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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

LRE-Finder, a Web tool for detection of the 23S rRNA mutations and the optrA, cfr, cfr(B) and... Abstract Objectives In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. Methods LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. Results LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. Conclusions A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online. Introduction Linezolid, an oxazolidinone, can be used for treatment of infections caused by VRE. Resistance to linezolid in enterococci is often due to mutations in the V domain of the 23S rRNA gene. In a recent literature review on linezolid-resistant enterococci (LRE), the G2576T mutation (Escherichia coli numbering) was the most common cause of the LRE phenotype.1 Another mutation in the 23S rRNA, G2505A (E. coli numbering), has also been reported for LRE, but seems to be less frequent than the G2576T mutation.2,3 Furthermore, transferable resistance genes [cfr, cfr(B), optrA and poxtA]4–7 encoding linezolid resistance have been described in enterococci, whereas amino acid substitutions in the ribosomal proteins L3, L4 and/or L22, suspected to cause decreased susceptibility to linezolid in staphylococci, are rare in enterococci.1,8 Real-time PCR methods for detection of the G2576T mutation have been published, but no method is currently available to analyse this mutation (or the G2505A mutation) in genome sequencing data.9,10 Beukers et al.11 recently described the need for such an online tool for easy analysis of raw WGS data to estimate the fraction of 23S alleles carrying the G2576T mutation. Herein, a Web tool (LRE-Finder) for detection of the 23S rRNA mutations (G2576T and G2505A), and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci, is presented and validated. Materials and methods Identifying resistance genes or mutations encoding linezolid resistance LRE-Finder (https://cge.cbs.dtu.dk/services/LRE-Finder/) detects the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the genes encoding linezolid resistance [cfr, cfr(B), optrA and poxtA] by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA).12 The different variants of the genes encoding linezolid resistance tested by LRE-Finder are listed in Table S1 (available as Supplementary data at JAC Online). The LRE-Finder program can be obtained from the BitBucket download page (https://bitbucket.org/genomicepidemiology/lre-finder/src/master/), where the gene sequences are contained in the ‘elm-fsa’ file included in the ‘elmDB.tar.gz’ archive. Table 1. Characterization of the 21 LRE and the 26 linezolid-susceptible enterococci Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolates that are mutated in position 2505 or 2576 are marked in bold. LSE, linezolid-susceptible enterococci; NA, not available. a Determined by MLST. View Large Table 1. Characterization of the 21 LRE and the 26 linezolid-susceptible enterococci Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolates that are mutated in position 2505 or 2576 are marked in bold. LSE, linezolid-susceptible enterococci; NA, not available. a Determined by MLST. View Large Evaluation of method For evaluation, fastq files from 16 linezolid-resistant Enterococcus faecium and five linezolid-resistant Enterococcus faecalis isolates were submitted to LRE-Finder. The LRE isolates were obtained from the Danish Departments of Clinical Microbiology from January 2015 through September 2018. Furthermore, fastq files from 1473 VRE isolates were submitted to LRE-Finder as negative controls. The VRE isolates were obtained from the national surveillance of VRE in Denmark from January 2015 through June 2018. All isolates had been sequenced on Illumina platforms using paired-end reads. For further confirmation of the ability of LRE-Finder to detect the resistance genes relevant for linezolid resistance, Illumina raw sequencing data from a Staphylococcus epidermidis 1B8 with cfr (accession number ERR2117874), from E. faecium UW10882 with cfr(B) (accession number SRR3870887) and E. faecium P36 with poxtA (accession number KP834591) were obtained from GenBank as none of the Danish LRE strains carried these genes. Susceptibility testing The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates (20 E. faecium and 6 E. faecalis) were additionally selected for MIC determination. The 20 E. faecium isolates were selected to represent the major complex types (CTs) detected in Denmark during 2015–17.13,14 Finally, six vancomycin-resistant E. faecalis, representing different STs, were selected for linezolid susceptibility testing. The MICs of linezolid (Fresenius Kabi, Bad Homburg, Germany) were determined using broth microdilution.15E. faecalis 29212 was used as a control strain. The clinical breakpoint for linezolid for enterococci is ≥8 mg/L.16 Data accession Sequencing data from the study are available in GenBank PRJEB29922. Results and discussion Resistance to linezolid in enterococci is often due to mutations in the V domain of the 23S rRNA gene. E. faecalis has four copies of the 23S rRNA gene and E. faecium has six. According to Boumghar-Bourtchaï et al.,17 one of four mutated 23S alleles in the 2576 position was sufficient for an MIC of 8 mg/L of linezolid in vitro for E. faecalis. In E. faecium, one of six mutated 23S gene alleles leads to linezolid resistance.18 Linezolid resistance related to the G2505A mutation has been reported previously in vivo for two E. faecalis strains2,19 and in vitro for one E. faecium strain.3 Here, linezolid resistance was associated with a single mutated 23S allele in E. faecalis, whereas the numbers of mutated alleles were not described for the E. faecium strain.19 Among the Danish LRE isolates, LRE-Finder detected five E. faecalis isolates with optrA, 14 E. faecium with the G2576T mutation and two E. faecium with the G2505A mutation (Table 1). The LRE genes for control isolates for cfr, cfr(B) and poxtA were also detected by LRE-Finder. For the 14 linezolid-resistant E. faecium with the G2576T mutation in our study, the fraction of Ts varied between 18.4% and 67.0% in position 2576, corresponding to one to four mutated alleles. For the two linezolid-resistant E. faecium strains with the G2505A mutation (from the same patient sampled at different timepoints), 48.8% and 67.1% As were detected in position 2505, equal to three and four mutated alleles, respectively (Table 1). This is, to our knowledge, the first report of a G2505A mutation detected in vivo in an E. faecium isolate from a patient. Marshall et al.18 reported for E. faecium a clear association between the number of 23S rRNA genes containing the G2576T mutation and the level of linezolid resistance expressed, an observation that was confirmed in our study (Table 1). Lobritz et al.19 described a similar observation for E. faecalis. None of the E. faecalis isolates in our study had the G2576T mutation. Therefore, it was not possible to test the association between the number of 23S rRNA genes containing the G2576T mutation and the level of linezolid resistance expressed for E. faecalis in the present study. To set a suitable lower cut-off for detecting at least one mutated 23S gene allele, 1473 previously sequenced VRE genomes were analysed using LRE-Finder. Here, the fraction of Ts was between 0% and 1.6% in position 2576 and the fraction of As was between 0% and 1.0% in position 2505, representing the random noise present in Illumina raw sequencing data. A subset of these consisting of 26 VRE isolates was selected for susceptibility testing and all had a linezolid MIC <8 mg/L (Table 1), thus confirming the predictions made based on the LRE-Finder analysis. A cut-off of 10% mutations in position 2576 and/or position 2505 was therefore set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both E. faecalis and E. faecium, while ignoring low-level sequencing noise. None of the four linezolid resistance genes was detected in the 1473 sequences from the VRE isolates. In summary, a Web tool for detection of the most common mutations and genes encoding linezolid resistance in enterococci is now available online and has been validated, showing 100% concordance with phenotypic susceptibility testing. LRE-Finder is also easy to use for individuals without advanced bioinformatics skills. However, it is important to emphasize that using draft assembly sequences is not recommended, as such usage will fail to detect mutations in 23S, when these mutations are constituting only a minority of the bases in the given position. Acknowledgements We thank Karin Sixhøj Pedersen for her excellent technical assistance. Funding Part of this work was supported by the Danish Ministry of Health. This study has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 643476 (COMPARE). Transparency declarations None to declare. References 1 Bi R , Qin T , Fan W et al. 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Linezolid update: stable in vitro activity following more than a decade of clinical use and summary of associated resistance mechanisms . Drug Resist Updat 2014 ; 17 : 1 – 12 . Google Scholar Crossref Search ADS PubMed 9 Woodford N , Tysall L , Auckland C et al. Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis . J Clin Microbiol 2002 ; 40 : 4298 – 300 . Google Scholar Crossref Search ADS PubMed 10 Werner G , Strommenger B , Klare I et al. Molecular detection of linezolid resistance in Enterococcus faecium and Enterococcus faecalis by use of 5' nuclease real-time PCR compared to a modified classical approach . J Clin Microbiol 2004 ; 42 : 5327 – 31 . Google Scholar Crossref Search ADS PubMed 11 Beukers AG , Hasman H , Hegstad K et al. Recommendations to address the difficulties encountered when determining linezolid resistance from whole-genome sequencing data . Antimicrob Agents Chemother 2018 ; 62 : e00613-18 . Google Scholar Crossref Search ADS PubMed 12 Clausen PTLC , Aarestrup FM , Lund O. Rapid and precise alignment of raw reads against redundant databases with KMA . BMC Bioinfomatics 2018 ; 19 : 307 . Google Scholar Crossref Search ADS 13 Høg BB , Bager F , Korsgaard H et al. DANMAP2017—Use of Antimicrobial Agents and Occurrence of Antimicrobial Resistance in Bacteria From Food Animals, Food and Humans in Denmark. https://www.danmap.org/-/media/arkiv/projekt-sites/danmap/danmap-reports/danmap-2017/danmap2017.pdf? la=en. 14 Hammerum AM , Baig S , Kamel Y et al. Emergence of vanA Enterococcus faecium in Denmark, 2005–15 . J Antimicrob Chemother 2017 ; 72 : 2184 – 90 . Google Scholar Crossref Search ADS PubMed 15 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Eleventh Edition: M07 . CLSI , Wayne, PA, USA , 2018 . 16 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing—Twenty-seventh Edition: M100 . CLSI , Wayne, PA, USA , 2017 . 17 Boumghar-Bourtchaï L , Dhalluin A , Malbruny B et al. Influence of recombination on development of mutational resistance to linezolid in Enterococcus faecalis JH2-2 . Antimicrob Agents Chemother 2009 ; 53 : 4007. Google Scholar Crossref Search ADS PubMed 18 Marshall SH , Donskey CJ , Hutton-Thomas R et al. Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis . Antimicrob Agents Chemother 2002 ; 46 : 3334 – 6 . Google Scholar Crossref Search ADS PubMed 19 Lobritz M , Hutton-Thomas R , Marshall S et al. Recombination proficiency influences frequency and locus of mutational resistance to linezolid in Enterococcus faecalis . Antimicrob Agents Chemother 2003 ; 47 : 3318 – 20 . Google Scholar Crossref Search ADS PubMed © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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

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Publisher
Oxford University Press
Copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
ISSN
0305-7453
eISSN
1460-2091
DOI
10.1093/jac/dkz092
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See Article on Publisher Site

Abstract

Abstract Objectives In enterococci, resistance to linezolid is often mediated by mutations in the V domain of the 23S rRNA gene (G2576T or G2505A). Furthermore, four genes [optrA, cfr, cfr(B) and poxtA] encode linezolid resistance in enterococci. We aimed to develop a Web tool for detection of the two mutations and the four genes encoding linezolid resistance in enterococci from whole-genome sequence data. Methods LRE-Finder (where LRE stands for linezolid-resistant enterococci) detected the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the cfr, cfr(B), optrA and poxtA genes by aligning raw sequencing reads (fastq format) with k-mer alignment. For evaluation, fastq files from 21 LRE isolates were submitted to LRE-Finder. As negative controls, fastq files from 1473 non-LRE isolates were submitted to LRE-Finder. The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates were additionally selected for linezolid MIC determination. Results LRE-Finder was validated and showed 100% concordance with phenotypic susceptibility testing. A cut-off of 10% mutations in position 2576 and/or position 2505 was set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both Enterococcus faecalis and Enterococcus faecium, while ignoring low-level sequencing noise. Conclusions A Web tool for detection of the 23S rRNA mutations (G2576T and G2505A) and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci is now available online. Introduction Linezolid, an oxazolidinone, can be used for treatment of infections caused by VRE. Resistance to linezolid in enterococci is often due to mutations in the V domain of the 23S rRNA gene. In a recent literature review on linezolid-resistant enterococci (LRE), the G2576T mutation (Escherichia coli numbering) was the most common cause of the LRE phenotype.1 Another mutation in the 23S rRNA, G2505A (E. coli numbering), has also been reported for LRE, but seems to be less frequent than the G2576T mutation.2,3 Furthermore, transferable resistance genes [cfr, cfr(B), optrA and poxtA]4–7 encoding linezolid resistance have been described in enterococci, whereas amino acid substitutions in the ribosomal proteins L3, L4 and/or L22, suspected to cause decreased susceptibility to linezolid in staphylococci, are rare in enterococci.1,8 Real-time PCR methods for detection of the G2576T mutation have been published, but no method is currently available to analyse this mutation (or the G2505A mutation) in genome sequencing data.9,10 Beukers et al.11 recently described the need for such an online tool for easy analysis of raw WGS data to estimate the fraction of 23S alleles carrying the G2576T mutation. Herein, a Web tool (LRE-Finder) for detection of the 23S rRNA mutations (G2576T and G2505A), and the optrA, cfr, cfr(B) and poxtA genes from whole-genome sequences from enterococci, is presented and validated. Materials and methods Identifying resistance genes or mutations encoding linezolid resistance LRE-Finder (https://cge.cbs.dtu.dk/services/LRE-Finder/) detects the fraction of Ts in position 2576 and the fraction of As in position 2505 of the 23S rRNA and the genes encoding linezolid resistance [cfr, cfr(B), optrA and poxtA] by aligning raw sequencing reads (fastq format) with k-mer alignment (KMA).12 The different variants of the genes encoding linezolid resistance tested by LRE-Finder are listed in Table S1 (available as Supplementary data at JAC Online). The LRE-Finder program can be obtained from the BitBucket download page (https://bitbucket.org/genomicepidemiology/lre-finder/src/master/), where the gene sequences are contained in the ‘elm-fsa’ file included in the ‘elmDB.tar.gz’ archive. Table 1. Characterization of the 21 LRE and the 26 linezolid-susceptible enterococci Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolates that are mutated in position 2505 or 2576 are marked in bold. LSE, linezolid-susceptible enterococci; NA, not available. a Determined by MLST. View Large Table 1. Characterization of the 21 LRE and the 26 linezolid-susceptible enterococci Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolate no. Year Species Linezolid resistance mechanism Position 2505 A% Position 2576 T% MIC (mg/L) STa CT LRE  3774 2018 E. faecium G2505A 48.8 0.0 32 ST80 16  3696 2018 E. faecium G2505A 67.1 0.0 >32 ST80 16  3565 2018 E. faecium G2576T 0.0 18.4 8 ST203 859  3617 2018 E. faecium G2576T 0.1 36.3 8 ST203 859  2764 2016 E. faecium G2576T 0.0 36.7 8 ST80 14  2446 2016 E. faecium G2576T 0.0 23.3 16 ST17 1000  2402 2016 E. faecium G2576T 0.0 30.6 16 ST192 15  2751 2016 E. faecium G2576T 0.1 33.6 16 ST203 859  2445 2016 E. faecium G2576T 0.0 67.0 16 ST17 1000  2762 2016 E. faecium G2576T 0.0 47.7 32 ST80 866  3350 2017 E. faecium G2576T 0.0 48.7 32 ST80 871  3318 2017 E. faecium G2576T 0.2 53.9 32 ST117 1543  2889 2016 E. faecium G2576T 0.0 61.1 32 ST117 24  2047 2015 E. faecium G2576T 0.1 66.5 32 ST80 871  3410 2017 E. faecium G2576T 0.0 66.6 32 ST203 1513  2926 2016 E. faecium G2576T 0.0 64.7 >32 ST203 859  2763 2016 E. faecalis optrA 0.1 0.1 8 ST16 NA  3655 2018 E. faecalis optrA 0.0 0.1 8 ST476 NA  3895 2018 E. faecalis optrA 0.0 0.2 8 ST480 NA  3936 2018 E. faecalis optrA 0.0 0.0 8 ST179 NA  3714 2018 E. faecalis optrA 0.0 0.1 16 ST16 NA LSE  3244 2017 E. faecium none 0.0 0.0 1 ST80 1160  2653 2016 E. faecium none 0.0 0.0 1 ST80 860  2981 2017 E. faecium none 0.2 0.0 1 ST80 14  1908 2015 E. faecalis none 0.3 0.0 2 ST6 NA  2375 2016 E. faecalis none 0.0 0.2 2 ST64 NA  2645 2016 E. faecalis none 0.0 0.0 2 ST28 NA  2845 2016 E. faecalis none 0.1 0.1 2 ST30 NA  3413 2017 E. faecalis none 0.0 0.3 2 ST774 NA  3540 2018 E. faecalis none 0.0 0.1 2 ST179 NA  2522 2016 E. faecium none 0.0 0.0 2 ST80 866  2948 2017 E. faecium none 0.0 0.0 2 ST203 859  3025 2017 E. faecium none 0.0 0.0 2 ST80 993  3414 2017 E. faecium none 0.0 0.0 2 ST203 1051  3510 2017 E. faecium none 0.0 0.0 2 ST80 1729  3104 2017 E. faecium none 0.0 0.0 4 ST203 859  1798 2015 E. faecium none 0.4 0.3 4 ST203 859  2393 2016 E. faecium none 0.1 0.0 4 ST117 873  2431 2016 E. faecium none 0.1 0.0 4 ST203 859  2766 2016 E. faecium none 0.0 0.3 4 ST203 859  2984 2017 E. faecium none 0.0 0.0 4 ST80 1064  2994 2017 E. faecium none 0.0 0.0 4 ST80 871  3087 2017 E. faecium none 0.1 0.0 4 ST80 1053  3312 2017 E. faecium none 0.0 0.0 4 ST117 1180  3383 2017 E. faecium none 0.0 0.0 4 ST117 24  3384 2017 E. faecium none 0.0 0.2 4 ST1421 1134  3406 2017 E. faecium none 0.0 0.0 4 ST203 1507  ATCC 29212 E. faecalis none 2 Isolates that are mutated in position 2505 or 2576 are marked in bold. LSE, linezolid-susceptible enterococci; NA, not available. a Determined by MLST. View Large Evaluation of method For evaluation, fastq files from 16 linezolid-resistant Enterococcus faecium and five linezolid-resistant Enterococcus faecalis isolates were submitted to LRE-Finder. The LRE isolates were obtained from the Danish Departments of Clinical Microbiology from January 2015 through September 2018. Furthermore, fastq files from 1473 VRE isolates were submitted to LRE-Finder as negative controls. The VRE isolates were obtained from the national surveillance of VRE in Denmark from January 2015 through June 2018. All isolates had been sequenced on Illumina platforms using paired-end reads. For further confirmation of the ability of LRE-Finder to detect the resistance genes relevant for linezolid resistance, Illumina raw sequencing data from a Staphylococcus epidermidis 1B8 with cfr (accession number ERR2117874), from E. faecium UW10882 with cfr(B) (accession number SRR3870887) and E. faecium P36 with poxtA (accession number KP834591) were obtained from GenBank as none of the Danish LRE strains carried these genes. Susceptibility testing The MICs of linezolid were determined for the 21 LRE isolates. As LRE-negative controls, 26 VRE isolates (20 E. faecium and 6 E. faecalis) were additionally selected for MIC determination. The 20 E. faecium isolates were selected to represent the major complex types (CTs) detected in Denmark during 2015–17.13,14 Finally, six vancomycin-resistant E. faecalis, representing different STs, were selected for linezolid susceptibility testing. The MICs of linezolid (Fresenius Kabi, Bad Homburg, Germany) were determined using broth microdilution.15E. faecalis 29212 was used as a control strain. The clinical breakpoint for linezolid for enterococci is ≥8 mg/L.16 Data accession Sequencing data from the study are available in GenBank PRJEB29922. Results and discussion Resistance to linezolid in enterococci is often due to mutations in the V domain of the 23S rRNA gene. E. faecalis has four copies of the 23S rRNA gene and E. faecium has six. According to Boumghar-Bourtchaï et al.,17 one of four mutated 23S alleles in the 2576 position was sufficient for an MIC of 8 mg/L of linezolid in vitro for E. faecalis. In E. faecium, one of six mutated 23S gene alleles leads to linezolid resistance.18 Linezolid resistance related to the G2505A mutation has been reported previously in vivo for two E. faecalis strains2,19 and in vitro for one E. faecium strain.3 Here, linezolid resistance was associated with a single mutated 23S allele in E. faecalis, whereas the numbers of mutated alleles were not described for the E. faecium strain.19 Among the Danish LRE isolates, LRE-Finder detected five E. faecalis isolates with optrA, 14 E. faecium with the G2576T mutation and two E. faecium with the G2505A mutation (Table 1). The LRE genes for control isolates for cfr, cfr(B) and poxtA were also detected by LRE-Finder. For the 14 linezolid-resistant E. faecium with the G2576T mutation in our study, the fraction of Ts varied between 18.4% and 67.0% in position 2576, corresponding to one to four mutated alleles. For the two linezolid-resistant E. faecium strains with the G2505A mutation (from the same patient sampled at different timepoints), 48.8% and 67.1% As were detected in position 2505, equal to three and four mutated alleles, respectively (Table 1). This is, to our knowledge, the first report of a G2505A mutation detected in vivo in an E. faecium isolate from a patient. Marshall et al.18 reported for E. faecium a clear association between the number of 23S rRNA genes containing the G2576T mutation and the level of linezolid resistance expressed, an observation that was confirmed in our study (Table 1). Lobritz et al.19 described a similar observation for E. faecalis. None of the E. faecalis isolates in our study had the G2576T mutation. Therefore, it was not possible to test the association between the number of 23S rRNA genes containing the G2576T mutation and the level of linezolid resistance expressed for E. faecalis in the present study. To set a suitable lower cut-off for detecting at least one mutated 23S gene allele, 1473 previously sequenced VRE genomes were analysed using LRE-Finder. Here, the fraction of Ts was between 0% and 1.6% in position 2576 and the fraction of As was between 0% and 1.0% in position 2505, representing the random noise present in Illumina raw sequencing data. A subset of these consisting of 26 VRE isolates was selected for susceptibility testing and all had a linezolid MIC <8 mg/L (Table 1), thus confirming the predictions made based on the LRE-Finder analysis. A cut-off of 10% mutations in position 2576 and/or position 2505 was therefore set in LRE-Finder for predicting a linezolid resistance phenotype. This cut-off allows for detection of a single mutated 23S allele in both E. faecalis and E. faecium, while ignoring low-level sequencing noise. None of the four linezolid resistance genes was detected in the 1473 sequences from the VRE isolates. In summary, a Web tool for detection of the most common mutations and genes encoding linezolid resistance in enterococci is now available online and has been validated, showing 100% concordance with phenotypic susceptibility testing. LRE-Finder is also easy to use for individuals without advanced bioinformatics skills. However, it is important to emphasize that using draft assembly sequences is not recommended, as such usage will fail to detect mutations in 23S, when these mutations are constituting only a minority of the bases in the given position. Acknowledgements We thank Karin Sixhøj Pedersen for her excellent technical assistance. Funding Part of this work was supported by the Danish Ministry of Health. This study has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 643476 (COMPARE). Transparency declarations None to declare. References 1 Bi R , Qin T , Fan W et al. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Jun 1, 2019

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