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Dissemination of linezolid-dependent, linezolid-resistant Staphylococcus epidermidis clinical isolates belonging to CC5 in German hospitals

Dissemination of linezolid-dependent, linezolid-resistant Staphylococcus epidermidis clinical... Abstract Objectives Linezolid-resistant Staphylococcus epidermidis (LRSE) and linezolid-dependent ST22 strains have been shown to predominate in tertiary care facilities all over Greece. We report herein the dissemination of ST22 but also ST2, ST5 and ST168 linezolid-dependent LRSE clones in four unrelated German hospitals. Methods Fourteen LRSE clinical isolates recovered during 2012–14 from five distantly located German hospitals were tested by for MIC determination broth microdilution and Etest, PCR/sequencing for cfr and for mutations in 23S rRNA, rplC, rplD and rplV genes, MLST, PFGE and growth curves without and with linezolid at 16 and 32 mg/L. Results Most (11, 78.6%) isolates had linezolid MICs >256 mg/L. Five isolates carried the cfr gene. Eight isolates belonged to ST22, two isolates each to ST168 and ST2 and one isolate each to ST5 and ST23. Ten isolates [seven belonging to ST22 and one to each of ST2, ST5 and ST168; all these STs belong to clonal complex (CC) 5] exhibited linezolid-dependent growth, growing significantly faster in linezolid-containing broth. Four isolates were non-dependent (one belonging to each of ST22, ST2, ST23 and ST168). Four isolates came from three different hospitals, whereas four and six isolates were recovered during outbreaks of LRSE in two distinct hospitals. Conclusions The multi-clonal dissemination of CC5 linezolid-dependent LRSE throughout German hospitals along with the clonal expansion of ST22 linezolid-dependent LRSE in Greek hospitals is of particular concern. It is plausible that this characteristic is inherent and provides a selective advantage to CC5 LRSE under linezolid pressure, contributing to their dissemination throughout hospitals in these countries. Introduction Linezolid, the first oxazolidinone drug, was launched in 2001 and is still displaying excellent in vitro activity against Staphylococcus epidermidis on a global scale,1 although outbreaks of linezolid-resistant S. epidermidis (LRSE) are occasionally reported.2,3 LRSE clinical isolates have been described to exhibit partial dependence on linezolid, with significant growth acceleration under linezolid exposure.4 This growth characteristic was attributed to structural and functional adaptations of ribosomes as a result of linezolid binding and confers a selective advantage on LRSE strains that are exposed to linezolid.5 We recently showed that LRSE became quite common in ICUs throughout Greece, with the vast majority of isolates exhibiting dependence on linezolid and belonging to ST22,6 which implies that the expansion of linezolid resistance was probably driven by this trait. LRSE emerged and were reported to cause outbreaks also in German hospitals, with most of the strains also belonging to ST22.7 The current study aimed to test the hypothesis that linezolid-dependent LRSE (ST22 or other lineages) also exist and may contribute to the dissemination of linezolid resistance in German hospitals. Materials and methods Bacterial isolates and susceptibility testing The study included 14 LRSE clinical isolates recovered between 2012 and 2014 from five distantly located German hospitals (Lower Saxony, hospital C; Saxony-Anhalt, hospital D; Saxony, hospitals E and H; North Rhine-Westphalia, hospital Y). Linezolid MICs were determined by broth microdilution according to EUCAST (www.eucast.org); linezolid Etest® (bioMérieux, Marcy-l'Étoile, France) was used to confirm the result. In all phenotypic assays, Staphylococcus aureus strain ATCC 29213 (linezolid MIC 0.5 mg/L) was used as a control strain. Growth characteristics of bacterial isolates Bacterial growth rates were determined in triplicate, as described previously.4 All 14 LRSE study isolates were tested without and with linezolid at 16 and 32 mg/L and at 0.25 mg/L for the strain ATCC 29213. These linezolid concentrations were selected according to previous studies on linezolid-dependent LRSE.4–6 The growth rates of the isolates without and with linezolid were statistically compared using the paired t-test and the χ2 test; P values <0.05 indicated significance and statistical analyses were performed with Minitab software (version 13.31) and SPSS software (version 17; SPSS Inc., Chicago, IL, USA). Typing and molecular testing for linezolid resistance mechanisms The 14 LRSE were genotyped by MLST as described previously.8 They were tested by PCR for the presence of the cfr gene;7 mutations in the peptidyl-transferase centre and in riboproteins L3, L4 and L22 possibly conferring linezolid resistance were identified by PCR/sequencing.8 Results Four and six LRSE isolates, respectively, were recovered during LRSE outbreaks in two distinct German hospitals. The larger outbreak, which involved six LRSE, was preceded by a considerable increase in linezolid consumption for the treatment of MRSA infections. The remaining four isolates came from three different hospitals. The phenotypic and genotypic characteristics of the study isolates are shown in Table 1. In brief, of the 14 LRSE clinical isolates tested, most (11, 78.6%) exhibited high-level resistance, with linezolid MICs being >256 mg/L. Five isolates carried the cfr gene. As for the clonal composition of the isolates, eight belonged to ST22, two isolates each to ST2 and ST168, one isolate to ST5 and one to ST23. Diverse and complex linezolid resistance mechanisms were identified among the LRSE, including new and already described mutations in 23S rRNA and mutations in the associated ribosomal proteins that may affect linezolid activity. In particular, all 14 isolates harboured at least one 23S rRNA mutation, 11 at least one mutation in L3, 10 at least one mutation in L4 and none displayed mutations in L22. Table 1. Characterization of LRSE isolates according to their origin, ST, linezolid MIC, putative linezolid resistance mechanisms, linezolid-dependent growth and data concerning linezolid treatment of the respective patient           Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no            Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no  NA, data not available; UTI, urinary tract infection. a ST2, ST5, ST22 and ST168 belong to CC5. Table 1. Characterization of LRSE isolates according to their origin, ST, linezolid MIC, putative linezolid resistance mechanisms, linezolid-dependent growth and data concerning linezolid treatment of the respective patient           Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no            Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no  NA, data not available; UTI, urinary tract infection. a ST2, ST5, ST22 and ST168 belong to CC5. Bacterial growth rates indicated that nine isolates (seven belonging to ST22 and one to each of ST2 and ST5) exhibited significant (P < 0.05) linezolid-dependent growth at both 16 and 32 mg/L linezolid and four isolates were linezolid-non-dependent (one belonging to each of ST22, ST2, ST23 and ST168). One isolate (14-02427, ST168) repeatedly exhibited an unusual mode of growth, being linezolid-non-dependent up to 12 h of growth, but clearly linezolid-dependent at 24–36 h of linezolid exposure, as indicated by significantly faster growth with than without linezolid exposure. Growth curves of a representative linezolid-dependent and a linezolid-non-dependent isolate are shown in Figure 1. Figure 1. View largeDownload slide (a) Growth curve of a representative linezolid-dependent isolate. (b) Growth curve of a representative linezolid-non-dependent isolate. Figure 1. View largeDownload slide (a) Growth curve of a representative linezolid-dependent isolate. (b) Growth curve of a representative linezolid-non-dependent isolate. Discussion Linezolid resistance among S. epidermidis is still uncommon worldwide; however, LRSE are increasingly reported from Europe4,6,7,9–12 and occasionally cause outbreaks. In a multicentre study from Greece, LRSE were quite common, particularly in ICUs, where during 2013 resistance rates reached 20.9%6 and, of note, the majority of LRSE belonged to ST22. The emergence of LRSE was reported to be preceded by increased linezolid consumption13 and is widely considered to be linked to heavy linezolid usage.6,13 In recent years, LRSE have been described to exhibit partial dependence on linezolid, which is apparently due to structural adaptations of the ribosomes leading to faster protein synthesis rates and remarkably accelerated growth.5 In Greece, linezolid-dependent isolates were shown to represent the majority of LRSE and to belong exclusively to ST22, implying that the common administration of linezolid might have facilitated the selection and spread of this LRSE clone. In the current study, LRSE from five distantly located German hospitals also included a high proportion (10/14, 71.4%) of linezolid-dependent strains, which belonged mainly to ST22 (n = 7) and to a lesser extent to ST2 (n = 1), ST5 (n = 1) and ST168 (n = 1); of note, all these STs are related (https://pubmlst.org/bigsdb?db=pubmlst_sepidermidis_isolates) and belong to clonal complex (CC) 5. This observation may imply the existence of inherent traits of this lineage that predispose to linezolid-dependent growth and remain to be discovered in future studies. In conclusion, the multi-clonal dissemination of CC5 linezolid-dependent LRSE in German hospitals, together with the predominance of ST22 linezolid-dependent LRSE in Greece, indicates that LRSE of CC5 and possibly other lineages commonly exhibit this phenotypic trait, which apparently provides selective advantage under linezolid exposure. Given that linezolid usage is quite common for the treatment of VRE, MRSA or vancomycin-intermediate S.aureus (VISA) infections, linezolid dependence may underlie the observed increasing number of isolations of LRSE in Europe. These observations necessitate further studies to identify the occurrence of linezolid-dependent LRSE worldwide. Finally, it appears that rational usage of linezolid is important to limit the selection of linezolid-dependent LRSE and preserve the activity of this drug. Funding This work was supported by internal funding. Transparency declarations None to declare. References 1 Mendes RE, Hogan PA, Jones RN et al.   Surveillance for linezolid resistance via the Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) programme (2014): evolving resistance mechanisms with stable susceptibility rates. J Antimicrob Chemother  2016; 71: 1860– 5. Google Scholar CrossRef Search ADS PubMed  2 Mendes RE, Deshpande LM, Jones RN. 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  3 Gu B, Kelesidis T, Tsiodras S et al.   The emerging problem of linezolid-resistant Staphylococcus. J Antimicrob Chemother  2013; 68: 4– 11. Google Scholar CrossRef Search ADS PubMed  4 Pournaras S, Ntokou E, Zarkotou O et al.   Linezolid dependence in Staphylococcus epidermidis bloodstream isolates. Emerg Infect Dis  2013; 19: 129– 32. Google Scholar CrossRef Search ADS PubMed  5 Kokkori S, Apostolidi M, Tsakris A et al.   Linezolid-dependent function and structure adaptation of ribosomes in a Staphylococcus epidermidis strain exhibiting linezolid dependence. Antimicrob Agents Chemother  2014; 58: 4651– 6. Google Scholar CrossRef Search ADS PubMed  6 Karavasilis V, Zarkotou O, Panopoulou M et al.   Wide dissemination of linezolid-resistant Staphylococcus epidermidis in Greece is associated with a linezolid-dependent ST22 clone. J Antimicrob Chemother  2015; 70: 1625– 9. Google Scholar PubMed  7 Bender J, Strommenger B, Steglich M et al.   Linezolid resistance in clinical isolates of Staphylococcus epidermidis from German hospitals and characterization of two cfr-carrying plasmids. J Antimicrob Chemother  2015; 70: 1630– 8. Google Scholar PubMed  8 Mendes RE, Deshpande LM, Farrell DJ et al.   Assessment of linezolid resistance mechanisms among Staphylococcus epidermidis causing bacteraemia in Rome, Italy. J Antimicrob Chemother  2010; 65: 2329– 35. Google Scholar CrossRef Search ADS PubMed  9 Decousser JW, Desroches M, Bourgeois-Nicolaos N et al.   Susceptibility trends including emergence of linezolid resistance among coagulase-negative staphylococci and meticillin-resistant Staphylococcus aureus from invasive infections. Int J Antimicrob Agents  2015; 46: 622– 30. Google Scholar CrossRef Search ADS PubMed  10 Russo A, Campanile F, Falcone M et al.   Linezolid-resistant staphylococcal bacteraemia: a multicentre case-case-control study in Italy. Int J Antimicrob Agents  2015; 45: 255– 61. Google Scholar CrossRef Search ADS PubMed  11 Barros M, Branquinho R, Grosso F et al.   Linezolid-resistant Staphylococcus epidermidis, Portugal, 2012. Emerg Infect Dis  2014; 20: 903– 5. Google Scholar CrossRef Search ADS PubMed  12 Mihaila L, Defrance G, Levesque E et al.   A dual outbreak of bloodstream infections with linezolid-resistant Staphylococcus epidermidis and Staphylococcus pettenkoferi in a liver intensive care unit. Int J Antimicrob Agents  2012; 40: 472– 4. Google Scholar CrossRef Search ADS PubMed  13 Mulanovich VE, Huband MD, McCurdy SP et al.   Emergence of linezolid-resistant coagulase-negative Staphylococcus in a cancer centre linked to increased linezolid utilization. J Antimicrob Chemother  2010; 65: 2001– 4. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. 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/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Dissemination of linezolid-dependent, linezolid-resistant Staphylococcus epidermidis clinical isolates belonging to CC5 in German hospitals

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Oxford University Press
Copyright
© The Author(s) 2018. 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.
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0305-7453
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1460-2091
DOI
10.1093/jac/dkx524
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Abstract

Abstract Objectives Linezolid-resistant Staphylococcus epidermidis (LRSE) and linezolid-dependent ST22 strains have been shown to predominate in tertiary care facilities all over Greece. We report herein the dissemination of ST22 but also ST2, ST5 and ST168 linezolid-dependent LRSE clones in four unrelated German hospitals. Methods Fourteen LRSE clinical isolates recovered during 2012–14 from five distantly located German hospitals were tested by for MIC determination broth microdilution and Etest, PCR/sequencing for cfr and for mutations in 23S rRNA, rplC, rplD and rplV genes, MLST, PFGE and growth curves without and with linezolid at 16 and 32 mg/L. Results Most (11, 78.6%) isolates had linezolid MICs >256 mg/L. Five isolates carried the cfr gene. Eight isolates belonged to ST22, two isolates each to ST168 and ST2 and one isolate each to ST5 and ST23. Ten isolates [seven belonging to ST22 and one to each of ST2, ST5 and ST168; all these STs belong to clonal complex (CC) 5] exhibited linezolid-dependent growth, growing significantly faster in linezolid-containing broth. Four isolates were non-dependent (one belonging to each of ST22, ST2, ST23 and ST168). Four isolates came from three different hospitals, whereas four and six isolates were recovered during outbreaks of LRSE in two distinct hospitals. Conclusions The multi-clonal dissemination of CC5 linezolid-dependent LRSE throughout German hospitals along with the clonal expansion of ST22 linezolid-dependent LRSE in Greek hospitals is of particular concern. It is plausible that this characteristic is inherent and provides a selective advantage to CC5 LRSE under linezolid pressure, contributing to their dissemination throughout hospitals in these countries. Introduction Linezolid, the first oxazolidinone drug, was launched in 2001 and is still displaying excellent in vitro activity against Staphylococcus epidermidis on a global scale,1 although outbreaks of linezolid-resistant S. epidermidis (LRSE) are occasionally reported.2,3 LRSE clinical isolates have been described to exhibit partial dependence on linezolid, with significant growth acceleration under linezolid exposure.4 This growth characteristic was attributed to structural and functional adaptations of ribosomes as a result of linezolid binding and confers a selective advantage on LRSE strains that are exposed to linezolid.5 We recently showed that LRSE became quite common in ICUs throughout Greece, with the vast majority of isolates exhibiting dependence on linezolid and belonging to ST22,6 which implies that the expansion of linezolid resistance was probably driven by this trait. LRSE emerged and were reported to cause outbreaks also in German hospitals, with most of the strains also belonging to ST22.7 The current study aimed to test the hypothesis that linezolid-dependent LRSE (ST22 or other lineages) also exist and may contribute to the dissemination of linezolid resistance in German hospitals. Materials and methods Bacterial isolates and susceptibility testing The study included 14 LRSE clinical isolates recovered between 2012 and 2014 from five distantly located German hospitals (Lower Saxony, hospital C; Saxony-Anhalt, hospital D; Saxony, hospitals E and H; North Rhine-Westphalia, hospital Y). Linezolid MICs were determined by broth microdilution according to EUCAST (www.eucast.org); linezolid Etest® (bioMérieux, Marcy-l'Étoile, France) was used to confirm the result. In all phenotypic assays, Staphylococcus aureus strain ATCC 29213 (linezolid MIC 0.5 mg/L) was used as a control strain. Growth characteristics of bacterial isolates Bacterial growth rates were determined in triplicate, as described previously.4 All 14 LRSE study isolates were tested without and with linezolid at 16 and 32 mg/L and at 0.25 mg/L for the strain ATCC 29213. These linezolid concentrations were selected according to previous studies on linezolid-dependent LRSE.4–6 The growth rates of the isolates without and with linezolid were statistically compared using the paired t-test and the χ2 test; P values <0.05 indicated significance and statistical analyses were performed with Minitab software (version 13.31) and SPSS software (version 17; SPSS Inc., Chicago, IL, USA). Typing and molecular testing for linezolid resistance mechanisms The 14 LRSE were genotyped by MLST as described previously.8 They were tested by PCR for the presence of the cfr gene;7 mutations in the peptidyl-transferase centre and in riboproteins L3, L4 and L22 possibly conferring linezolid resistance were identified by PCR/sequencing.8 Results Four and six LRSE isolates, respectively, were recovered during LRSE outbreaks in two distinct German hospitals. The larger outbreak, which involved six LRSE, was preceded by a considerable increase in linezolid consumption for the treatment of MRSA infections. The remaining four isolates came from three different hospitals. The phenotypic and genotypic characteristics of the study isolates are shown in Table 1. In brief, of the 14 LRSE clinical isolates tested, most (11, 78.6%) exhibited high-level resistance, with linezolid MICs being >256 mg/L. Five isolates carried the cfr gene. As for the clonal composition of the isolates, eight belonged to ST22, two isolates each to ST2 and ST168, one isolate to ST5 and one to ST23. Diverse and complex linezolid resistance mechanisms were identified among the LRSE, including new and already described mutations in 23S rRNA and mutations in the associated ribosomal proteins that may affect linezolid activity. In particular, all 14 isolates harboured at least one 23S rRNA mutation, 11 at least one mutation in L3, 10 at least one mutation in L4 and none displayed mutations in L22. Table 1. Characterization of LRSE isolates according to their origin, ST, linezolid MIC, putative linezolid resistance mechanisms, linezolid-dependent growth and data concerning linezolid treatment of the respective patient           Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no            Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no  NA, data not available; UTI, urinary tract infection. a ST2, ST5, ST22 and ST168 belong to CC5. Table 1. Characterization of LRSE isolates according to their origin, ST, linezolid MIC, putative linezolid resistance mechanisms, linezolid-dependent growth and data concerning linezolid treatment of the respective patient           Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no            Linezolid             Index no./ hospital  Culture site  Infection  MLSTa  cfr gene  MIC (mg/L)  treatment  23S rRNA  Protein L3  Protein L4  Protein L22  Linezolid dependence  12-00322/C  blood  sepsis  ST22  +  16  NA  C2161T  Leu101Val  WT  WT  no  Ala157Arg  12-01569/D  urine  UTI  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-02439/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03718/D  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  12-03723/D  blood  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-00905/E  blood  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  InsHis138  13-01045/E  NA  NA  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  13-01084/H  NA  sepsis  ST22  −  >256  NA  C2161T  Leu101Val  Glu128Ala  WT  yes  T2502A  Gly152Asp  Gln174Arg  C2532T  Asp159Tyr  Arg182Gln  14-03576/Y  blood  NA  ST2  −  >256  NA  C2190T  −  Ser61Arg  WT  yes  C2561T  Asn158Ser  G2603T  14-03623/Y  NA  decubitus  ST2  +  12  +  C2190T  −  −  WT  no  14-01514/Y  blood  endocarditis  ST5  +  >256  +  C2190T  His 146Gln  Ser61Arg  WT  yes  Val154Leu  Asn158Ser  Ala157Arg  14-00183/Y  NA  NA  ST23  −  8  +  C2190T  −  Ser61Arg  WT  no  Asn158Ser  14-02427/Y  blood  NA  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  yes  13-04707/Y  blood  sepsis  ST168  +  >256  +  C2190T  Gly152Asp  −  WT  no  NA, data not available; UTI, urinary tract infection. a ST2, ST5, ST22 and ST168 belong to CC5. Bacterial growth rates indicated that nine isolates (seven belonging to ST22 and one to each of ST2 and ST5) exhibited significant (P < 0.05) linezolid-dependent growth at both 16 and 32 mg/L linezolid and four isolates were linezolid-non-dependent (one belonging to each of ST22, ST2, ST23 and ST168). One isolate (14-02427, ST168) repeatedly exhibited an unusual mode of growth, being linezolid-non-dependent up to 12 h of growth, but clearly linezolid-dependent at 24–36 h of linezolid exposure, as indicated by significantly faster growth with than without linezolid exposure. Growth curves of a representative linezolid-dependent and a linezolid-non-dependent isolate are shown in Figure 1. Figure 1. View largeDownload slide (a) Growth curve of a representative linezolid-dependent isolate. (b) Growth curve of a representative linezolid-non-dependent isolate. Figure 1. View largeDownload slide (a) Growth curve of a representative linezolid-dependent isolate. (b) Growth curve of a representative linezolid-non-dependent isolate. Discussion Linezolid resistance among S. epidermidis is still uncommon worldwide; however, LRSE are increasingly reported from Europe4,6,7,9–12 and occasionally cause outbreaks. In a multicentre study from Greece, LRSE were quite common, particularly in ICUs, where during 2013 resistance rates reached 20.9%6 and, of note, the majority of LRSE belonged to ST22. The emergence of LRSE was reported to be preceded by increased linezolid consumption13 and is widely considered to be linked to heavy linezolid usage.6,13 In recent years, LRSE have been described to exhibit partial dependence on linezolid, which is apparently due to structural adaptations of the ribosomes leading to faster protein synthesis rates and remarkably accelerated growth.5 In Greece, linezolid-dependent isolates were shown to represent the majority of LRSE and to belong exclusively to ST22, implying that the common administration of linezolid might have facilitated the selection and spread of this LRSE clone. In the current study, LRSE from five distantly located German hospitals also included a high proportion (10/14, 71.4%) of linezolid-dependent strains, which belonged mainly to ST22 (n = 7) and to a lesser extent to ST2 (n = 1), ST5 (n = 1) and ST168 (n = 1); of note, all these STs are related (https://pubmlst.org/bigsdb?db=pubmlst_sepidermidis_isolates) and belong to clonal complex (CC) 5. This observation may imply the existence of inherent traits of this lineage that predispose to linezolid-dependent growth and remain to be discovered in future studies. In conclusion, the multi-clonal dissemination of CC5 linezolid-dependent LRSE in German hospitals, together with the predominance of ST22 linezolid-dependent LRSE in Greece, indicates that LRSE of CC5 and possibly other lineages commonly exhibit this phenotypic trait, which apparently provides selective advantage under linezolid exposure. Given that linezolid usage is quite common for the treatment of VRE, MRSA or vancomycin-intermediate S.aureus (VISA) infections, linezolid dependence may underlie the observed increasing number of isolations of LRSE in Europe. These observations necessitate further studies to identify the occurrence of linezolid-dependent LRSE worldwide. Finally, it appears that rational usage of linezolid is important to limit the selection of linezolid-dependent LRSE and preserve the activity of this drug. Funding This work was supported by internal funding. Transparency declarations None to declare. References 1 Mendes RE, Hogan PA, Jones RN et al.   Surveillance for linezolid resistance via the Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) programme (2014): evolving resistance mechanisms with stable susceptibility rates. J Antimicrob Chemother  2016; 71: 1860– 5. Google Scholar CrossRef Search ADS PubMed  2 Mendes RE, Deshpande LM, Jones RN. 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Google Scholar CrossRef Search ADS PubMed  9 Decousser JW, Desroches M, Bourgeois-Nicolaos N et al.   Susceptibility trends including emergence of linezolid resistance among coagulase-negative staphylococci and meticillin-resistant Staphylococcus aureus from invasive infections. Int J Antimicrob Agents  2015; 46: 622– 30. Google Scholar CrossRef Search ADS PubMed  10 Russo A, Campanile F, Falcone M et al.   Linezolid-resistant staphylococcal bacteraemia: a multicentre case-case-control study in Italy. Int J Antimicrob Agents  2015; 45: 255– 61. Google Scholar CrossRef Search ADS PubMed  11 Barros M, Branquinho R, Grosso F et al.   Linezolid-resistant Staphylococcus epidermidis, Portugal, 2012. Emerg Infect Dis  2014; 20: 903– 5. Google Scholar CrossRef Search ADS PubMed  12 Mihaila L, Defrance G, Levesque E et al.   A dual outbreak of bloodstream infections with linezolid-resistant Staphylococcus epidermidis and Staphylococcus pettenkoferi in a liver intensive care unit. Int J Antimicrob Agents  2012; 40: 472– 4. Google Scholar CrossRef Search ADS PubMed  13 Mulanovich VE, Huband MD, McCurdy SP et al.   Emergence of linezolid-resistant coagulase-negative Staphylococcus in a cancer centre linked to increased linezolid utilization. J Antimicrob Chemother  2010; 65: 2001– 4. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. 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/about_us/legal/notices)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 1, 2018

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