scholarly journals Effect of vancomycin on cytoplasmic peptidoglycan intermediates and van operon mRNA levels in VanA-type vancomycin resistant Enterococcus faecium

2021 ◽  
Author(s):  
Shivani Gargvanshi ◽  
Harika Vemula ◽  
William G. Gutheil
Keyword(s):  
Enterococcus Faecium ◽  
Protein Biosynthesis ◽  
Alternative Pathway ◽  
Resistance Mechanism ◽  
Gene Cassette ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Liquid Chromatograph ◽  
Vana Gene ◽  
Vancomycin Resistant

Resistance in VanA-type vancomycin resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins ( vanRSHAXYZ ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby d- Alanyl- d- Alanine ( d -Ala- d -Ala) is replaced with d- Alanyl- d- Lactate ( d -Ala- d -Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatograph tandem mass spectrometry before and after vancomycin exposure, and to correlate these changes with changes in vanA operon mRNA levels measured by RT-qPCR. Normal pathway intermediates predominate in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18 hr) vancomycin exposure resulted in a mix of the terminal normal (UDP-Penta) and alternative (UDP-Pentadepsi) pathway intermediates (2:3 ratio). Time courses reveal normal pathway intermediates responding rapidly (peaking in 3-10 minutes), and alternative pathway intermediates responding more slowly (peaking in 15-45 minutes). RT-qPCR demonstrated that vanA operon mRNA transcript levels increase rapidly after exposure, reaching maximal levels in 15 minutes. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin sensitive Enterococcus faecium (VSEfm), and methicillin resistant Staphylococcus aureus (MRSA) after vancomycin exposure demonstrate substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistance to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode for enzymes that provide for an alternative peptidoglycan (PG) biosynthesis pathway. In VanA-type resistance these alternative pathway enzymes replace the d -Ala- d -Ala terminus of normal PG intermediates with d -Ala- d -Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, VanA gene induction, and changes in the normal and alternative pathway intermediate levels has not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture

2020 ◽  
Author(s):  
Kyriaki Xanthopoulou ◽  
Julia Wille ◽  
Janine Zweigner ◽  
Kai Lucaßen ◽  
Thorsten Wille ◽  
...  
Keyword(s):  
Blood Culture ◽  
Enterococcus Faecium ◽  
Core Genome ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Vana Gene ◽  
Plasmid Analysis ◽  
Vancomycin Resistant ◽  
Faecium Isolate

Abstract Objectives To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. Methods The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Results Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Conclusions Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

scholarly journals Detection of clonally related vanB2-containing Enterococcus faecium strains in two Spanish hospitals

2006 ◽  
Vol 55 (9) ◽  
pp. 1237-1243 ◽  
Author(s):  
Carmen Torres ◽  
Susanna Escobar ◽  
Aránzazu Portillo ◽  
Luis Torres ◽  
Antonio Rezusta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Intergenic Region ◽  
Enterococcus Faecium ◽  
Resistance Mechanism ◽  
Vancomycin Resistance ◽  
Pfge Pattern ◽  
Genetic Environment ◽  
Vancomycin Resistant

The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16–32 and 0.5 μg ml−1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanS B–Y B region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanX B–ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanX B gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6′)-Ia, aph(3′)-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.

scholarly journals Complete Genome Sequences of Four Isolates of Vancomycin-Resistant Enterococcus faecium with the vanA Gene and Two Daptomycin Resistance Mutations, Obtained from Two Inpatients with Prolonged Bacteremia

2020 ◽  
Vol 9 (6) ◽  
Author(s):  
Piroon Jenjaroenpun ◽  
Thidathip Wongsurawat ◽  
Zulema Udaondo ◽  
Courtney Anderson ◽  
James Lopez ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Complete Genome ◽  
De Novo ◽  
Sequence Data ◽  
Resistance Mutations ◽  
Genome Sequences ◽  
Content Type ◽  
Vana Gene ◽  
Illumina Sequence ◽  
Vancomycin Resistant

Here, we present complete genome sequences of four Enterococcus faecium isolates, obtained from two patients with apparent vancomycin-resistant Enterococcus faecium bacteremia; these isolates also carried two mutations known to be associated with daptomycin resistance. Sequences were obtained using de novo and hybrid assembly of Oxford Nanopore and Illumina sequence data.

scholarly journals Whole Genome Sequencing of Vancomycin Resistant Enterococcus faecium Isolated from Saudi Arabia

2018 ◽  
Vol 15 (4) ◽  
pp. 791-797
Author(s):  
Mushtaq Ahmad Khan ◽  
Fauwaz Al-Rashid ◽  
Mamdoh Meqdam ◽  
Hisham Al-Ajlan ◽  
Mohammed Al-Mogbel
Keyword(s):  
Saudi Arabia ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Enterococcus Faecium ◽  
Gene Cassette ◽  
Multidrug Resistant ◽  
Maternity Hospital ◽  
Whole Genome ◽  
Healthcare Facilities ◽  
Vancomycin Resistant

Enterococcus faecium are one of the most prevalent species cultured from humans and they have become increasingly common cause of infections in the hospital settings globally. The objective of current study was to characterize 26 E. faecium isolates collected from different patients attending Maternity Hospital in Ha’il, Saudi Arabia. The bacterial isolates were identified by MALDI-TOF-MS and the antibiotic susceptibility was performed by Microscan. Whole genome sequencing of a single vancomycin resistant E. faecium (VRE) was performed using MiSeq. The results of antimicrobial susceptibility revealed that, 99%, 90%, 83% and 73% of isolates were resistant to Clindamycin, Gentamicin, Oxacillin and Tetracycline respectively. One (3%) among 26 E. faecium isolates was found to produce resistance to vancomycin. The WGS analysis of VRE showed that it belonged to ST280 and was found to harbor vanB gene cassette. This is the first report of VRE from the Ha’il region of Saudi Arabia. VRE may act as a reservoir for multidrug resistant genes and other important virulence factors that favor the dissemination of antimicrobial resistance. Therefore, the surveillance studies to prevent dissemination of VRE shall be implemented in the healthcare facilities all across Saudi Arabia.

scholarly journals 1629. Vancomycin Resistance in Enterococcus faecium Clinical Isolates Responsible for Bloodstream Infections in US Hospitals Over Ten Years (2010-2019) and Activity of Oritavancin

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S806-S807
Author(s):  
Cecilia G Carvalhaes ◽  
Helio S Sader ◽  
Jennifer M Streit ◽  
Mariana Castanheira ◽  
Rodrigo E Mendes
Keyword(s):  
Enterococcus Faecium ◽  
Bloodstream Infections ◽  
Chemical Diversity ◽  
Intrinsic Resistance ◽  
Services Research ◽  
Vana Gene ◽  
Department Of Health ◽  
Common Phenotype ◽  
Center Research ◽  
Research Grant

Abstract Background Enterococcus faecium (EFM) causes difficult-to-treat infections due to its intrinsic resistance (R) and ability to acquire R to many antimicrobials. This study evaluated the vancomycin (VAN)-R rates over time and the activity of oritavancin (ORI) against a collection of EFM causing bloodstream infections (BSI). Methods A total of 1,081 BSI EFM isolates collected from 36 US hospitals in a prevalence mode design during 2010-2019 were evaluated. Bacterial identification was confirmed by MALDI-TOF MS. Susceptibility testing was performed by reference broth microdilution. For comparison, the ORI breakpoint for VAN-susceptible E. faecalis was applied to EFM. Isolates were characterized as VanA or VanB phenotypes based on their susceptibility (S) to VAN and teicoplanin (TEC). The VanB phenotype was confirmed by PCR and/or whole genome sequencing. Results Overall, 72.3% (782/1,081) of EFM were VAN-R (Table). VanA was the most common phenotype (97.7%; 764/782). The yearly VAN-R rates decreased from 81.8% in 2010 to 58.7% in 2019. A total of 18 (2.3%) isolates exhibited a VanB phenotype (TEC MIC, 0.5-8 mg/L); however, the vanB gene only was confirmed in 9 EFM isolates (TEC MIC, 0.5-1 mg/L), which were all collected in 2010-2012. The remaining 9 (50.0%) VanB phenotype EFM isolates carried a vanA gene (TEC MIC, 4-8 mg/L). ORI was very active against VAN-susceptible EFM (MIC50/90, ≤ 0.008/≤0.008/mg/L), VanA (MIC50/90, 0.03/0.12 mg/L; MIC100, 0.5 mg/L), and VanB (MIC50/90, ≤ 0.008/0.015 mg/L; MIC100, 0.03 mg/L) subsets. Only linezolid (LZD) and ORI (MIC, ≤ 0.12 mg/L) showed > 95.0%S against EFM and VAN-R subsets. Daptomycin (DAP)-R rarely was observed (0.8%), but it was more frequently found in the last 5 years. However, 49.9% of EFM isolates showed elevated DAP MICs (2 and 4 mg/L). ORI inhibited 77.8%, and 100.0% of DAP-R and LZD-nonsusceptible EFM isolates at ≤ 0.12 mg/L, respectively. Conclusion VAN-R rates among EFM causing BSI in the US decreased during 2010-2019. VanA remains the most common phenotype, whereas vanB-carrying isolates became rarer in later years. Interestingly, half of VanB-phenotype isolates carried a vanA gene. ORI was very active against EFM causing BSI, including isolates R to VAN, DAP, and/or nonsusceptible to LZD. Table 1 Disclosures Cecilia G. Carvalhaes, MD, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Pfizer (Research Grant or Support) Helio S. Sader, MD, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Melinta (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support) Jennifer M. Streit, BS, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support) Mariana Castanheira, PhD, 1928 Diagnostics (Research Grant or Support)A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Merck & Co, Inc. (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support)Pfizer (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Rodrigo E. Mendes, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Basilea Pharmaceutica International, Ltd (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Pfizer (Research Grant or Support)

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