Continuous Assay for VanX, the d-Alanyl–d-Alanine Dipeptidase Required for High-Level Vancomycin Resistance

1999 ◽  
Vol 272 (1) ◽  
pp. 94-99 ◽  
Author(s):  
Jeffrey J. Brandt ◽  
Lisa L. Chatwood ◽  
Ke-Wu Yang ◽  
Michael W. Crowder
Keyword(s):  
Vancomycin Resistance ◽  
High Level ◽  
Continuous Assay

scholarly journals Lack of Relationship between Purine Biosynthesis and Vancomycin Resistance in Staphylococcus aureus: a Cautionary Tale for Microarray Interpretation

2007 ◽  
Vol 51 (4) ◽  
pp. 1274-1280 ◽  
Author(s):  
Paige M. Fox ◽  
Michael W. Climo ◽  
Gordon L. Archer
Keyword(s):  
Staphylococcus Aureus ◽  
Vancomycin Resistance ◽  
Purine Metabolism ◽  
Purine Biosynthesis ◽  
Cautionary Tale ◽  
Apparent Relationship ◽  
Purine Pathway ◽  
High Level ◽  
Microarray Studies

ABSTRACT Previous microarray data (E. Mongodin, J. Finan, M. W. Climo, A. Rosato, S. Gill, and G. L. Archer, J. Bacteriol. 185:4638-4643, 2003) noted an association in two vancomycin-intermediate Staphylococcus aureus (VISA) strains between high-level, passage-induced vancomycin resistance, a marked increase in the transcription of purine biosynthetic genes, and mutation of the putative purine regulator purR. Initial studies to report on the possible association between vancomycin resistance and alterations in purine metabolism in one of these strains (VP-32) confirmed, by Western analysis, an increase in the translation of PurH and PurM, two purine pathway enzymes. In addition, PurR was identified, by knockout and complementation in a vancomycin-susceptible strain, as a repressor of the purine biosynthetic operon in S. aureus, and the PurR missense mutation was shown to inactivate the repressor. However, despite the apparent relationship between increased purine biosynthesis and increased vancomycin resistance in VP-32, neither the addition of exogenous purines to a defined growth medium nor the truncation or inactivation of purR improved the growth of vancomycin-susceptible S. aureus in the presence of vancomycin. Furthermore, the passage of additional vancomycin-susceptible and VISA strains to high-level vancomycin resistance occurred without changes in cellular purine metabolism or mutation of purR despite the development of thickened cell walls in passaged strains. Thus, we could confirm neither a role for altered purine metabolism in the development of vancomycin resistance nor its requirement for the maintenance of a thickened cell wall. The failure of biochemical and physiological studies to support the association between transcription and phenotype initially found in careful microarray studies emphasizes the importance of follow-up investigations to confirm microarray observations.

scholarly journals Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255187
Author(s):  
Mushtaq T. S. AL Rubaye ◽  
Jessin Janice ◽  
Jørgen Vildershøj Bjørnholt ◽  
Aleksandra Jakovljev ◽  
Maria Elisabeth Hultström ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Vancomycin Resistance ◽  
Genomic Islands ◽  
Vancomycin Resistant Enterococci ◽  
Chromosomal Site ◽  
Enterococcus Casseliflavus ◽  
Temporal Occurrence ◽  
Vancomycin Resistant ◽  
High Level

Background Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. Methods The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. Results The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64—>256 mg/L) and various levels of teicoplanin susceptibility (MIC 2—>256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. Conclusions We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.

scholarly journals Vancomycin and high-level aminoglycoside resistance in Enterococcus species

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Seyda Ozarslan Kurtgoz ◽  
Burcin Ozer ◽  
Melek Inci ◽  
Nizami Duran ◽  
Erkan Yula
Keyword(s):  
Vancomycin Resistance ◽  
Automated System ◽  
Diffusion Method ◽  
Beta Lactamase ◽  
Aminoglycoside Resistance ◽  
Disk Diffusion Method ◽  
Gradient Diffusion ◽  
Gentamicin Resistance ◽  
Polymerase Chain ◽  
High Level

The aim of the study was to investigate vancomycin and high-level aminoglycoside resistance (HLAR) in <em>Enterococcus</em> species by phenotypic and genotypic methods. A hundred <em>Enterococcus</em> strains were included in the study. Antimicrobial susceptibilities of strains were investigated by automated system, betalactamase production was investigated by nitrocefin disks, vancomycin resistance and HLAR were investigated by gradient diffusion method (GDM) and disk diffusion method, respectively. For detection of vancomycin and high-level gentamicin resistance (HLGR) genes, polymerase chain reaction was used. Teicoplanin linezolid, vancomycin, ampicillin, penicillin are the most susceptible antibiotics and strains were detected not to produce beta lactamase. Vancomycin resistance was detected in ten isolates by automated system and in only five isolates by GDM. Five isolates carrying <em>VanA</em> gene were determined. The ratio of HLGR and high-level streptomycin resistance was found 40 and 63% respectively. <em>aac (6’)-1eaph (2’’)-1a</em> gene was detected in 58% of strains. <em>E. faecium</em> strains were found more resistant to the antibiotics than the other species. Beta lactamase was detected in none of strains. The automated system detected vancomycin resistance in more strains than GDM. Therefore it is concluded that strains, which were detected to be resistant to vancomycin, should be confirmed by GDM. The ratio of <em>VanA</em> gene in strains is consistent with other studies. The HLAR ratio was found in about half of strains. The ratio of<em> aac(6’)-1e-aph(2’’)-1a</em> gene, which is the most reported gene in our country and other countries and one of the HLGR genes investigated in our study, was detected 58%.

scholarly journals Penicillin-Binding Protein 2 Is Essential for Expression of High-Level Vancomycin Resistance and Cell Wall Synthesis in Vancomycin- Resistant Staphylococcus aureus Carrying the Enterococcal vanA Gene Complex

2004 ◽  
Vol 48 (12) ◽  
pp. 4566-4573 ◽  
Author(s):  
Anatoly Severin ◽  
Shang Wei Wu ◽  
Keiko Tabei ◽  
Alexander Tomasz
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Binding Protein ◽  
Inducible Promoter ◽  
Protein Product ◽  
Vancomycin Resistance ◽  
Penicillin Binding Protein ◽  
Oxacillin Resistance ◽  
Vancomycin Resistant ◽  
High Level

ABSTRACT A combination of biochemical and genetic experiments were performed in order to better understand the mechanism of expression of high-level vancomycin resistance in Staphylococcus aureus. The transcription of pbp2 of the highly vancomycin- and oxacillin-resistant strain COLVA200 and its mutant derivative with inactivated mecA were put under the control of an inducible promoter, and the dependence of oxacillin and vancomycin resistance and cell wall composition on the concentration of the isopropyl-β-d-thiogalactopyranoside inducer was determined. The results indicate that mecA—the genetic determinant of oxacillin resistance—while essential for oxacillin resistance, is not involved with the expression of vancomycin resistance. Penicillin binding protein 2A, the protein product of mecA, appears to be unable to utilize the depsipeptide cell wall precursor produced in the vancomycin-resistant cells for transpeptidation. The key penicillin binding protein essential for vancomycin resistance and for the synthesis of the abnormally structured cell walls characteristic of vancomycin-resistant S. aureus (A. Severin, K. Tabei, F. Tenover, M. Chung, N. Clarke, and A. Tomasz, J. Biol. Chem. 279:3398-3407, 2004) is penicillin binding protein 2.

scholarly journals High-level vancomycin-resistant enterococci causing hospital infections

1989 ◽  
Vol 103 (1) ◽  
pp. 173-181 ◽  
Author(s):  
A. H. C. Uttley ◽  
R. C. George ◽  
J. Naidoo ◽  
N. Woodford ◽  
A. P. Johnson ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Vancomycin Resistance ◽  
Recipient Strain ◽  
Hospital Infections ◽  
Minimal Inhibitory Concentrations ◽  
Vancomycin Resistant Enterococci ◽  
Vancomycin Resistant ◽  
Many Sources ◽  
High Level ◽  
Level Resistance

SUMMARYNosocomial infection or colonization due to enterococci with high-level resistance to vancomycin (minimal inhibitory concentrations [MICs] between 64 and > 2000 mg/L) has occurred in 41 patients with renal disease. These vancomycin-resistant enterococci were cultured from many sources including blood. All but one strain contained one or more plasmids ranging in molecular weight from 1·0 to 40 Megadaltons (MDa). Vancomycin resistance was transferable by conjugation to a susceptible recipient strain ofEnterococcus faecalisbut this was not always associated with plasmid DNA. The emergence of transferable high-level vancomycin resistance in enterococci causing significant clinical infections is of particular importance since vancomycin is widely regarded as a reserve drug for the management of infections with multi-resistant Gram-positive organisms.

scholarly journals Failure of VITEK2 to reliably detect vanB- mediated vancomycin resistance in Enterococcus faecium

2020 ◽  
Author(s):  
Sarah V. Walker ◽  
Martina Wolke ◽  
Georg Plum ◽  
Robert E. Weber ◽  
Guido Werner ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Vancomycin Resistance ◽  
Bacterial Suspension ◽  
Initial Growth ◽  
Low Level ◽  
Test Kit ◽  
Before And After ◽  
Vancomycin Resistant ◽  
High Level ◽  
Level Resistance

AbstractObjectivesThe increasing prevalence of vancomycin resistant enterococci (VRE) necessitates a reliable detection of VRE especially for low level resistance mediated by vanB in Enterococcus faecium. In this prospective study we analyzed if vanB mediated vancomycin resistance can be reliably detected by Vitek2.Methods1344 enterococcal isolates from routine clinical specimens were tested by Vitek2 (bioMérieux, Nürtingen, Germany). Additionally, a bacterial suspension (0.5 McFarland) was inoculated on a chromID VRE screening agar (bioMérieux) and incubated for 48 hours. If vancomycin was tested susceptible by Vitek2 but growth was detected on the screening agar a PCR for vanA/vanB was performed (GeneXpert vanA/B test kit, Cepheid, Frankfurt, Germany). MICs of vancomycin susceptible by Vitek but vanA/B positive isolates were determined before and after cultivation in a broth with increasing concentration of vancomycin.Results156/492 of E. faecium were VRE, predominantly vanB (87.0%) of which 14 were not identified as VRE by Vitek2 (sensitivity 91.0%). The majority (9/14) demonstrated high-level MICs by broth dilution. Even after exposure to increasing vancomycin concentrations MICs remained nearly identical. Three of the undetected isolates demonstrated initial growth on chromID VRE, after the vancomycin exposure additional 7 isolates demonstrated growth on chromID VRE.ConclusionsVitek2 fails to detect vanB mediated vancomycin resistance consistently, especially but not limited to low-level resistance. As this may lead to treatment failure and further dissemination of vanB VRE, additional methods (e.g. culture on VRE screening agar or PCR) are necessary to reliably identify vanB-positive enterococci in clinical routine.

scholarly journals Characterization of an Enterococcus gallinarum Isolate Carrying a DualvanAandvanBCassette

2015 ◽  
Vol 53 (7) ◽  
pp. 2225-2229 ◽  
Author(s):  
Alireza Eshaghi ◽  
Dea Shahinas ◽  
Aimin Li ◽  
Ruwandi Kariyawasam ◽  
Philip Banh ◽  
...  
Keyword(s):  
Clinical Isolate ◽  
Resistance Mechanism ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Enterococcal Species ◽  
Enterococcus Gallinarum ◽  
High Level ◽  
Identification And Characterization ◽  
Level Resistance

The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate ofEnterococcus gallinarumexhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence ofvanAandvanBgenes in addition to the naturally carriedvanCgene.vanAwas identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. ThevanBoperon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn1549and Tn5382. To the best of our knowledge, this is the first report ofE. gallinarumcarrying bothvanAandvanBoperons, indicating the importance of identifying the vancomycin resistance mechanism in non-E. faeciumand non-E. faecalisenterococcal species.

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