Regulated interactions between partner and non-partner sensors and response regulators that control glycopeptide resistance gene expression in enterococci

Arthur, Michel; Depardieu, Florence; Courvalin, Patrice

doi:10.1099/13500872-145-8-1849

Cited by 52 publications

(65 citation statements)

References 20 publications

(16 reference statements)

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“…The H1-H2 DNA fragment used for deletion of pbp5 was constructed by two sequential amplifications with partially complementary primers as previous described (1). In the first step, the H1 and H2 fragments were separately amplified (primers H1F, 5Ј-AGAATCATTTTTGACTG-3Ј, and H1R, 5Ј-CAAATGGTT CGCTGGGTTTCAATAATCCCCTAAC-3Ј, for H1; primers H2Fb, 5Ј-ACCCA GCGAACCATTTGAAAAGAGAAAATGAACG-3Ј, and H2R, 5Ј-AGGGAA ATATGTTGGTC-3Ј, for H2).…”

Section: Methodsmentioning

confidence: 99%

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis

et al. 2004

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Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classesA and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of ␤-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 ⌬ponA ⌬pbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of ␤-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

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Section: Methodsmentioning

confidence: 99%

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis

et al. 2004

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“…Treatment of one of the patients with teicoplanin was probably responsible for the selection of a VREM strain variant, which due to the deletion of a large part of the vanB gene cluster was also resistant to teicoplanin. This finding, together with data from other laboratories, indicates that various genetic changes may determine resistance to teicoplanin in VanB enterococci and thus limit its use against these microorganisms (2,6,33,36).…”

Section: Discussionmentioning

confidence: 83%

“…that probably resulted from taking over the VanS B functions by a host kinase (2,4,5,6,7). Deletion mutants lacking the whole VanR B -VanS B regulatory system and the P YB promoter have not been, however, observed to date.…”

Section: Discussionmentioning

confidence: 99%

“…However, the protein was not studied in detail, and it is not known what modifications of the vanB gene cluster were responsible for the phenotype. On the other hand such mutants have been often obtained in the laboratory, including selection in an animal model, and all those studied down to the molecular level were found to result from point mutations in the vanS B gene (2,4,5,6,7,25,33). Some of these changes caused the VanS B ability to recognize teicoplanin as the expression inducer, whereas others abolished its phosphatase activity, which led to the permanent phosphorylation of VanR B and constitutive transcription of the resistance genes.…”

Section: Discussionmentioning

confidence: 99%

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Selection of a Teicoplanin-Resistant Enterococcus faecium Mutant during an Outbreak Caused by Vancomycin-Resistant Enterococci with the VanB Phenotype

Kawalec

Gniadkowski²,

Kędzierska³

et al. 2001

J Clin Microbiol

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Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR B and -S B , and the vancomycin-inducible promoter P YB . Expression of the resistance genes vanH B , -B, and -X B was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

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“…The ligase gene was identical to that of VRE, with no gross structural changes in the vanB operon based on the size of the PCR product obtained. Subtle sequence or functional changes in the operon, particularly in the vanS/vanR regulatory system, may account for the vancomycin-susceptible phenotype observed with LM-VRE (4), as might operon copy number and the presence of remnant fragments within the genome of these isolates (3,11,21).…”

Section: Discussionmentioning

confidence: 99%

Improved Detection of vanB2 -Containing Enterococcus faecium with Vancomycin Susceptibility by Etest Using Oxgall Supplementation

Grabsch¹,

Chua

Xie³

et al. 2008

J Clin Microbiol

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We have isolated a number of vanB-containing Enterococcus faecium isolates on bile esculin screening agar containing 6 mg/liter vancomycin, which on subsequent susceptibility testing using Etest have repeatedly demonstrated vancomycin MICs of <4 mg/liter. To investigate this genotype-phenotype incongruence of "low-MIC vancomycin-resistant enterococci" (LM-VRE), we examined the molecular characteristics of these isolates, including the presence of the vanB operon, using PCR amplification and DNA sequencing. All LM-VRE isolates contained vanB associated with the transposon Tn1549 and were polyclonal. Sequencing of the vanB ligase gene showed no differences from the prototype vanB2. In addition, we examined supplemented media to improve phenotypic detection of these isolates. Etest detection of LM-VRE improved when Mueller-Hinton agar (MHA) and brain heart infusion agar (BHIA) were supplemented with 10 g/liter oxgall (MHA-Oxg and BHIA-Oxg, respectively). We assessed the sensitivity and specificity of these media to detect vancomycin resistance using vancomycin-resistant vanB-containing E. faecium (n ‫؍‬ 11), vancomycin-susceptible (van negative) E. faecium (n ‫؍‬ 11), vancomycinsusceptible (van negative) E. faecalis (n ‫؍‬ 11), and our LM-VRE (n ‫؍‬ 23) isolates. After 48 h of incubation, both MHA-Oxg and BHIA-Oxg were 100% (34/34) sensitive and 100% (22/22) specific in the identification of vancomycin resistance. These findings suggest that supplementation of MHA or BHIA with 10 g/liter oxgall should be considered in laboratories where VRE detection protocols rely primarily on strain phenotype rather than early vanB gene detection by PCR.Vancomycin-resistant enterococci (VRE) are significant nosocomial pathogens worldwide, and both vanA and vanB strains of VRE may result in serious infections, including bacteremia (15, 23). In Australia, unlike the United States and Europe, vanB-containing Enterococcus faecium predominates (7,18). vanB-containing isolates show variable phenotypic resistance to vancomycin (MIC, 4 to 1,024 mg/liter) and can therefore be more difficult to detect than vanA-containing VRE that readily demonstrate high-level resistance to vancomycin (9,10,14,20).Both the vanA and vanB operons are associated with transposons, and transfer of resistance among enterococci appears to be mediated via acquisition of these elements (22). The vanA operon is located on the transposon Tn1546, while three transposons, Tn1547, Tn5382, and Tn1549, have been described which contain the vanB operon; sequencing data suggest Tn1549 is essentially identical to Tn5382 (22).Using Enterococcosel agar (BBL, Sparks, MD) containing 6 mg/liter vancomycin (EVA) for VRE screening, we have identified a number of vanB-containing E. faecium isolates that on subsequent susceptibility testing using the standard Etest (AB Biodisk, Solna, Sweden) method (inoculum of 0. We present here a description of the resistance genotypephenotype incongruence of these variants of VRE, including molecular characteristics. These LM-VRE strains can be ...

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Regulated interactions between partner and non-partner sensors and response regulators that control glycopeptide resistance gene expression in enterococci

Cited by 52 publications

References 20 publications

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis

Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis

Selection of a Teicoplanin-Resistant Enterococcus faecium Mutant during an Outbreak Caused by Vancomycin-Resistant Enterococci with the VanB Phenotype

Improved Detection of vanB2 -Containing Enterococcus faecium with Vancomycin Susceptibility by Etest Using Oxgall Supplementation

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