vanD and vanG -Like Gene Clusters in a Ruminococcus Species Isolated from Human Bowel Flora

Abstract

A vancomycin-resistant, anaerobic, gram-positive coccus containing the vanD and vanG-like genes (strain CCRI-16110) was isolated from a human fecal specimen during a hospital surveillance program to detect carriers of vancomycin-resistant enterococci. Comparison of the 16S rRNA gene sequence of strain CCRI-16110 with databases revealed a potentially novel Ruminococcus species that was most similar (<94% identity) to Clostridium and Ruminococcus species. Strain CCRI-16110 was highly resistant to vancomycin and teicoplanin (MICs of >256 g/ml). The complete DNA sequence of the vanD cluster was most similar (98.2% identity) to that of Enterococcus faecium BM4339, containing the vanD1 allele. An intD gene with 99% identity with that of this E. faecium strain was found to be associated with the vanD gene cluster of this novel anaerobic bacterium. Strain CCRI-16110 also harbors genes encoding putative VanS G , VanG, and VanT G proteins displaying 56, 73.6, and 55% amino acid sequence identity, respectively, compared to the corresponding proteins encoded by the vanG1 and vanG2 operons of Enterococcus faecalis BM4518 and N03-0233. This study reports for the first time an anaerobic bacterium containing the vanD gene cluster. This strain also harbors a partial vanG-like gene cluster. The presence of vanD-and vanG-containing anaerobic bacteria in the human bowel flora suggests that these bacteria may serve as a reservoir for the vanD and vanG vancomycin resistance genes.The glycopeptide antibiotics vancomycin and teicoplanin interfere with cell wall synthesis in gram-positive bacteria by binding to the terminal dipeptide D-alanyl-D-alanine regions of the pentapeptide precursors of peptidoglycan side chains. Resistance to glycopeptide antibiotics was first described for enterococci (23, 35) and has now spread worldwide. Six different genes (vanA, vanB, vanC, vanD, vanE, and vanG) have been shown to confer glycopeptide resistance in enterococci (7).The VanD-type resistance is constitutively expressed in enterococci (8, 11) and is characterized by moderate MICs of vancomycin (64 to 128 g/ml) and teicoplanin (4 to 64 g/ml). This VanD-type resistance results from the acquisition of the vanR D , vanS D , vanY D , vanH D , vanD, and vanX D cluster of genes, which directs synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (11). The vanD gene cluster is located on the chromosome and is not transferable to other enterococci by conjugation in vitro (11). The VanD-type resistance in enterococci is also characterized by the presence of an impaired D-alanyl-D-alanine (Ddl) ligase due to mutations in the chromosomal ligase-encoding gene ddl (10). Although the Ddl ligase is inactive, the VanD-type enterococci are able to grow even in the absence of glycopeptide because the vanD cluster is expressed constitutively as a result of mutations in the VanS D sensor or in the VanR D regulator (10).The VanG-type resistance in enterococci is characterized by low-level resistance to vancomycin (MIC, 16 g/ml) and susceptibility t...

New Combinations of Mutations in VanD-Type Vancomycin-Resistant Enterococcus faecium , Enterococcus faecalis , and Enterococcus avium Strains

“…In E. gallinarum N04-0414 (8) and a Ruminococcus sp. (17), the vanD clusters are, respectively, 94% and 98.2% identical with that of BM4339, whereas in E. raffinosus GV5 (37), the vanD cluster is highly similar (99.5% identity) to that in 10/96A, except for the presence of a frameshift mutation in vanY D in the latter strain. However, E. faecalis BM4654 and E. faecium N03-0072 (7), which are identical, harbor hybrid operons: the predicted VanS D , VanY D , and VanD proteins are more similar to their counterparts in 10/96A than in BM4339 and its derivatives, whereas the opposite is true for the remaining proteins (VanR D , VanH D , and VanX D ) ( Table 3).…”

“…VanD has been found only in a few strains of E. faecium (6,7,10,13,16,31), two E. faecalis strains (13), one Enterococcus gallinarum strain (8), and one Enterococcus raffinosus strain (37). Recently, the vanD operon was detected in a Ruminococcus sp., an anaerobic gram-positive bacterium from human bowel flora, suggesting a potential reservoir for vancomycin resistance genes in commensals of the digestive tract (17).…”

Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth, and the BD GeneOhm™ VanR Assay for identification of vancomycin-resistant enterococci in screening specimens

“…Since genotypes vanE to vanG (and most probably the previously identified vanL and vanM, too; [Boyd et al, 2008] and [Xu et al, 2010]) still remain rare among clinical enterococcal isolates encountered in hospitals worldwide (Werner et al, 2008), this does not complicate substantially the performance and predictive value of the present assay in detecting and identifying VRE colonizers. Quite contrarily, the prevalence of vanB, vanD, vanE, and vanG genes among nonenterococcal, intestinal bacteria may thwart the good performance of a putative assay capable of detecting all acquired van genotypes ( [Domingo et al, 2005], [Domingo et al, 2007], [Graham et al, 2008], [Launay et al, 2006] and [Tsvetkova et al, 2010]). Performance of the assay in identifying a set of 20 reference and test strains (vanA/vanB positive and van negative) performed at 3 laboratories showed 100% sensitivity and specificity.…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…and others (Patel 2000;Stinear et al, 2001;Domingo et al, 2005;Ballard et al, 2005b;Domingo et al, 2007). One of these isolates was investigated further and a new, naturally vancomycinresistant species, Ruminococcus gauvreauii, was identified possessing a vanD gene cluster (Domingo et al, 2008).…”