VanA-type Enterococcus Faecium Research Articles

Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediate Levels In Resistant Enterococcus faecium

Vancomycin is a natural glycopeptide antibiotic and is used for the treatment of multi-drug resistant Gram positive bacteria. However, resistance to vancomycin has emerged. Vancomycin resistance in VanA-type Enterococcus faecium is due to a vancomycin resistance cassette comprised of vanH, vanX, vanY, vanZ, vanR and vanS genes. These genes encode for an alternative peptidoglycan biosynthesis pathway in which D-Ala-D-Ala is replaced with D-Ala-D-Lac. Vancomycin binds with a lower affinity to D-Ala-D-Lac as it does to the nomal dipeptide. In this study, several of the unique UDP-linked intermediates from VanA-type vancomycin resistant Enterococcus faecium (VRE) were preparatively purified from vancomycin treated cultures and used to optimize their LC-MS/MS quantification. Cytoplasmic pool levels were then measured in VRE before and after vancomycin exposure. VRE in the absence of vancomycin had low but readily detectible basal levels of VanA pathway intermediates. After a 15 min vancomycin exposure, normal pathway intermediates were only modestly affected, whereas alternative pathway intermediates increased substantially. Alternative pathway intermediates then decreased back toward unexposed levels after longer vancomycin exposure. A vancomycin concentration profile demonstrated a midpoint of vancomycin effect between 1 and 4 μg/mL. Time course experiments revealed a complex response to vancomycin exposure, with most normal pathway intermediates responding rapidly (minutes) to vancomycin exposure, and alternative pathway intermediates increasing for 15-20 min and then decreasing partially back toward normal levels. qRT-PCR study also demonstrated that mRNA levels of VanA pathway genes also took about 15 min to reach their maximal level of induction.

DdcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism to reduce vancomycin MIC levels. Overall, our work identified factors that alter chlorhexidine and vancomycin synergism in a model VanA-type VRE strain.

In vitro activity of oritavancin alone or in combination against vancomycin-susceptible and -resistant enterococci.

The optimal treatment for serious infections due to Enterococcus spp. is unknown although combination antimicrobial therapy is often recommended for invasive infections to achieve bactericidal activity and improve clinical outcomes. Oritavancin is a novel lipoglycopeptide agent with in vitro activity against enterococci, including vancomycin-resistant VanA-type Enterococcus faecium. Data on its activity in combination with other antibacterials are limited. The objective of this study was to evaluate the activity of oritavancin alone and in combination with ceftriaxone, daptomycin, gentamicin, linezolid and rifampicin against vancomycin-susceptible and -resistant enterococci in in vitro time-kill analyses. Five enterococcal strains were used for all experiments: three vancomycin-resistant VanA-type E. faecium clinical bloodstream isolates, vancomycin-resistant VanA-type E. faecium ATCC 700221 and vancomycin-susceptible Enterococcus faecalis ATCC 29212. Individual drugs were tested at ¼, ½, 1, 2 and 4× MIC. Oritavancin combination experiments were performed with each agent at ¼× MIC. Daptomycin was the most active single agent and was bactericidal against all strains at 4× MIC, followed by oritavancin, which was bactericidal against all three clinical VRE strains at ≥2× MIC. In combination experiments at ¼× MIC, oritavancin was synergistic with gentamicin against strains not displaying high-level aminoglycoside resistance. No other synergy against VRE strains was observed in any experiment. Strain- and drug-dependent antagonism was observed for many combinations. These in vitro data do not support the routine use of combination therapy with oritavancin in the treatment of infections due to VRE.

Diversity of vancomycin-resistant enterococci in a low endemicity area.

Although vancomycin-resistant enterococci (VRE) are frequent nosocomial pathogens worldwide, in Japan their prevalence is low and their molecular epidemiology remains unclear. In The University of Tokyo Hospital (Tokyo, Japan), only five isolates of VRE (vanA-type Enterococcus faecium) were identified in the 20 years before 2010; however, nine isolates of vanB-type E. faecium were identified from 2011 to 2012. Multilocus sequence typing (MLST) revealed that they belonged to five sequence types (ST18, ST78, ST203, ST412 and ST612). Despite the limited isolation, including one from a medical tourist, there was significant diversity in genotypes. Increasing medical travel might enhance the diversity of VRE.

Persistence ofvanA-TypeEnterococcus faeciumin Korean Livestock After Ban on Avoparcin

Prevalence of vancomycin-resistant enterococci (VRE) was investigated in Korean livestock 4 years after the ban of avoparcin in feed additives. VRE were isolated from approximately 16.7% of the chicken samples (57 strains from 342 meat samples) and 1.9% of the pig samples (4 from 214 fecal samples). No VRE, however, was isolated from 110 bovine fecal samples. All the 61 VRE isolates were vanA-type Enterococcus faecium expressing a high-level resistance to vancomycin, and showed resistance to teicoplanin as well except two poultry isolates. In addition, the VRE isolates had heterogeneous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, although identical or closely related profiles were observed among strains isolated from the same farm. Although the chicken isolates were all poultry type with G at position 8,234 of the vanX gene, the pig isolates were all swine type with T at position 8,234 of the vanX gene.

Outbreak of Vancomycin-Resistant Enterococci (VRE) in a Hematological Oncology Ward and Hygienic Preventive Measures. A Long-Term Study

Background: We report on two endemic outbreaks ofVRE (vancomycin-resistant enterococci, vanA type Ente-rococcusfaecium) on a hematological oncology ward ina university hospital and a new strategic concept to fightagainst spread of VRE. Patients and Methods: Duringmanagement of the outbreak, a total of 1,124 patientsand 1,700 specimens were investigated from June 1999to December 2001. Protective measures were instituted.Intensive prophylactic infection control was practiceduntil December 2003. Results: Of the 1,124 patients,44 were VRE-positive. From 1,700 environment speci-mens,110 VRE isolates were obtained from the inani-mateenvironment, 5 from the living environment. Mole-cularbiological typing identified 5 different clones whichhad spread on the ward in different locations. Conclu-sion:The study shows the need for prophylactic microbi-ologicalscreening investigations in the risk group ofhematological oncology patients and consistent imple-mentationof protective hygiene measures to prevent thespread of multiresistant infectious pathogens on riskwards. It is also shown that intervention can to controlsuch an endemic outbreak.

A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand.

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.