Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Flipse J

PROVIDER: S-EPMC6502811 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Flipse Jacky J von Wintersdorff Christian J H CJH van Niekerk Julius M JM Jamin Casper C van Tiel Frank H FH Hasman Henrik H van Alphen Lieke B LB

Scientific reports 20190506 1

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 ...[more]

PMID: 31061446

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Project description:Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Project description:We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.

Project description:A new open reading frame, encoding a putative integrase-like protein, was detected downstream from the six genes of the vanD glycopeptide resistance cluster in Enterococcus faecium BM4339 (B. Casadewall and P. Courvalin, J. Bacteriol. 181:3644-3648, 1999). In this cluster, genes coding for the VanR(D)-VanS(D) two-component regulatory system were cotranscribed from the P(R(D)) promoter, whereas transcription of the vanY(D), vanH(D), vanD, vanX(D), and intD genes was initiated from the P(Y(D)) promoter located between vanS(D) and vanY(D) (the D subscript indicates that the gene is part of the vanD operon). The VanR(D)-VanS(D) regulatory system is likely to activate transcription of the resistance genes from the promoter P(Y(D)). Glycopeptide-susceptible derivatives of BM4339 were obtained by trans complementation of the frameshift mutation in the ddl gene, restoring functional D-alanine:D-alanine ligase activity in this strain. The glycopeptide-susceptible transformant BM4409, producing only D-alanyl-D-alanine-terminating peptidoglycan precursors, did not express the resistance genes encoding the VanY(D) D,D-carboxypeptidase, the VanH(D) dehydrogenase, the VanD ligase, the VanX(D) D,D-dipeptidase, and also the IntD integrase, although the regulatory region of the vanD cluster was still transcribed. In BM4409, the absence of VanR(D)-VanS(D), apparently dependent, transcription from promoter P(Y(D)) correlated with the lack of D-alanyl-D-lactate-terminating precursors. The vanX(D) gene was transcribed in BM4339, but detectable amounts of VanX(D) D,D-dipeptidase were not synthesized. However, the gene directed synthesis of an active enzyme when cloned on a multicopy plasmid in Escherichia coli, suggesting that the enzyme was unstable in BM4339 or that it had very low activity that was detectable only under conditions of high gene dosage. This activity is not required for glycopeptide resistance in BM4339, since this strain cannot synthesize D-alanyl-D-alanine.

Dataset Information

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

Publications

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

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