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Antimicrobial Resistance and Infectious Diseases Laboratory, Murdoch University, Perth, WA, AustraliaPathWest Laboratory Medicine WA, Fiona Stanley Hospital, Perth, WA, AustraliaSchool of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia
Antimicrobial Resistance and Infectious Diseases Laboratory, Murdoch University, Perth, WA, AustraliaPathWest Laboratory Medicine WA, Fiona Stanley Hospital, Perth, WA, Australia
Linezolid (LNZ) is an oxazolidinone antibiotic and a drug of last resort used to treat infections caused by Gram-positive pathogens that are resistant to multiple classes of antibiotics. In Australia, LNZ was introduced in 2002 and is currently reserved to treat suspected or proven infections caused by methicillin-resistant staphylococci or vancomycin-resistant enterococci. Despite restricted use, LNZ resistance has emerged worldwide, threatening the effective treatment of multidrug-resistant bacterial infections.
In Australia, LNZ-resistant enterococci (LRE) are monitored and reported by each state and territory to the National Alert System for Critical Antimicrobial Resistances (CARAlert), as part of the AURA (Antimicrobial Use and Resistance in Australia) surveillance system.
LNZ interferes with bacterial protein synthesis and prevents the formation of the translation initiation complex by binding to the 23S rRNA on the 50S ribosomal subunit. In enterococci, resistance to LNZ is often caused by point mutations in the bacterial 23S rRNA gene; the most common mutation is G2576T. Enterococcus faecalis has four copies of the 23S rRNA gene and there is a direct correlation between the number of gene copies carrying the G2576T mutation and the phenotypic level of LNZ resistance.
LRE-Finder, a web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.
Although an increasing number of LNZ-resistant E. faecalis (LREfs) has been reported worldwide, little is known about the genetic diversity and resistance mechanism of LREfs in Australia. In this study, we used whole genome sequencing to characterise and determine the mechanism of LNZ resistance in 25 clinical isolates of LREfs isolated in Western Australia (WA) from 2016 to 2021.
Isolates were identified as E. faecalis using matrix-assisted laser desorption ionisation (MALDI Biotyper; Bruker Daltonics, Germany). Antimicrobial susceptibility testing was initially performed on the VITEK 2 (bioMérieux, France) automated system using the AST-P612 susceptibility panel. Resistance to LNZ was confirmed by the Etest method (bioMérieux) as per the manufacturer's instructions and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints [susceptible (S) ≤2 mg/L; intermediate (I) 4 mg/L; resistant (R) ≥8 mg/L]. Whole genome sequencing was performed on a NextSeq 500 platform (Illumina, USA) using 150 bp paired-end chemistry. LNZ resistance determinants were identified using the LRE-Finder 1.0 tool
LRE-Finder, a web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.
which accurately detects the most common mutations associated with LNZ resistance in enterococci. LRE-Finder aligns raw sequence reads (fastq) with k-mer alignment and can detect the number of 23S rRNA gene copies harbouring the relevant mutations. Genomes were aligned and a phylogenetic tree was constructed using the neighbour-joining algorithm. Genetic environments were annotated and visualised using the Geneious Prime software (Biomatters, New Zealand).
Of the 25 isolates, 72% (n=18) had a LNZ MIC of 8 mg/L and the remainder had a MIC of 4 mg/L (n=3), 16 mg/L (n=3) and 64 mg/L (n=1). Although 10 unique STs were identified, 48% (n=12) of isolates belonged to ST16 (Fig. 1). Most ST16 isolates had a LNZ MIC of 8 mg/L and were phylogenetically related (Fig. 1). Except for LREfs-WA-08, all isolates possessed four wild-type copies of the 23S rRNA gene. LREfs-WA-08, which had the highest LNZ MIC (64 mg/L), harboured the G2576T mutation in three copies of the 23S rRNA gene. Resistance to LNZ in the remaining isolates was due to the presence of the optrA gene. The cfr, cfr(B) and poxtA genes were not identified.
Fig. 1Phylogeny and resistance determinants of linezolid-resistant E. faecalis isolated in Western Australia in 2016–2021. The tree was constructed using the neighbour-joining algorithm and 200 bootstrapping values. MIC (minimum inhibitory concentration) values were determined using the E-test. LNZ, linezolid; ST, sequence type; WT, wild type; Year, year of isolation. optrA-1 (Accession No. KP399637); optrA-5 (Accession No. KT862783); optrA-6 (Accession No. KT862784); optrA-7 (Accession No. KT862775).
Four different optrA allele sequences (1968 bp in length) that differed by a maximum of 6 nucleotides were identified: optrA-1 (Accession No. KP399637), optrA-5 (Accession No. KT862783), optrA-6 (Accession No. KT862784), and optrA-7 (Accession No. KT862775). The peptide sequences encoded by optrA-1 and optrA-5 are identical and differ by two amino acid residues from the peptide sequences encoded by optrA-6 and optrA-7. The optrA gene was chromosomally encoded in 22 isolates and located on a plasmid in the remaining two optrA-positive isolates. The chromosome-encoded optrA was located on a 12.9 kb transposon, recently characterised as Tn6674,
which integrated into the radC gene (Fig. 2A). In addition to the tnpA, tnpB, and tnpC transposase genes, the optrA-carrying transposon also carried the ant(9)-Ia (also called spc), erm(A) and fexA resistance genes which confer resistance to spectinomycin, erythromycin and chloramphenicol, respectively. The two isolates harbouring a plasmid-encoded optrA, LREfs-WA-01 and LREfs-WA-04, were ST16 and were the only isolates harbouring the optrA-7 variant. The genetic environment of the plasmid-encoded optrA was identical (100% nucleotide identity) to the genetic environment of optrA in p10-2-2 (Accession No. KT862775.1), a plasmid harboured by the ST59 LREfs strain 10-2-2 recovered from a pig in China.
The fexA and erm(A) genes were identified upstream and downstream of the plasmid-encoded optrA, respectively (Fig. 2B).
Fig. 2Schematic representation of the genetic environment of optrA in E. faecalis investigated in this study. (A) The optrA gene is chromosomally encoded and located on a Tn6674 transposon which also carries transposase genes (tnpA, tnpB, and tnpC), the spectinomycin resistance gene ant(9)-Ia (also called spc), the macrolide-lincosamide-streptogramin B resistance gene erm(A), and the chloramphenicol-florfenicol resistance gene fexA. (B) The optrA gene is plasmid-encoded and is flanked by the fexA and erm(A). met encodes an S-adenosylmethionine (SAM)-dependent methyltransferase; folC encodes a dihydrofolate synthase; radC encodes a DNA repair protein; cspC encodes a cold shock protein; rnjA encodes ribonuclease J; impB is predicted to encode a plasmid-associated type VI secretion protein.
all optrA-positive isolates in our collection harboured only one copy of the gene. The optrA gene encodes an ATP-binding cassette (ABC)-F protein which binds to the large (50S) ribosomal subunit and protects the ribosome by interfering with the actions of ribosome-targeted antibiotics such as oxazolidinones and phenicols.
A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin.
The gene was originally identified on a plasmid, pE349 (Accession No. KP399637), harboured by an ST116 LREfs of human origin. The pE349 plasmid contained the same impB-fexA-optrA fragment identified in our optrA-carrying plasmids, but it did not harbour erm(A).
The optrA gene has further been detected in various countries in enterococci isolated from food animals, animal carcasses, animal food products and wastewater, highlighting the importance of this resistance gene in a One Health context.
In our collection, optrA was predominantly chromosomal and the gene was carried on the Tn6674 transposon in conjunction with fexA, erm(A) and ant(9)-Ia. Tn6674 has previously been detected in circular intermediate forms,
suggesting chromosomally encoded optrA is transposable, which could explain the presence of Tn6674 in the genetically diverse E. faecalis isolates in our study. Tn6674 was always identified within the radC gene which is considered a hotspot for the chromosomal integration of optrA-carrying genetic elements. In addition to hospitalised patients, the Tn6674 transposon has previously been identified in healthy humans and food-producing animals across different continents,
Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes.
highlighting the importance of monitoring this mobile multi-resistance genetic element.
In conclusion, LNZ resistance in our collection was predominantly mediated by the presence of optrA. The optrA gene clusters harboured other resistance genes such as fexA and erm(A), and in the case of chromosomally encoded optrA, also harboured ant(9)-Ia (or spc). However, the isolate with the highest LNZ MIC was optrA-negative and resistance was due to the G2576T mutation in three of its four 23S rRNA gene copies. Although optrA can be detected using de novo draft assemblies, mutations in 23S must be determined by analysing the raw sequence reads using a tool such as LRE-Finder because consensus sequences produced by assemblers ignore less abundant mutations. Based on our findings, similar genomic characterisation studies on LREfs in other parts of Australia should be conducted. Furthermore, as LNZ resistance has been associated with food-producing animals in other countries, a One Health surveillance approach is warranted.
Conflicts of interest and sources of funding
The authors state that there are no conflicts of interest to disclose.
References
Turner A.M.
Lee J.Y.H.
Gorrie C.L.
et al.
Genomic insights into last-line antimicrobial resistance in multidrug-resistant Staphylococcus and vancomycin-resistant Enterococcus.
LRE-Finder, a web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and poxtA genes encoding linezolid resistance in enterococci from whole-genome sequences.
A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin.
Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes.
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