding binary toxin (cdtA ϩ cdtB ϩ ) (5).
Whole-genome sequencing (WGS) of AI0499 was performed in duplicate at two independent institutions. Genomic DNA (gDNA) was extracted using a Gentra Puregene kit (Qiagen, Hilden, Germany), and libraries were created using Nextera XT protocols (Illumina, Inc., San Diego, CA). The first sequence run was performed on an Illumina MiSeq sequencer with 250-bp paired-end (PE) chemistry, generating 406,204 reads and 36ϫ coverage. The second was performed on an Illumina HiSeq sequencer with 100-bp PE chemistry, generating 3,684,407 reads and 131ϫ coverage. Multilocus sequence typing (MLST) and antimicrobial gene profiling were performed using SRST2 (6). Genomes were assembled, annotated, and curated using a pipeline comprising SPAdes, Prokka, Artemis, and Easyfig (7)(8)(9)(10). In vitro susceptibility to vancomycin was investigated in triplicate using the CLSI agar dilution methodology as previously described (11) and in triplicate using Etest methodology.
WGS and de novo assembly of the AI0499 genome revealed a single chromosome of 4,095,918 bp and 28.75% GC with 3,960 coding sequences (CDS) and an overall coverage of~130ϫ. Strain AI0499 was characterized as sequence type (ST) 11 (MLST clade 5) and harbored a complete binary toxin locus comprising cdtR, cdtA, and cdtB genes. Strain AI0499 possessed an uncommon pathogenicity locus identified as toxinotype XI and defined by the complete absence of tcdB, a fragmented and truncated tcdA gene (A2 fragment, 3,231 bp; A3 fragment, 915 bp), and a variant tcdC gene (allele tcdC-A1 as described by Curry et al.) (12,13). SRST2 identified seven vancomycin resistance genes with Ͼ99% sequence identity to vanXB, vanB, vanHB, vanW, vanYB, vanSB, and vanRB. AI0499 was negative for vanG Cd .
In Gram-positive bacteria, vancomycin resistance is mediated by several van operons and arises as a result of both (i) biosynthesis of modified peptidoglycan precursors ending in D-Ala-D-Lac or D-Ala-D-Ser to which vancomycin shows reduced binding and (ii) the elimination of high-affinity natural D-Ala-D-Ala precursors (13). Tn1549 (GenBank accession no. AF192329) is a member of the Tn916 family of conjugative transposons and harbors a vanB subtype 2 operon (vanB2) comprising genes encoding a dipeptidase (vanXB, 609 bp), a ligase (vanB, 1,029 bp), a dehydrogenase (vanHB, 972 bp), a putative hydrolase (vanW, 828 bp), and a carboxypeptidase (vanYB, 807 bp). Two further genes, vanSB (1,344 bp) and vanRB (663 bp), also colocated within the vanB2 operon, play a crucial role in the phenotypic expression of vancomycin resistance (14).
Sequence analysis of regions flanking the vanB2 gene cluster in AI0499 revealed an element of 42,375 bp showing significant sequence identity and synteny with the prototypical Tn1549 (GenBank sequence accession no. AF192329) (Fig. 1). Notably, this element differed markedly, particularly in its accessory region, from other putative Tn1549-like CTns, CTn2, CTn4, and CTn5, previously described in C. difficile (data not shown) (15,16).
The element designated Tn1549-like contained 38 open reading frames (ORFs) and, like Tn1549, was organized into transposition, accessory (antimicrobial resistance), and conjugation regions (Fig. 1). Defining the left and right terminal ends of the element were 11-bp inverted repeats matching those found in Tn1549 and likely representing excision/integration sites (14). Comparing the vanB2 operon in AI0499 to that of Tn1549 revealed significant homology in vanXB, vanB, vanHB, vanW, vanYB, and vanSB (Fig. 1). However, in AI0499 vanRB was fragmented into a 525-bp fragment located adjacent to vanSB and a 134-bp fragment some 2.1 kb away (Fig. 1). Notably, two CDS present in strain AI0499 but absent in Tn1549 were found interrupting the vanRB gene. Bm3R1 (582 bp) and KU558763_7 (1,032 bp) encode a transcriptional repressor and decarboxylase originating from Bacillus megaterium and Bacillus cereus, respectively. The Tn1549like element contained four additional CDS completely absent from Tn1549 (Fig. 1). KU558763_5 (684 bp) and KU558763_6 (702 bp), colocated between the transposition and vancomycin resistance regions, encode hypothetical proteins originating from Clostridium clostridioforme. KU558763_10 (1,803 bp), located~3 kb into the conjugation region, and KU558763_25 (1,665 bp), located near the far left extremity, both encode group II introns originating from an unidentified Clostridiales member and C. clostridioforme, respectively.
Several clostridial species, including C. bolteae, C. hathewayi, C. innocuum, C. clostridioforme, and C. symbiosum, harbor vanB-like elements and demonstrate vancomycin resistance in vitro (17)(18)(19). Notably, strain AI0499 did not show any reduced susceptibility to vancomycin in vitro (MIC, 1 mg/liter), most likely due to the fragmentation of vanRB; however, this first description of a phenotypically silent vanB2 operon in C. difficile further confirms that anaerobes of the animal gut microbiota are a reservoir of clinically important vanB-like resistance operons. Accession number(s).The nucleotide sequence of the Tn1549-like element from strain AI0499 has been submitted to GenBank (accession no. KU558763). The HiSeq PE sequence reads have been deposited in the NCBI Short Read Archive under accession no. SRP067713.