John Merlino1,2*, Kiora Pillay1, Sophia Rizzo1, Daniel Seed1, Steven Siarakas1 and Timothy Gray1,3
1Department of Microbiology and Infectious Diseases, NSW Health Pathology, Concord Hospital, Australia
2Department of Infectious Disease and Immunology, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Australia
3Concord Clinical School, Faculty of Medicine and Health, University of Sydney, Australia
*Corresponding Author: John Merlino, Department of Microbiology and Infectious Diseases, NSW Health Pathology, Concord Hospital, Australia
Received: August 27, 2024; Published: September 17, 2024
Citation: John Merlino., et al. “Vancomycin-Variable Enterococcus faecium ST1424: Containing the vanA gene with the vanS, vanR and vanH gene without vanX, vanY and vanZ Gene in a Clinical Isolate in Australia". Acta Scientific Microbiology 7.10 (2024):40-43.
In this article we describe a new vancomycin variable Enterococcus faecium clone (VVE) isolated from a blood cultured specimen in central Sydney, Australia. The E. faecium isolated contained the vanA gene, and was susceptible to vancomycin and teicoplanin in vitro. Whole genomic sequencing (WGS) revealed that this isolate contained vanR, vanS, vanH and vanA genes but did not contain, vanX, vanY, or vanZ genes. Multi-locus sequence (MLTS) revealed the isolate to be E. faecium ST1424. The E. faecium was susceptible to daptomycin. Newly emerging VVE sequence types of E. faecium such as the VVE ST1424 described poses challenges to the control of health associated infections and infection control practices in hospitals.
Keywords: Vancomycin Variable Enterococcus faecium; vanR; vanS; vanA; vanX; vanY; vanZ; ST1424; ST1421; VVE; VRE
Enterococci infections, particularly those caused by antibiotic-resistant strains, are associated with significant mortality and have been designated as a high priority pathogen by the World Health Organization [1]. Glycopepetide-susceptible vanA-bearing E. faecium have been described previously, first isolated in Canada in 2011 [2]. Clinical cases of isolation have been reported in the literature, their predominant mechanism of resistance is encoded on transposon Tn1546 which is carried on a plasmid and includes the two component regulator gene (vanRS) and a gene cluster (vanHAXYZ) encoding the resistant mechanisms [3]. Biochemically, the vanA gene encodes a ligase that catalyzes the linkage of D-alanine and D-lactate, which replaces the typical D-alanine D-alanine precursor for peptidoglycan, therefore decreasing the affinity of glycopeptide antibiotics for their target sites [3,4]. VVE previously examined did not contain the vanR or vanS genes, which are believed to be essential to the expression of the vanHAX gene cluster. The vanS gene is a membrane-bound histidine kinase that senses the presence of vancomycin, resulting in ATP-dependent autophosphorylation. The phosphor-vanS then transfers phosphate to the response regulator vanR in the cytoplasm. Phospho-vanR binds to intergenic region upstream of the vanHAX operon and facilitates transcription, resulting in drug resistances [3-5]. While vanH, vanA, vanX, vanY are associated with vancomycin resistance, vanZ is associated with teicoplanin resistance. Thaker., et al. reported the underlaying molecular mechanism for the variable resistance phenotype in VVE strains and showed that various changes to the region upstream of vanHAX can result in the constitutive expression of gene cassette, conferring resistance in the absence of vanRS genes [5]. Current studies have shown that a complete vanH/vanA/vanX operon is required for the development of a vancomycin-resistant phenotype [6,7].
This mechanism of resistance in enterococci is a concern since VVE are vancomycin-susceptible enterococci containing a silenced vanA gene cluster that has been shown to revert to a resistant phenotype through genetic rearrangements occurring at low frequencies [6]. The presence of such VVEs in hospitals has clinical implications, the resistant subpopulation may be selected for during antibiotic exposure, causing therapeutic failure [5,8,9]. This article describes a newly emerging VVE sequence type of E. faecium vanA ST1424 which may pose challenges to the control of health associated infections and infection control practices.
The Enterococcus faecium clone (VVE) described here (MB20075945) was isolated at Concord Hospital from a blood cultured specimen in central Sydney, Australia.
Identification was performed by Matrix-Assisted Laser Desorption-Ionization Time of Flight mass spectrometry (MALDI-TOF MS Biotyper sirius one) (Bruker, Germany) in accordance with the manufacture instructions.
Antimicrobial susceptibility testing of the E. faecium isolates was determined by the automated VITEK 2 XL microbiology analyzer (BioMérieux Inc.) using antimicrobial susceptibility testing (AST) AST-P643 card (BioMérieux, Australia). Results were interpreted as susceptible or resistant based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [10]. The isolate was also examined using the 5µg Vancomycin disc as described by EUCAST. The isolate was categorized as resistant if the zone diameter was less than 12 mm. Resistance was suspected when the vancomycin zone edge was fuzzy or colonies were growing within the inhibition zone. Enterococcus faecalis ATCC 29212 vancomycin susceptible and Enterococcus faecalis ATCC 51299 VanB vancomycin resistant were used as controls.
DNA extraction was performed with single colonies into 200μL sterile saline and boiled for 10 minutes at 95°C. After boiling, the liquid contained bacteria was centrifuged for 30 seconds with 12000r/sec. The supernatant was used as the template for real-time PCR as previously described by Merlino., et al. [11,12]. Enterococcus faecalis ATCC 29212 vancomycin susceptible and Enterococcus faecalis ATCC 51299 VanB vancomycin resistant were used as controls.
MLST and WGS was performed by a reference laboratory at Royal Prince Alfred Hospital. Seven housekeeping genes (adk, atpA, ddl, gyd, gdh, purK, and pstS) were selected for the MLST analysis of the E. faecium isolate (Table 1). PCR conditions used for MLST and WGS (illumina MiSeq) were performed as previously described [13].
Enterococcus faecium was isolated from a blood cultured specimen on horse blood agar and identified by MALDI-TOF with a score of 2.53. Molecular investigation of the Enterococcus faecium was found to contain the vanA gene, however the isolate was susceptible to vancomycin and teicoplanin in vitro on the Vitek 2 XL [6]. The isolate was further examined using the 5 µg Vancomycin disc as described by EUCAST. The isolate was categorized as susceptible with a zone diameter of greater than 12mm. No resistance was suspected since the vancomycin zone edge was not fuzzy and no colonies were growing within the inhibition zone. Further genomic investigations of the Enterococcus faecium containing the vanA gene by whole genomic sequencing (illumina MiSeq) revealed that this isolate contained vanR, vanS, vanH and vanA genes but did not contain, vanX, vanY, or vanZ genes. The resistome of the VVE E. faecium isolate from whole genome sequencing is shown in table 1. The isolate was multi-locus sequenced and revealed to be E. faecium ST1424. The Enterococcus faecium was susceptible to daptomycin. The Enterococcus faecium was not isolated again from blood cultures or any rectal swabs once daptomycin was used in therapy.
Table 1: Resistome of the VVE E. faecium isolate from the whole genome sequencing and the results of the multi-locus sequencing results of the VVE E. faecium isolate MB20075945.
Antibiotic class: AG: aminoglycosides, MA: macrolides, OTH: various antibiotics, TE: tetracyclines, TMP: trimethoprim, VAN: vancomycin. Gene present at >90% identity (red) or >60% (orange) coverage.
Herein, we report a VVE ST1424 containing vanR, vanS, vanH and vanA genes and the deletion of vanX, vanY, and vanZ genes. Therefore, confirming that expression of vancomycin resistance in some VVE may not be completely dependent on the absence of the vanS and vanR genes. Further genome analysis of our clinical isolate also revealed partial deletion of the vanA gene (83bp deletion; 92% coverage). The vanA gene present in this isolate is truncated by insertion of IS1216E element together with a complete copy of the vanH gene of the assembled genome sequence (figure 1).
Figure 1: The vanA gene present in the isolate MB20075945 truncated by insertion of IS1216E element together with a complete copy of the vanH, vanS and vanR gene.
While in Australia VVE ST1421 has been extensively studied, very little is known about VVE ST1424 [6]. Further research is required involving both in vitro and in vivo studies to determine whether vancomycin exposure induces resistances to vancomycin in VVE strains with both vanS and vanR and partial deletion of the vanX, vanY, and vanZ. Studies in Australia with VVE ST1421 has shown that vanX a D-Ala-DAla dipeptidase is required for vancomycin resistance, and the deletion in vanX results the VVE phenotypically susceptible to vancomycin [6,8]. VVE ST1424 unlike VVE ST1421 has an additional deletion of both vanY and vanZ and the presence of both vanS and vanR. Both vanS and vanR genes have been found in vanA VRE but seldom in VVE susceptible isolates such as ST1421. The potential for vancomycin resistance to rise following vancomycin exposure creates a risk of treatment failure in such isolates. Our initial attempt failed to grow the organism in different concentrations of vancomycin of commercial chromogenic culture media containing 4 µg/ml (Brilliance VRE agar, Thermofisher-Australia) and 8 µg/ml (CHROMID VRE agar, Edwards – Australia) of vancomycin, concentrations used by many commercial companies to detect VRE in hospital infection control screening. Transmission is a major issue with such VVE isolates, the ability of the partial described vanA gene being transmitted in enterococci from one patient to another in a hospital setting without infection control detection.
Newly emerging VVE sequence types of E. faecium such as VVE ST1424 pose challenges to the control of health associated infections in hospitals an area that requires further investigation in clinical settings and infection control [8].
The authors would like to thank the Royal Prince Alfred Hospital Microbiology for performing Whole Genome Sequencing and MLST on the isolate.
The authors state no conflicts of interest exist.
Copyright: © 2024 John Merlino., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Open Access by
Acta Scientific is licensed under a Creative Commons Attribution 4.0 International License
Based on a work at https://actascientific.com
ff
