vanD Gene Research Articles

Virulence genes in Enterococcus faecium and Enterococcus faecalis isolated from blood culture in haematological patients

Objective. To study virulence genes in E. faecium and E. faecalis isolated from the blood cultures of patients with hematological diseases. Materials and Methods. Virulence genes were studied in E. faecium and E. faecalis strains isolated from blood culture from hematological patients in four Russian hospitals (2002–2020). Susceptibility to vancomycin was determined by broth microdilution method (CLSI, 2022). Virulence genes (esp, hyl, asa1, cylA and gelE) in E. faecium and E. faecalis as well as vancomycin resistance genes (vanA, vanB and vanD) in Enterococcus spp. were detected by multiplex PCR. Results. A total of 551 Enterococcus spp. strains were studied, of them 440 (79.9%) were E. faecium and 111 (20.1%) E. faecalis. Resistance to vancomycin was detected in 86 (19.5%) E. faecium, of them 62 (72.1%) carried vanA and 24 (27.9%) vanB genes. One (1.1%) of 111 E. faecalis was vancomycinintermediate (MIC 16 μg/ml) with vanD gene. Virulence genes were detected in 86.2% of Enterococcus spp., significantly more often among E. faecalis (95.5%) compared to E. faecium (83.9%, p = 0.003). The predominant genes in E. faecium were esp (70.2%) and hyl (52.1%), the detection of the asa1, cylA and gelE genes was minimal. Other genes dominated in E. faecalis: gelE (66.7%), asa1 (65.8%), cylA (36.9%). Statistically significant differences between E. faecium and E. faecalis were determined for all studied virulence genes (p < 0.0001). A combination of three or more virulence genes was detected significantly more often among E. faecalis in comparison with E. faecium (45% vs. 2.5%, p < 0.0001), whereas the presence of one gene or their absence prevailed in E. faecium (40.2% vs. 17.1%, p < 0.0001; 16.1% vs. 4.5%, p = 0.003, respectively). When comparing the two study periods (2002–2010 and 2011–2020), E. faecalis showed a significant increase in the proportion of strains with a combination of three or more virulence genes from 33.3% to 55% (p = 0.03), in E. faecium – with one virulence gene (from 31.7% to 46.3%, p = 0.002) and no genes (from 6.6% to 23%, p < 0.0001). Conclusions. Differences in the frequency of detection of virulence genes and their spectrum have been identified between E. faecium and E. faecalis strains. Virulence genes are significantly more often detected in E. faecalis with a predominance of combinations of three or more genes.

Molecular characterization of vancomycin-resistant Staphylococcus aureus isolated from bovine milk.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a zoonotic life-threatening pathogen. Vancomycin exhibits anti-bacterial activity by inhibiting peptidoglycan synthesis by binding to the D-ala-D-ala terminus of the peptidoglycan. But in VRSA, D-ala-D-ala is replaced by D-ala-D-lactate due to the presence of vanA, vanB or vanD genes. This study was intended to identify the molecular prevalence of VRSA in 768 bovine milk samples, risk factor association, antibiogram profile and bioinformatics analysis of VRSA by targeting vanB gene. Out of a total of 248 S. aureus isolates from mastitic milk samples, the phenotypic and genotypic prevalence of VRSA was estimated to be 17.74% and 10.89%, respectively. Farm-level risk factors including use of improper milking technique, lack of milker's care during milking, unhygienic conditions during milking and no dry cow therapy were found to be significantly associated (p < 0.05). Anti-microbial susceptibility testing of VRSA isolates exhibited the highest resistance to oxytetracycline, followed by oxacillin and Trimethoprim+sulfamethoxazole. The current study sequences showed more resemblance with reported sequences from Iraq (MN747834) and Egypt (MK095504, MK087830), which belong to vanB gene from S. aureus as compared to sequences from other countries, which belong to vanB gene from the genus Enterococcus. The Genetic Algorithm for Recombination Detection (GARD) found 234 potential breakpoints, translating into a search room of 123,883,305 models with up to 4 breakpoints. The phylogenetic motif profiling method discovered evolutionarily conserved residues across target genes' homologous protein sequences. These residues were discovered to be conserved in drug-resistant target proteins over the evolutionary process and may play a key role in their function. The current study revealed a molecular prevalence of VRSA in dairy animals, along with molecular analysis of vancomycin resistance in S. aureus by targeting the vanB gene using standard bioinformatics tools. The occurrence of VRSA in animals requires serious attention because this pathogen has zoonotic potential, so it can become a greater risk to consumer health.

Characterization of vancomycin-resistance vanD gene clusters in the human intestinal microbiota by metagenomics and culture-enriched metagenomics.

To characterize vancomycin-resistance vanD gene clusters and potential vanD-carrying bacteria in the intestinal microbiota of healthy volunteers exposed or not to β-lactam antibiotics. Stool samples were collected before and after 7 days of cefprozil β-lactam antibiotic exposure of 18 participants and six control participants who were not exposed to the antibiotic at the same time points. Metagenomic sequencing and culture-enriched metagenomic sequencing (with and without β-lactam selection) were used to characterize vanD gene clusters and determine potential vanD-carrying bacteria. Alteration by antimicrobials was also examined. Culture enrichment allowed detection of vanD genes in a large number of participants (11/24; 46%) compared to direct metagenomics (2/24; 8%). vanD genes were detected in stool cultures only following β-lactam exposure, either after β-lactam treatment of participants or after culture of stools with β-lactam selection. Six types of vanD gene clusters were identified. Two types of vanD cluster highly similar to those of enterococci were found in two participants. Other vanD genes or vanD clusters were nearly identical to those identified in commensal anaerobic bacteria of the families Lachnospiraceae and Oscillospiraceae and/or bordered by genomic sequences similar or related to these anaerobes, suggesting that they are the origin or carriers of vanD. This study showed that culture-enriched metagenomics allowed detection of vanD genes not detected by direct metagenomics and revealed collateral enrichment of bacteria containing vancomycin-resistance vanD genes following exposure to β-lactams, with a higher prevalence of the most likely gut commensal anaerobes carrying vanD. These commensal anaerobes could be the reservoir of vanD genes carried by enterococci.

Elucidation of host diversity of the VanD-carrying genomic islands in enterococci and anaerobes.

BackgroundVanD is a rare type of vancomycin resistance worldwide. However, the host diversity of the vanD gene cluster and the structural similarity of their genomic islands are not well understood.MethodsThree VanD-type Enterococcus faecium strains (AA620, AA622 and AA624) isolated from a Japanese patient who underwent vancomycin treatment in 2017 were analysed. This study utilized WGS analysis to characterize the three VanD-type E. faecium strains and describes the diversity of hosts possessing VanD-carrying genomic islands.ResultsThe three isolates exhibited variable MICs of vancomycin. In the relatively vancomycin-resistant AA620, mutations were identified in vanSD and ddl. The strains AA622 and AA624 had intact ddl and harboured two vanD gene clusters. qRT-PCR results revealed the ddl mutation to be a factor affecting the high vancomycin resistance range of AA620. WGS data showed the 155 kb and 185 kb genomic islands harbouring the vanD gene cluster inserted in the coding region of the lysS gene, located in the chromosome in AA620 and AA622/624, respectively. Comparing the VanD-carrying genomic islands to available sequences of other enterococci and enteric anaerobes revealed how the genomic islands of these organisms isolated worldwide shared similar core genes and backbones. These anaerobes belonged to various genera within the order Eubacteriales. The phylogenetic cluster of the genomic island core genome alignment did not correlate with the host-species lineage, indicating horizontal gene transfer in the gut microbiota.ConclusionsBy horizontal gene transfer, various bacteria forming the gut microbiota maintain VanD-carrying genomic islands.

Emergence of vancomycin- and teicoplanin-resistant Enterococcus faecium via vanD5-harbouring large genomic island.

Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.

Appearance of vanD-positive Enterococcus faecium in a tertiary hospital in the Netherlands: prevalence of vanC and vanD in hospitalized patients

Vancomycin-resistant enterococci (VRE) can rapidly spread through hospitals. Therefore, our hospital employs a screening program whereby rectal swabs are screened for the presence of vanA and vanB, and only PCR-positive broths are cultured on VRE selection agar. Early November 2016, a clinical vanA-/vanB-negative VRE isolate was detected in a vanA/vanB-screening-negative patient, giving the possibility that an undetected VRE might be spreading within our hospital. Whole-genome-sequencing of the isolate showed that resistance was vanD-mediated and core genome multilocus sequence typing showed it was a rare type: ST17/CT154. To determine the prevalence of vanA/B/C/D-carrying enterococci, we designed a real-time PCR for vanC1/2/3 and vanD and screened rectal swabs from 360 patients. vanD was found in 27.8% of the patients, yet culture demonstrated only E. faecium from vanA-positive broths and E. gallinarum from vanC1-positive broths. No vanD-positive VRE were found, limiting the possibility of nosocomial spread of this VRE. Moreover, the high prevalence of non-VRE vanD in rectal swabs makes it unfeasible to include the vanD PCR in our VRE screening. However, having validated the vanC1/2/3 and vanD PCRs allows us to rapidly check future vanA/B-negative VRE for the presence of vanC and vanD genes.

Antimicrobial resistance of nosocomial Enterococcus spp. isolated from blood culture in patients with hematological malignancies

Objective. To evaluate antimicrobial susceptibility of Enterococcus spp. isolated from blood culture in patients with hematological malignancies. Materials and Methods. Antimicrobial susceptibility of 427 Enterococcus spp. collected from 10 hospitals in 8 cities of Russia in 2002-2016 as part of the multicenter study was tested by the broth microdilution method [CLSI 2015]. Results. Among bloodstream pathogens there was a prevalence of E. faecium (78.2%), followed by E. faecalis (19.7%) and other Enterococcus spp. (2.1%). A total of 50 (15%) vancomycin-resistant E. faecium (Vancomycin-resistant Enterococcus – VRE) was detected, of them 33 (66%) were harboring vanA gene, 17 (34%) – vanB gene. In 2012 one linezolid resistant E. faecium (MIC = 16 µg/mL) was detected. Linezolid-resistant E. faecium contained the G2576T 23S rRNA mutation. All VRE faecium including the one linezolid-resistant isolate were susceptible to daptomycin. All E. faecium were susceptible to tigecycline, 78.7% – to chloramphenicol, 74.9% – to tetracycline. Proportion of E. faecium with high level resistance to gentamicin was 85%, to streptomycin – 60%, to both aminoglycosides – 45%. All E. faecalis were susceptible to linezolid, teicoplanin and tigecycline; 97.6% – to ampicillin. One isolate of E. faecalis (1.2%) with intermediate susceptibility to vancomycin (MIC = 16 µg/mL) harboring vanD gene and one isolate of E. gallinarum resistant to vancomycin, carrying vanC1 and vanB genes were detected. Conclusions. Isolates of E. faecalis had more favorable profile of antimicrobial susceptibility comparing to E. faecium. A total of 15% E. faecium were vancomycin resistant and one of them had resistance to linezolid. In this study one E. faecalis and one E. gallinarum isolates were non-susceptible to vancomycin.

Emergence of VanD-type vancomycin-resistant Enterococcus faecium in Stockholm, Sweden

A vancomycin-resistant Enterococcus faecium isolate from the urine of a liver transplant patient in Stockholm was found to contain a vanD gene. The sequence of the vanD PCR product shared 100% identity with the vanD5 allele. The isolate was resistant to a relatively high level of vancomycin (128 mg/L) and a low level of teicoplanin (4 mg/L). This is the first VanD-type vancomycin-resistant E. faecium isolate reported in Sweden. The emergence of this strain reinforces the necessity of infection control efforts to interrupt the spread of these organisms.