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Emergence and control of linezolid-resistant Staphylococcus epidermidis in an ICU of a German hospital

Emergence and control of linezolid-resistant Staphylococcus epidermidis in an ICU of a German... Abstract Objectives To investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE) in an interdisciplinary ICU, linezolid consumption and infection control measures taken. Methods Routine surveillance of nosocomial infections revealed colonization and infection with LRSE affecting 14 patients during a 15 month period. LRSE isolates were analysed with respect to their clonal relatedness, antimicrobial susceptibility, the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. cfr plasmids were characterized by Illumina sequencing. Medical records were reviewed and antibiotic consumption was determined. Results Molecular typing identified the presence of three different LRSE clusters: PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Ten strains harboured the cfr gene; we also detected mutations in the respective ribosomal protein genes. WGS revealed an almost identical 39 kb cfr plasmid obtained from strains of different genetic background (ST2, ST5, ST168) that shows high similarity to the recently published LRSE plasmid p12-02300. Due to an increase in the number of patients treated for infections with MRSA, a significant increase in linezolid usage was noted from January to July 2014 (from 5.55 to 20.41 DDDs/100 patient-days). Conclusions Here, we report the molecular epidemiology of LRSE in an ICU. Our results suggest the selection of resistant mutants under linezolid treatment as well as the spread of cfr-carrying plasmids. The reduction of linezolid usage and the strengthening of contact precautions proved to be effective infection control measures. Introduction Oxazolidinones represent a class of synthetic bacterial agents with activity against Gram-positive organisms. Linezolid was the first oxazolidinone introduced for clinical use. Indications comprise the treatment of uncomplicated and complicated skin and skin structure infections and hospital- and community-acquired pneumonia. It is also used as a last-resort antibiotic to treat infections with MDR bacteria including some Staphylococcus species.1 Oxazolidinones disrupt bacterial protein synthesis by binding to the peptidyl transferase centre of the ribosome, thereby inhibiting transition of aminoacyl-tRNA to the A site.2 Development of linezolid resistance among staphylococci is based on multiple mechanisms: (i) mutations in the linezolid 23S rRNA binding site; (ii) mutations in genes encoding the 50S ribosomal proteins L3, L4 and L22 of the peptide translocation centre (PTC) of the ribosome; and (iii) acquisition of a plasmid-encoded, transferable methyltransferase cfr and/or the ABC transporter optrA.3 While optrA confers non-susceptibility to oxazolidinones and phenicols, the methyltransferase cfr mediates resistance to at least five antimicrobial classes, resulting in a phenotype referred to as PhLOPSA (phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics).4–8 Among CoNS, Staphylococcus epidermidis causes the greatest number of infections. S. epidermidis are commensal bacteria; therefore, infections in humans with these opportunistic bacteria are often linked to implanted medical devices or to immune-suppression therapy.9,10 Especially implant-associated infections are difficult to treat due to the fact that clinical S. epidermidis isolates very often exhibit an MDR phenotype.11 Recent surveillance studies using large collections of Gram-positive bacteria indicate that linezolid-non-susceptible isolates are rare.1,12,13 However, outbreaks with linezolid-resistant S. epidermidis (LRSE) have been recently reported from countries worldwide.11,14–18 Some reports could link the prolonged or increased linezolid utilization to the emergence of linezolid-resistant strains.10,17,19 Linezolid-dependent growth of S. epidermidis strains, as shown for ST22 clones from Greece, was speculated to be causative for this observation.18,20 Here, we report the molecular epidemiology, control and management of LRSE in an interdisciplinary ICU of a secondary care hospital in North Rhine-Westphalia, Germany. The detection of the cfr gene in highly similar extrachromosomal DNA in different genetic backgrounds of LRSE suggested transmissibility of the resistance gene cfr. Methods Ethics approval All patient data used in this scientific work were anonymized. According to the German Protection against Infection Act [Infektionsschutzgesetz (IfSG)], infection control units are obliged not only to investigate possible nosocomial outbreaks but also to intervene with adequate infection control measures deduced from results of outbreak investigations.21 Therefore, no ethics approval was necessary. Outbreak setting The emergence of LRSE affected an interdisciplinary ICU of a 465 bed secondary care hospital in North Rhine-Westphalia. The 21 bed ICU included the following disciplines: internal medicine, surgery, urology, gynaecology and otorhinolaryngology. Patients were admitted from other hospitals, the community and from within the hospital. Cases and bacterial isolates Colonization was defined as detection of S. epidermidis without any sign of infection. We defined infections according to the current surveillance definitions for nosocomial infections of the CDC published by the National Reference Center for the Surveillance of Nosocomial Infections in 2011.22 Linezolid resistance was defined as MIC >4 mg/L (according to EUCAST guidelines). S. epidermidis was cultured according to routine diagnostic procedures. Screening for LRSE was performed using blood agar plates with linezolid discs to isolate staphylococci from the inhibition zone. Antibiotic susceptibility was determined using VITEK II. For environmental samples, swabs were cultured on blood agar plates and in tryptic soy broth. Identification of suspicious colonies was performed by MALDI-TOF. The S. epidermidis strains isolated were sent to the German National Reference Centre (NRC) for Staphylococci and Enterococci at the Robert Koch Institute (Wernigerode, Germany) for further characterization (n = 16; Table S1, available as Supplementary data at JAC Online). The isolates were cultured on sheep blood agar and S. epidermidis species were confirmed by biochemical reactions and by the negative tube coagulase test using human plasma. Antimicrobial susceptibility testing At the NRC the antimicrobial susceptibility of the isolates was determined by broth microdilution applying EUCAST breakpoints (www.eucast.org); Etest® for linezolid was used to confirm preliminary results. The following antibiotics were tested: penicillin, oxacillin, cefoxitin, fosfomycin, gentamicin, linezolid, erythromycin, clindamycin, tetracycline, tigecycline, vancomycin, teicoplanin, ciprofloxacin, mupirocin, moxifloxacin, daptomycin, fusidic acid, rifampicin and trimethoprim/sulfamethoxazole. DNA extraction Strains were grown overnight in tryptic soy broth at 37°C. DNA was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Lysostaphin (100 mg/L; Sigma, Munich, Germany) was added to the cell-lysis step. Detection of the cfr gene and analysis of ribosomal target site mutations Isolates were screened for the presence of the cfr gene by PCR as described previously.23 To analyse possible linezolid resistance-mediating mutations, the rplC, rplD and rplV genes and partial sequences of the 23S rRNA were amplified by PCR.5,6 Amplicons were sequenced and the software package Geneious 7.1 was used to analyse the nucleotide and derived amino acid sequences. Results were compared with the S. epidermidis reference strain ATCC 12228. Molecular typing SmaI macrorestriction analysis was performed according to the HARMONY protocol.24 Cluster analysis was performed with the BioNumerics software package (Applied Maths, Sint-Martens-Latem, Belgium), using the Dice coefficient, and visualized as a dendrogram by unweighted pair group method with arithmetic mean (UPGMA) with 1% tolerance and 1% optimization settings. A similarity cut-off of 90% was used to define clusters, which were designated by roman numbers. MLST for S. epidermidis was carried out as published elsewhere.25 WGS and bioinformatics analyses WGS was performed on three cfr-carrying strains, representing the different clonal lineages: 13-04707 (ST168), 14-01514 (ST5) and 15-00164 (ST2). One nanogram of genomic DNA was used for library preparation utilizing the Nextera XT Library Prep Kit according to the manufacturer’s instructions (Illumina). WGS was performed on a MiSeq instrument and in paired-end mode with a maximum readout of 2 × 300 bp. De novo assembly of the reads was carried out using the assembly algorithm a5-miseq and default parameters.26 Obtained cfr-containing contigs produced overlapping sequences for isolate 14-01514 only, of which a preliminary plasmid sequence of 38.550 bp was extracted. Closed circle confirmation of all three putative plasmids was verified by PCR. Oligonucleotides 13-04707cc_fw and 13-04707cc_rv were used to amplify the contig ends of p13-04707 and p15-00164 or primers 14-01514cc_fw and 14-01514cc_rv to verify ring closure of p14-01514. A reaction consisting of 12.5 μL of 2 × DreamTaq Green Master Mix, 0.1 μM of each primer and 0.5 μL of whole genomic DNA in a total volume of 25 μL was subjected to the following PCR protocol: 94°C for 120 s, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 120 s with a final elongation for 240 s at 72°C. If necessary, Sanger sequencing of the PCR product was carried out. According to these results, isolate 14-01514 revealed an additional 693 bp compared with the consensus sequence extracted from WGS data, which were manually added to the final plasmid sequence (39.243 bp). Plasmid p14-01514 was annotated utilizing the automatic annotation tool provided by NCBI. Amplification of the repetitive region of p13-04707 and p15-00164 was carried out using oligonucleotides 14-01514cc_fw and 14-01514cc_rv in a reaction volume and a PCR profile as stated above. Fragments were analysed by Sanger sequencing and evaluated in Geneious. Accession numbers Plasmid p14-01514 was deposited in GenBank under accession number KX520649. Raw reads of all three plasmids are available from SRA under accession number SRP078124. Recording of linezolid consumption The monthly linezolid use in the ICU since 2013 was recorded in DDDs/100 patient-days with special emphasis on the usage between the end of 2013 and the beginning 2015. Linezolid doses of 600 mg/day were considered 1 DDD. Data were benchmarked to linezolid prescriptions of 2013–15. Additionally, the occurrence of patients with infections due to MRSA was investigated to explain the consumption of linezolid in 2014. Results Description of the outbreak, infection control measures and interventions During November 2013 and January 2015 we detected LRSE in 14 patients (Table 1). The median age of the patients was 76 (57–90) years with predominance of the male gender (10/4). Table 1. Characteristics of patients with LRSE in chronological order Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      ERCP, endoscopic retrograde cholangiopancreatography; CPR, cardiopulmonary resuscitation; SU, surgical unit; BSI, bloodstream infection; UTI, urinary tract infection; LZD-R, linezolid resistant. a Infection: cases were defined as described elsewhere.22 b Linezolid consumption of the respective patient before the first LRSE was cultured. Linezolid doses of 600 mg/day were considered 1 DDD. c For Patient 2, three LRSE isolates were sent to the NRC for further analysis. Table 1. Characteristics of patients with LRSE in chronological order Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      ERCP, endoscopic retrograde cholangiopancreatography; CPR, cardiopulmonary resuscitation; SU, surgical unit; BSI, bloodstream infection; UTI, urinary tract infection; LZD-R, linezolid resistant. a Infection: cases were defined as described elsewhere.22 b Linezolid consumption of the respective patient before the first LRSE was cultured. Linezolid doses of 600 mg/day were considered 1 DDD. c For Patient 2, three LRSE isolates were sent to the NRC for further analysis. In July 2014 LRSE were identified in an intraoperative wound swab, a secretion drainage and in a blood culture of three patients (Figure 1; Patients 6, 7 and 8). In retrospect five further patients with LRSE in blood cultures, urine and central venous catheter tips were detected from November 2013 to May 2014 (Patients 1–5). An outbreak with LRSE was hypothesized and clonal transmissions were suspected. From August to January 2015 we identified prospectively four patients with LRSE in clinical samples (Patients 9–11 and 14), including one case of a bloodstream infection (Patient 11) (Figure 1). To assess the spread of this outbreak the first infected patient (Patient 11) was screened for further colonization and tested positive in several screening cultures (axilla, rectal, wound swab). To evaluate the extent of colonization of other patients in this ward, axilla, rectum and wounds of the remaining patients were also screened. Two more patients (12 and 13) colonized with LRSE were detected. To evaluate contamination of the patient-near environment, an environmental screening of the room of Patient 11 was conducted, which revealed no LRSE. Nevertheless, all surfaces in the patient room were disinfected and all disposable medical products exchanged. Figure 1. View largeDownload slide Time list of patients positive for LRSE and strain characteristics. Data in light grey bars display the length of stay in the ICU of the respective patient. The dark grey bars indicate the months in which patients received linezolid therapy. LRSE were obtained from the following material: blood culture (B), central line tip (C), wound intraoperative (W), secretion drainage (D), decubitus swab (DS), urine (U) and screening (S). Strains indicated with an asterisk were sent to the NRC for further characterization. LZD, linezolid; LZD-S, linezolid susceptible. Figure 1. View largeDownload slide Time list of patients positive for LRSE and strain characteristics. Data in light grey bars display the length of stay in the ICU of the respective patient. The dark grey bars indicate the months in which patients received linezolid therapy. LRSE were obtained from the following material: blood culture (B), central line tip (C), wound intraoperative (W), secretion drainage (D), decubitus swab (DS), urine (U) and screening (S). Strains indicated with an asterisk were sent to the NRC for further characterization. LZD, linezolid; LZD-S, linezolid susceptible. In order to prevent further transmission, we implemented contact isolation measures for all patients positive for LRSE. Contact isolation precautions included wearing gloves and aprons while entering the patient’s room in compliance with the recommendations of the CDC and the Robert Koch Institute to manage patients with MRSA.27,28 LRSE-positive patients were labelled in the hospital’s alert system, including additional information about recommended isolation precautions. The ICU staff were trained again in hand hygiene and the management of medical devices and got timely feedback information about LRSE-positive patients. In addition, linezolid consumption of the ICU was reviewed. The annual data corresponded to 7.0 DDDs/100 patient-days in 2013, 11.5 DDDs/100 patient-days in 2014 and 6.7 DDDs/100 patient-days in 2015. A significant increase in linezolid usage was observed, which started at the end of 2013 with a peak of 20.4 DDDs/100 patient-days in July 2014 (Figure 2). This peak correlated with the treatment of 10 patients with MRSA infections between February and July 2014, whereby linezolid was frequently part of the empirical choice of antibiotics, especially if MRSA pneumonia was suspected. After a maximum in monthly consumption in July 2014 linezolid use declined to a minimum of 2.02 DDDs/100 patient-days in October 2014. Again, in December 2014 linezolid use increased due to the treatment of MRSA patients. Both hospital and ICU consumption of linezolid and other antibiotic classes exceeded different reference levels, as indicated in Table S2. Figure 2. View largeDownload slide Linezolid usage data of the ICU by month from July 2013 to August 2015 versus the number of LRSE-positive patients. Linezolid consumption is calculated in DDDs/100 patient-days; linezolid doses of 600 mg/day were considered 1 DDD. Bars indicate the number of patients with first detection of LRSE in the respective month. Figure 2. View largeDownload slide Linezolid usage data of the ICU by month from July 2013 to August 2015 versus the number of LRSE-positive patients. Linezolid consumption is calculated in DDDs/100 patient-days; linezolid doses of 600 mg/day were considered 1 DDD. Bars indicate the number of patients with first detection of LRSE in the respective month. All except one patient (Patient 12) with LRSE had received linezolid, prior to colonization or infection with the resistant strain, as empirical treatment or for treatment of infections caused by MRSA or VRE (Table 2). To reduce linezolid use, prescriptions were discussed and the indications were double-checked by the infection control team and the physicians in charge. Alternative antibiotics were recommended to treat infections due to multiresistant Gram-positive organisms. A report was issued to the public health department. Table 2. Characterization of S. epidermidis strains exhibiting increased linezolid MICs; strains are grouped according to their ST, linezolid MIC and putative linezolid-resistance mechanisms No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  a The nucleotide positions of the mutations are listed according to Escherichia coli numbering. b The nucleotide positions of the mutations are listed according to S. aureus numbering. c Linezolid-susceptible S. epidermidis control. Table 2. Characterization of S. epidermidis strains exhibiting increased linezolid MICs; strains are grouped according to their ST, linezolid MIC and putative linezolid-resistance mechanisms No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  a The nucleotide positions of the mutations are listed according to Escherichia coli numbering. b The nucleotide positions of the mutations are listed according to S. aureus numbering. c Linezolid-susceptible S. epidermidis control. Molecular typing of bacterial isolates The entire set of 16 LRSE isolates was examined by macrorestriction analysis, which yielded three distinct clusters (Table 2): PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Antimicrobial susceptibility and putative linezolid-resistance mechanisms The isolates showed decreased susceptibility to linezolid, exhibiting MICs in the range of 8 to >256 mg/L (Table 1). They displayed a multiresistant phenotype, also being resistant to oxacillin (n = 15, 100%), gentamicin (n = 11, 73.3%), ciprofloxacin (n = 15, 100%), moxifloxacin (n = 15, 100%), co-trimoxazole (n = 14, 93.3%), mupirocin (n = 3, 20%), clindamycin (n = 9, 60%), erythromycin (n = 12, 80%), rifampicin (n = 10, 66.7%), tetracycline (n = 4, 26.67%) and teicoplanin (n = 2, 13.33%). Resistance to fusidic acid, tigecycline, vancomycin and daptomycin was not observed. Table 2 summarizes the results with respect to the presence of the cfr gene, mutations in the 23S rRNA and in genes encoding ribosomal proteins for all the isolates and also for the linezolid-susceptible control strain ATCC 12228. C2190T was the only modification found in the linezolid 23S rRNA binding site. Sequence chromatograms showed no evidence of nucleotide mixtures at the respective position. Amino acid alterations in L3 comprised Leu101Val, His146Gln, Gly152Asp, Val154Leu and Ala157Arg. Amino acid changes in L4 were found at the following positions: Ser81Arg; Asn158Ser; and a Gly71 insertion. Mutations in rplV were synonymous when detected. Ten strains (62.5%) harboured the cfr gene. In summary, all strains exhibiting PFGE type I/ST168 and PFGE type III/ST2 tested positive for cfr, whereas only four strains of the PFGE type II/ST5 cluster harboured the resistance determinant (Table 1). No apparent correlation was observed between the occurrence of certain nucleotide mutations, the presence of cfr and the level of linezolid resistance. Description of cfr-carrying plasmids Each cfr-positive isolate carried the cfr gene on an ∼40 kb plasmid as determined by S1 nuclease PFGE and Southern hybridization analysis using a cfr-specific probe (data not shown). De novo assembly and manual polishing of the contigs received by WGS produced a 39.243 bp plasmid for strain 14-01514 (termed p14-01514), which harboured cfr and the phenicol exporter fexA and had features highly similar to the previously published vector p12-02300.15 Mapping of reads from S. epidermidis 14-01514 to p12-02300 revealed 99 SNPs. In addition, p12-2300 is 379 bp shorter compared with p14-01514. A BLAST search on this particular variable region yielded homologue sequences from plasmids of Staphylococcus aureus, S. epidermidis (p12-02300) and Staphylococcus cohnii, all of which were located in non-coding regions (not shown). Further examination of the sequence utilizing the Tandem Repeats Finder program29 revealed a conserved repetitive region of 63 bp, which was present 8.7 times in p12-2300 and 14.7 times in p14-01514. The repetitive region was also investigated in S. epidermidis 13-04707 and 15-00164. PCR and subsequent Sanger sequencing showed that both cfr plasmids exhibit 189 bp less than p14-01514, which corresponds to three tandem repeats. This is in accordance with the results from Tandem Repeats Finder, indicating that the 63 bp repetitive sequence was present 11.7 times in p13-04707 and p15-00164, respectively. With a final cfr-containing contig size of 38.273 and 38.450 bp after de novo assembly, no overlapping sequences representing putative plasmids were obtained for S. epidermidis isolates 13-04707 and 15-00164. A comparison of these contigs with p14-01514 yielded 100% identity of the truncated sequences from 13-04707 and 15-00164 to the novel vector, hence suggesting a highly similar plasmid content of these isolates. Taking into account that cfr plasmids from S. epidermidis 13-04707 and 15-00164 differ in the total amount of the 63 bp tandem repeats (see above) but are otherwise identical to p14-01514, the putative final plasmid sizes for p13-04707 and p15-00164 were set to 39.054 bp each. We herein identified three almost identical cfr plasmids with high similarity to the recently described vector from S. epidermidis 12-02300.15 Discussion Management of the outbreak in the ICU There is a plethora of articles describing the emergence and outbreaks with linezolid-resistant staphylococci worldwide.11,14–18,30–32 However, there are just a few detailed reports including data on linezolid consumption, infection control measures and interventions.10,17,19,30,31,33 To control the outbreak we implemented contact isolation measures of LRSE-positive patients. Training courses on hand hygiene were organized for the ICU staff and data concerning the outbreak were regularly presented and discussed. A previous report from O’Connor et al.17 showed that during a cfr-mediated LRSE outbreak the staff lacked knowledge concerning resistance mechanisms and ways of transmission. They implemented a hospital-wide education programme for healthcare staff thenceforward.17 Another group from Spain reported an outbreak of linezolid-resistant S. aureus (LRSA) in 2008 and performed staff screening. While no staff member was found to be colonized, they could isolate LRSA from environmental surfaces.33 We did not perform screening of staff for LRSE carriage and only conducted environmental screening of the room in the case of the bacteraemia patient, where no LRSE was detected. Overlapping stays of patients in the ICU with LRSE ST5 and ST168, respectively, suggested the dissemination of two endemic clones. This spread could have been caused by transmission directly via the hands of healthcare workers (HCWs), indirectly via environmental contamination or both. Whether or not the skin flora of HCWs serves as a source for LRSE is unclear and we did not investigate a potential impact. However, it is well known that HCWs serve as vectors for strain transmission from patient to patient via contaminated hands, skin and environments. Empirical therapy of severe infections in MRSA-positive and MRSA-negative patients with linezolid seemed to facilitate the concomitant selection and spread of LRSE on the ward. Treatment with linezolid can suppress the susceptible cutaneous flora of patients. Hence, resistant strains may become predominant. Even if colonization of patients with LRSE is microbiologically not yet detectable, therapy with linezolid could initiate selection of the earlier-transmitted resistant strain. Therefore, our infection control measures included the restriction of linezolid prescription, which has already been published by Mulanovich et al.19 and others.30–34 They proposed a threshold of ≥13 DDDs/100 patient-days being associated with the development of an outbreak of LRSE.19 In accordance with the present study, the increased use of linezolid seemed efficient in promoting the emergence and persistence of linezolid-resistant clones in ICU patients.20,35 In conclusion, prospective surveillance, screening procedures, isolation precautions, education of ICU staff and providing them with information, control of MRSA and the restriction of linezolid use were efficient in controlling the emergence and transmission of LRSE. Molecular epidemiology of LRSE Worldwide, the nosocomial S. epidermidis population is dominated by strains that belong to the MLST clonal complex 5 (CC5). These epidemic clones are multiresistant, can persist in hospitals over a long period of time and seem to adapt quickly to different environments through recombination and frequent exchange of genetic mobile elements.36–39 In the present study three different STs were identified: ST2, ST5 and ST168 (a single-locus variant of ST2), all of them belonging to CC5. SmaI macrorestriction analysis confirmed the presence of three distinct clusters; no further differentiation was possible. Ribosomal mutations were analysed, thereby confirming an identical pattern of ribosomal protein alterations within the respective clade and indicating the spread of LRSE ST5 and ST168 on the ward. LRSE ST2 was only detected once, in January 2015, from the blood culture of the last defined case. LRSE ST168 and ST2 and 4/10 LRSE ST5 carried a cfr plasmid. Due to the sequencing of one plasmid from each clonal lineage, we confirmed the presence of a highly similar cfr plasmid in all three genetic backgrounds. The acquisition of this plasmid via horizontal gene transfer is possible, as similar scenarios with the respective conjugative cfr vectors are described elsewhere.15,40 There are only a few reports dealing with outbreaks caused by LRSE in Europe.34 In 2015 we published data concerning clusters of LRSE ST2 and ST22 in German hospitals, where we showed evidence for the acquisition of cfr plasmids via horizontal gene transfer during spread of the respective strains.15 The same year, O’Connor et al.17 described the first cfr-mediated LRSE outbreak in the Republic of Ireland. Several European studies present evidence for the presence of endemic LRSE clones that circulate in hospital settings.18,41,42 Similar to our study, these strains differ from commensal S. epidermidis isolates by exhibiting MDR and biofilm-forming capabilities, therefore being probably more successful in the hospital environment.39,43,44 Linezolid-resistance mechanisms We examined the isolates for molecular mechanisms that may contribute to linezolid resistance in CoNS. All strains showed a C2190T modification of the 23S rRNA that is occasionally described in linezolid-resistant CoNS.34 In contrast to G2576T, its association with increased linezolid MICs has not been functionally verified so far.1,7,34 Mutations in rplC and rplD genes, encoding 50S ribosomal proteins of the PTC of the ribosome, revealed non-synonymous mutations in the 50S ribosomal proteins L3 and L4. The impact of these alterations on the structure of the PTC and linezolid binding remains to be determined, as the influence of L3 mutations on its tertiary folding was shown in silico.45 An earlier study using laboratory-derived linezolid-resistant strains confirmed a correlation for selected L3 and L4 mutations.46 Although many articles report multiple ribosomal mutations in clinical LRSE, data concerning the functional proof of the putative resistance mechanisms are rare.1,8,15,47 However, a combination of mutations leads to functional and structural adaptation to linezolid and promotes a faster growth rate of linezolid-dependent strains.20,35 Ten (62.5%) of our isolates additionally carried a cfr-positive plasmid. In all cases cfr was detected in combination with alterations in ribosomal genes. WGS showed that all cfr plasmids were highly similar to the recently described cfr plasmid p12-02300 from a clinical S. epidermidis isolate in Germany15 and differed only by varying numbers of tandem repeats. The function of these sequences requires further investigation. As a result of highly similar plasmid content, the spread of endemic clones in addition to dissemination of the cfr plasmid(s) within the hospital environment can be suspected. Acknowledgements We acknowledge Dr Helmers and Dr Téllez-Castillo from MVZ Synlab Leverkusen for microbial routine diagnostics and for providing LRSE isolates and the pharmacist Hartmut Paul for providing us with monthly data on antibiotic use at the investigated ICU. We thank the staff at the NRC for excellent technical assistance. Funding The study used core funding from the hospitals of the city of Cologne. The German Reference Centre for Staphylococci and Enterococci is funded by the German Federal Ministry of Health. Transparency declarations None to declare. Supplementary data Tables S1 and S2 are available as Supplementary data at JAC Online. References 1 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  2 Swaney SM, Aoki H, Ganoza MC et al.   The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrob Agents Chemother  1998; 42: 3251– 5. Google Scholar PubMed  3 Fan R, Li D, Wang Y et al.   Presence of the optrA gene in methicillin-resistant Staphylococcus sciuri of porcine origin. Antimicrob Agents Chemother  2016; 60: 7200– 5. Google Scholar PubMed  4 Toh SM, Xiong L, Arias CA et al.   Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. 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Google Scholar PubMed  16 Cui L, Wang Y, Li Y et al.   Cfr-mediated linezolid-resistance among methicillin-resistant coagulase-negative staphylococci from infections of humans. PLoS One  2013; 8: e57096. Google Scholar CrossRef Search ADS PubMed  17 O’Connor C, Powell J, Finnegan C et al.   Incidence, management and outcomes of the first cfr-mediated linezolid-resistant Staphylococcus epidermidis outbreak in a tertiary referral centre in the Republic of Ireland. J Hosp Infect  2015; 90: 316– 21. Google Scholar CrossRef Search ADS PubMed  18 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  19 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  20 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  21 Infektionsschutzgesetz vom 20. Juli 2000 (BGBl. I S. 1045), das zuletzt durch Artikel 4 Absatz 20 des Gesetzes vom 18. Juli 2016 (BGBl. I S. 1666) geändert worden ist. BGBl I 2000: 1045. 22 Definitionen nosokomialer Infektionen (CDC Definitionen). 7. Auflage/Robert Koch-Institut, Nationales Referenzzentrum für Surveillance von nosokomialen Infektionen. Berlin, 2011. 23 Kehrenberg C, Schwarz S. Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant Staphylococcus isolates. Antimicrob Agents Chemother  2006; 50: 1156– 63. 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Google Scholar CrossRef Search ADS PubMed  43 Cherifi S, Byl B, Deplano A et al.   Genetic characteristics and antimicrobial resistance of Staphylococcus epidermidis isolates from patients with catheter-related bloodstream infections and from colonized healthcare workers in a Belgian hospital. Ann Clin Microbiol Antimicrob  2014; 13: 20. Google Scholar CrossRef Search ADS PubMed  44 Cherifi S, Byl B, Deplano A et al.   Comparative epidemiology of Staphylococcus epidermidis isolates from patients with catheter-related bacteremia and from healthy volunteers. J Clin Microbiol  2013; 51: 1541– 7. Google Scholar CrossRef Search ADS PubMed  45 Ikonomidis A, Grapsa A, Pavlioglou C et al.   Accumulation of multiple mutations in linezolid-resistant Staphylococcus epidermidis causing bloodstream infections; in silico analysis of L3 amino acid substitutions that might confer high-level linezolid resistance. J Chemother  2016; 28: 465– 8. Google Scholar CrossRef Search ADS PubMed  46 Locke JB, Hilgers M, Shaw KJ. Novel ribosomal mutations in Staphylococcus aureus strains identified through selection with the oxazolidinones linezolid and torezolid (TR-700). Antimicrob Agents Chemother  2009; 53: 5265– 74. Google Scholar CrossRef Search ADS PubMed  47 Tewhey R, Gu B, Kelesidis T et al.   Mechanisms of linezolid resistance among coagulase-negative staphylococci determined by whole-genome sequencing. MBio  2014; 5: e00894– 14. 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. 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Emergence and control of linezolid-resistant Staphylococcus epidermidis in an ICU of a German hospital

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Oxford University Press
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© 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/dky010
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Abstract

Abstract Objectives To investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE) in an interdisciplinary ICU, linezolid consumption and infection control measures taken. Methods Routine surveillance of nosocomial infections revealed colonization and infection with LRSE affecting 14 patients during a 15 month period. LRSE isolates were analysed with respect to their clonal relatedness, antimicrobial susceptibility, the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. cfr plasmids were characterized by Illumina sequencing. Medical records were reviewed and antibiotic consumption was determined. Results Molecular typing identified the presence of three different LRSE clusters: PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Ten strains harboured the cfr gene; we also detected mutations in the respective ribosomal protein genes. WGS revealed an almost identical 39 kb cfr plasmid obtained from strains of different genetic background (ST2, ST5, ST168) that shows high similarity to the recently published LRSE plasmid p12-02300. Due to an increase in the number of patients treated for infections with MRSA, a significant increase in linezolid usage was noted from January to July 2014 (from 5.55 to 20.41 DDDs/100 patient-days). Conclusions Here, we report the molecular epidemiology of LRSE in an ICU. Our results suggest the selection of resistant mutants under linezolid treatment as well as the spread of cfr-carrying plasmids. The reduction of linezolid usage and the strengthening of contact precautions proved to be effective infection control measures. Introduction Oxazolidinones represent a class of synthetic bacterial agents with activity against Gram-positive organisms. Linezolid was the first oxazolidinone introduced for clinical use. Indications comprise the treatment of uncomplicated and complicated skin and skin structure infections and hospital- and community-acquired pneumonia. It is also used as a last-resort antibiotic to treat infections with MDR bacteria including some Staphylococcus species.1 Oxazolidinones disrupt bacterial protein synthesis by binding to the peptidyl transferase centre of the ribosome, thereby inhibiting transition of aminoacyl-tRNA to the A site.2 Development of linezolid resistance among staphylococci is based on multiple mechanisms: (i) mutations in the linezolid 23S rRNA binding site; (ii) mutations in genes encoding the 50S ribosomal proteins L3, L4 and L22 of the peptide translocation centre (PTC) of the ribosome; and (iii) acquisition of a plasmid-encoded, transferable methyltransferase cfr and/or the ABC transporter optrA.3 While optrA confers non-susceptibility to oxazolidinones and phenicols, the methyltransferase cfr mediates resistance to at least five antimicrobial classes, resulting in a phenotype referred to as PhLOPSA (phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics).4–8 Among CoNS, Staphylococcus epidermidis causes the greatest number of infections. S. epidermidis are commensal bacteria; therefore, infections in humans with these opportunistic bacteria are often linked to implanted medical devices or to immune-suppression therapy.9,10 Especially implant-associated infections are difficult to treat due to the fact that clinical S. epidermidis isolates very often exhibit an MDR phenotype.11 Recent surveillance studies using large collections of Gram-positive bacteria indicate that linezolid-non-susceptible isolates are rare.1,12,13 However, outbreaks with linezolid-resistant S. epidermidis (LRSE) have been recently reported from countries worldwide.11,14–18 Some reports could link the prolonged or increased linezolid utilization to the emergence of linezolid-resistant strains.10,17,19 Linezolid-dependent growth of S. epidermidis strains, as shown for ST22 clones from Greece, was speculated to be causative for this observation.18,20 Here, we report the molecular epidemiology, control and management of LRSE in an interdisciplinary ICU of a secondary care hospital in North Rhine-Westphalia, Germany. The detection of the cfr gene in highly similar extrachromosomal DNA in different genetic backgrounds of LRSE suggested transmissibility of the resistance gene cfr. Methods Ethics approval All patient data used in this scientific work were anonymized. According to the German Protection against Infection Act [Infektionsschutzgesetz (IfSG)], infection control units are obliged not only to investigate possible nosocomial outbreaks but also to intervene with adequate infection control measures deduced from results of outbreak investigations.21 Therefore, no ethics approval was necessary. Outbreak setting The emergence of LRSE affected an interdisciplinary ICU of a 465 bed secondary care hospital in North Rhine-Westphalia. The 21 bed ICU included the following disciplines: internal medicine, surgery, urology, gynaecology and otorhinolaryngology. Patients were admitted from other hospitals, the community and from within the hospital. Cases and bacterial isolates Colonization was defined as detection of S. epidermidis without any sign of infection. We defined infections according to the current surveillance definitions for nosocomial infections of the CDC published by the National Reference Center for the Surveillance of Nosocomial Infections in 2011.22 Linezolid resistance was defined as MIC >4 mg/L (according to EUCAST guidelines). S. epidermidis was cultured according to routine diagnostic procedures. Screening for LRSE was performed using blood agar plates with linezolid discs to isolate staphylococci from the inhibition zone. Antibiotic susceptibility was determined using VITEK II. For environmental samples, swabs were cultured on blood agar plates and in tryptic soy broth. Identification of suspicious colonies was performed by MALDI-TOF. The S. epidermidis strains isolated were sent to the German National Reference Centre (NRC) for Staphylococci and Enterococci at the Robert Koch Institute (Wernigerode, Germany) for further characterization (n = 16; Table S1, available as Supplementary data at JAC Online). The isolates were cultured on sheep blood agar and S. epidermidis species were confirmed by biochemical reactions and by the negative tube coagulase test using human plasma. Antimicrobial susceptibility testing At the NRC the antimicrobial susceptibility of the isolates was determined by broth microdilution applying EUCAST breakpoints (www.eucast.org); Etest® for linezolid was used to confirm preliminary results. The following antibiotics were tested: penicillin, oxacillin, cefoxitin, fosfomycin, gentamicin, linezolid, erythromycin, clindamycin, tetracycline, tigecycline, vancomycin, teicoplanin, ciprofloxacin, mupirocin, moxifloxacin, daptomycin, fusidic acid, rifampicin and trimethoprim/sulfamethoxazole. DNA extraction Strains were grown overnight in tryptic soy broth at 37°C. DNA was extracted using the DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Lysostaphin (100 mg/L; Sigma, Munich, Germany) was added to the cell-lysis step. Detection of the cfr gene and analysis of ribosomal target site mutations Isolates were screened for the presence of the cfr gene by PCR as described previously.23 To analyse possible linezolid resistance-mediating mutations, the rplC, rplD and rplV genes and partial sequences of the 23S rRNA were amplified by PCR.5,6 Amplicons were sequenced and the software package Geneious 7.1 was used to analyse the nucleotide and derived amino acid sequences. Results were compared with the S. epidermidis reference strain ATCC 12228. Molecular typing SmaI macrorestriction analysis was performed according to the HARMONY protocol.24 Cluster analysis was performed with the BioNumerics software package (Applied Maths, Sint-Martens-Latem, Belgium), using the Dice coefficient, and visualized as a dendrogram by unweighted pair group method with arithmetic mean (UPGMA) with 1% tolerance and 1% optimization settings. A similarity cut-off of 90% was used to define clusters, which were designated by roman numbers. MLST for S. epidermidis was carried out as published elsewhere.25 WGS and bioinformatics analyses WGS was performed on three cfr-carrying strains, representing the different clonal lineages: 13-04707 (ST168), 14-01514 (ST5) and 15-00164 (ST2). One nanogram of genomic DNA was used for library preparation utilizing the Nextera XT Library Prep Kit according to the manufacturer’s instructions (Illumina). WGS was performed on a MiSeq instrument and in paired-end mode with a maximum readout of 2 × 300 bp. De novo assembly of the reads was carried out using the assembly algorithm a5-miseq and default parameters.26 Obtained cfr-containing contigs produced overlapping sequences for isolate 14-01514 only, of which a preliminary plasmid sequence of 38.550 bp was extracted. Closed circle confirmation of all three putative plasmids was verified by PCR. Oligonucleotides 13-04707cc_fw and 13-04707cc_rv were used to amplify the contig ends of p13-04707 and p15-00164 or primers 14-01514cc_fw and 14-01514cc_rv to verify ring closure of p14-01514. A reaction consisting of 12.5 μL of 2 × DreamTaq Green Master Mix, 0.1 μM of each primer and 0.5 μL of whole genomic DNA in a total volume of 25 μL was subjected to the following PCR protocol: 94°C for 120 s, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 120 s with a final elongation for 240 s at 72°C. If necessary, Sanger sequencing of the PCR product was carried out. According to these results, isolate 14-01514 revealed an additional 693 bp compared with the consensus sequence extracted from WGS data, which were manually added to the final plasmid sequence (39.243 bp). Plasmid p14-01514 was annotated utilizing the automatic annotation tool provided by NCBI. Amplification of the repetitive region of p13-04707 and p15-00164 was carried out using oligonucleotides 14-01514cc_fw and 14-01514cc_rv in a reaction volume and a PCR profile as stated above. Fragments were analysed by Sanger sequencing and evaluated in Geneious. Accession numbers Plasmid p14-01514 was deposited in GenBank under accession number KX520649. Raw reads of all three plasmids are available from SRA under accession number SRP078124. Recording of linezolid consumption The monthly linezolid use in the ICU since 2013 was recorded in DDDs/100 patient-days with special emphasis on the usage between the end of 2013 and the beginning 2015. Linezolid doses of 600 mg/day were considered 1 DDD. Data were benchmarked to linezolid prescriptions of 2013–15. Additionally, the occurrence of patients with infections due to MRSA was investigated to explain the consumption of linezolid in 2014. Results Description of the outbreak, infection control measures and interventions During November 2013 and January 2015 we detected LRSE in 14 patients (Table 1). The median age of the patients was 76 (57–90) years with predominance of the male gender (10/4). Table 1. Characteristics of patients with LRSE in chronological order Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      ERCP, endoscopic retrograde cholangiopancreatography; CPR, cardiopulmonary resuscitation; SU, surgical unit; BSI, bloodstream infection; UTI, urinary tract infection; LZD-R, linezolid resistant. a Infection: cases were defined as described elsewhere.22 b Linezolid consumption of the respective patient before the first LRSE was cultured. Linezolid doses of 600 mg/day were considered 1 DDD. c For Patient 2, three LRSE isolates were sent to the NRC for further analysis. Table 1. Characteristics of patients with LRSE in chronological order Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      Patient  Sex  Age (years)  Ward  Culture site  Date of culture  Infection due to LRSEa  Infectiona  MRSA (culture site)  Underlying disease and/or condition  Linezolid consumption (DDDs)b  VRE (culture site)  LZD-R VRE (culture site)  1  male  57  ICU  blood  22/11/2013      negative  haemorrhage after ERCP  15      2c  male  71  ICU  central line tip  02/01/2014    BSI with Enterococcus faecium  negative  fracture of trochanter major  48  positive (urine)    blood  07/03/2014  central line tip  18/03/2014  3  male  87  ICU  urine  12/12/2013      negative  cardiac decompensation, pneumonia  56      4  female  66  ICU  central line tip  15/05/2014    BSI due to UTI and BSI with MRSA  positive (blood, tracheal secretion)  ileus  55      5  male  61  ICU  blood  16/05/2014    BSI with MRSA  positive (blood)  myocardial infarction, CPR  26  positive (wound)    6  male  72  ICU  wound  21/07/2014      negative  ulcus duodeni haemorrhage  17  positive (secretion drainage)    7  male  64  ICU  secretion drainage  22/07/2014    pneumonia with MRSA  positive (punctate)  pancreatic cancer  13      8  female  78  SU  blood  26/07/2014      positive (bronchial secretion)  pneumonia  14      9  female  76  ICU  secretion drainage  01/08/2014      negative  peritonitis  32      10  male  90  ICU  blood  04/08/2014    pneumonia  negative  colon cancer  15      11  male  77  ICU  blood  07/09/2014  BSI    positive (wound)  renal failure  23      12  male  76  ICU  axilla  19/09/2014      negative  media infarction  0      13  female  79  ICU  decubitus  18/09/2014    UTI  negative  incarcerated incisional hernia  65    positive (wound)  14  male  77  ICU  blood  08/01/2015      positive (screening)  pneumonia  14      ERCP, endoscopic retrograde cholangiopancreatography; CPR, cardiopulmonary resuscitation; SU, surgical unit; BSI, bloodstream infection; UTI, urinary tract infection; LZD-R, linezolid resistant. a Infection: cases were defined as described elsewhere.22 b Linezolid consumption of the respective patient before the first LRSE was cultured. Linezolid doses of 600 mg/day were considered 1 DDD. c For Patient 2, three LRSE isolates were sent to the NRC for further analysis. In July 2014 LRSE were identified in an intraoperative wound swab, a secretion drainage and in a blood culture of three patients (Figure 1; Patients 6, 7 and 8). In retrospect five further patients with LRSE in blood cultures, urine and central venous catheter tips were detected from November 2013 to May 2014 (Patients 1–5). An outbreak with LRSE was hypothesized and clonal transmissions were suspected. From August to January 2015 we identified prospectively four patients with LRSE in clinical samples (Patients 9–11 and 14), including one case of a bloodstream infection (Patient 11) (Figure 1). To assess the spread of this outbreak the first infected patient (Patient 11) was screened for further colonization and tested positive in several screening cultures (axilla, rectal, wound swab). To evaluate the extent of colonization of other patients in this ward, axilla, rectum and wounds of the remaining patients were also screened. Two more patients (12 and 13) colonized with LRSE were detected. To evaluate contamination of the patient-near environment, an environmental screening of the room of Patient 11 was conducted, which revealed no LRSE. Nevertheless, all surfaces in the patient room were disinfected and all disposable medical products exchanged. Figure 1. View largeDownload slide Time list of patients positive for LRSE and strain characteristics. Data in light grey bars display the length of stay in the ICU of the respective patient. The dark grey bars indicate the months in which patients received linezolid therapy. LRSE were obtained from the following material: blood culture (B), central line tip (C), wound intraoperative (W), secretion drainage (D), decubitus swab (DS), urine (U) and screening (S). Strains indicated with an asterisk were sent to the NRC for further characterization. LZD, linezolid; LZD-S, linezolid susceptible. Figure 1. View largeDownload slide Time list of patients positive for LRSE and strain characteristics. Data in light grey bars display the length of stay in the ICU of the respective patient. The dark grey bars indicate the months in which patients received linezolid therapy. LRSE were obtained from the following material: blood culture (B), central line tip (C), wound intraoperative (W), secretion drainage (D), decubitus swab (DS), urine (U) and screening (S). Strains indicated with an asterisk were sent to the NRC for further characterization. LZD, linezolid; LZD-S, linezolid susceptible. In order to prevent further transmission, we implemented contact isolation measures for all patients positive for LRSE. Contact isolation precautions included wearing gloves and aprons while entering the patient’s room in compliance with the recommendations of the CDC and the Robert Koch Institute to manage patients with MRSA.27,28 LRSE-positive patients were labelled in the hospital’s alert system, including additional information about recommended isolation precautions. The ICU staff were trained again in hand hygiene and the management of medical devices and got timely feedback information about LRSE-positive patients. In addition, linezolid consumption of the ICU was reviewed. The annual data corresponded to 7.0 DDDs/100 patient-days in 2013, 11.5 DDDs/100 patient-days in 2014 and 6.7 DDDs/100 patient-days in 2015. A significant increase in linezolid usage was observed, which started at the end of 2013 with a peak of 20.4 DDDs/100 patient-days in July 2014 (Figure 2). This peak correlated with the treatment of 10 patients with MRSA infections between February and July 2014, whereby linezolid was frequently part of the empirical choice of antibiotics, especially if MRSA pneumonia was suspected. After a maximum in monthly consumption in July 2014 linezolid use declined to a minimum of 2.02 DDDs/100 patient-days in October 2014. Again, in December 2014 linezolid use increased due to the treatment of MRSA patients. Both hospital and ICU consumption of linezolid and other antibiotic classes exceeded different reference levels, as indicated in Table S2. Figure 2. View largeDownload slide Linezolid usage data of the ICU by month from July 2013 to August 2015 versus the number of LRSE-positive patients. Linezolid consumption is calculated in DDDs/100 patient-days; linezolid doses of 600 mg/day were considered 1 DDD. Bars indicate the number of patients with first detection of LRSE in the respective month. Figure 2. View largeDownload slide Linezolid usage data of the ICU by month from July 2013 to August 2015 versus the number of LRSE-positive patients. Linezolid consumption is calculated in DDDs/100 patient-days; linezolid doses of 600 mg/day were considered 1 DDD. Bars indicate the number of patients with first detection of LRSE in the respective month. All except one patient (Patient 12) with LRSE had received linezolid, prior to colonization or infection with the resistant strain, as empirical treatment or for treatment of infections caused by MRSA or VRE (Table 2). To reduce linezolid use, prescriptions were discussed and the indications were double-checked by the infection control team and the physicians in charge. Alternative antibiotics were recommended to treat infections due to multiresistant Gram-positive organisms. A report was issued to the public health department. Table 2. Characterization of S. epidermidis strains exhibiting increased linezolid MICs; strains are grouped according to their ST, linezolid MIC and putative linezolid-resistance mechanisms No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  a The nucleotide positions of the mutations are listed according to Escherichia coli numbering. b The nucleotide positions of the mutations are listed according to S. aureus numbering. c Linezolid-susceptible S. epidermidis control. Table 2. Characterization of S. epidermidis strains exhibiting increased linezolid MICs; strains are grouped according to their ST, linezolid MIC and putative linezolid-resistance mechanisms No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  No. of isolates  ST  PFGE type  MIC of linezolid (mg/L)  cfr  23SrRNAa  Mutations in ribosomal genes/proteinsb   rplC/L3  rplD/L4  rplV/L22  5  ST168  I  16 to >256  +  C2190T  C301G/Leu 101Val    G455A/Gl y152Asp        WT  WT  6  ST5  II  >256  −  C2190T  C301G/Leu 101Val  T438G/His14 6Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/ Ser81 Arg    A138G/–  C141T/–  G165A/–  4  ST5  II  >256  +  C2190T  C301G/Leu 101Val  T438G/ His146 Gln    C456T/–  G460T/ Val154 Leu  G469A, C470G/ Ala157 Arg  C78T/–  T102C/–  213-GGT- 214/71- Gly-72  T240G/Ser81 Arg  A473G/ Asn158 Ser  A138G/–  C141T/–  G165A/–  1  ST2  III  8  +  C2190T  C301G/Leu 101Val    WT  WT  ATCC 12228c  ≤1  −  WT  WT  WT  WT  a The nucleotide positions of the mutations are listed according to Escherichia coli numbering. b The nucleotide positions of the mutations are listed according to S. aureus numbering. c Linezolid-susceptible S. epidermidis control. Molecular typing of bacterial isolates The entire set of 16 LRSE isolates was examined by macrorestriction analysis, which yielded three distinct clusters (Table 2): PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Antimicrobial susceptibility and putative linezolid-resistance mechanisms The isolates showed decreased susceptibility to linezolid, exhibiting MICs in the range of 8 to >256 mg/L (Table 1). They displayed a multiresistant phenotype, also being resistant to oxacillin (n = 15, 100%), gentamicin (n = 11, 73.3%), ciprofloxacin (n = 15, 100%), moxifloxacin (n = 15, 100%), co-trimoxazole (n = 14, 93.3%), mupirocin (n = 3, 20%), clindamycin (n = 9, 60%), erythromycin (n = 12, 80%), rifampicin (n = 10, 66.7%), tetracycline (n = 4, 26.67%) and teicoplanin (n = 2, 13.33%). Resistance to fusidic acid, tigecycline, vancomycin and daptomycin was not observed. Table 2 summarizes the results with respect to the presence of the cfr gene, mutations in the 23S rRNA and in genes encoding ribosomal proteins for all the isolates and also for the linezolid-susceptible control strain ATCC 12228. C2190T was the only modification found in the linezolid 23S rRNA binding site. Sequence chromatograms showed no evidence of nucleotide mixtures at the respective position. Amino acid alterations in L3 comprised Leu101Val, His146Gln, Gly152Asp, Val154Leu and Ala157Arg. Amino acid changes in L4 were found at the following positions: Ser81Arg; Asn158Ser; and a Gly71 insertion. Mutations in rplV were synonymous when detected. Ten strains (62.5%) harboured the cfr gene. In summary, all strains exhibiting PFGE type I/ST168 and PFGE type III/ST2 tested positive for cfr, whereas only four strains of the PFGE type II/ST5 cluster harboured the resistance determinant (Table 1). No apparent correlation was observed between the occurrence of certain nucleotide mutations, the presence of cfr and the level of linezolid resistance. Description of cfr-carrying plasmids Each cfr-positive isolate carried the cfr gene on an ∼40 kb plasmid as determined by S1 nuclease PFGE and Southern hybridization analysis using a cfr-specific probe (data not shown). De novo assembly and manual polishing of the contigs received by WGS produced a 39.243 bp plasmid for strain 14-01514 (termed p14-01514), which harboured cfr and the phenicol exporter fexA and had features highly similar to the previously published vector p12-02300.15 Mapping of reads from S. epidermidis 14-01514 to p12-02300 revealed 99 SNPs. In addition, p12-2300 is 379 bp shorter compared with p14-01514. A BLAST search on this particular variable region yielded homologue sequences from plasmids of Staphylococcus aureus, S. epidermidis (p12-02300) and Staphylococcus cohnii, all of which were located in non-coding regions (not shown). Further examination of the sequence utilizing the Tandem Repeats Finder program29 revealed a conserved repetitive region of 63 bp, which was present 8.7 times in p12-2300 and 14.7 times in p14-01514. The repetitive region was also investigated in S. epidermidis 13-04707 and 15-00164. PCR and subsequent Sanger sequencing showed that both cfr plasmids exhibit 189 bp less than p14-01514, which corresponds to three tandem repeats. This is in accordance with the results from Tandem Repeats Finder, indicating that the 63 bp repetitive sequence was present 11.7 times in p13-04707 and p15-00164, respectively. With a final cfr-containing contig size of 38.273 and 38.450 bp after de novo assembly, no overlapping sequences representing putative plasmids were obtained for S. epidermidis isolates 13-04707 and 15-00164. A comparison of these contigs with p14-01514 yielded 100% identity of the truncated sequences from 13-04707 and 15-00164 to the novel vector, hence suggesting a highly similar plasmid content of these isolates. Taking into account that cfr plasmids from S. epidermidis 13-04707 and 15-00164 differ in the total amount of the 63 bp tandem repeats (see above) but are otherwise identical to p14-01514, the putative final plasmid sizes for p13-04707 and p15-00164 were set to 39.054 bp each. We herein identified three almost identical cfr plasmids with high similarity to the recently described vector from S. epidermidis 12-02300.15 Discussion Management of the outbreak in the ICU There is a plethora of articles describing the emergence and outbreaks with linezolid-resistant staphylococci worldwide.11,14–18,30–32 However, there are just a few detailed reports including data on linezolid consumption, infection control measures and interventions.10,17,19,30,31,33 To control the outbreak we implemented contact isolation measures of LRSE-positive patients. Training courses on hand hygiene were organized for the ICU staff and data concerning the outbreak were regularly presented and discussed. A previous report from O’Connor et al.17 showed that during a cfr-mediated LRSE outbreak the staff lacked knowledge concerning resistance mechanisms and ways of transmission. They implemented a hospital-wide education programme for healthcare staff thenceforward.17 Another group from Spain reported an outbreak of linezolid-resistant S. aureus (LRSA) in 2008 and performed staff screening. While no staff member was found to be colonized, they could isolate LRSA from environmental surfaces.33 We did not perform screening of staff for LRSE carriage and only conducted environmental screening of the room in the case of the bacteraemia patient, where no LRSE was detected. Overlapping stays of patients in the ICU with LRSE ST5 and ST168, respectively, suggested the dissemination of two endemic clones. This spread could have been caused by transmission directly via the hands of healthcare workers (HCWs), indirectly via environmental contamination or both. Whether or not the skin flora of HCWs serves as a source for LRSE is unclear and we did not investigate a potential impact. However, it is well known that HCWs serve as vectors for strain transmission from patient to patient via contaminated hands, skin and environments. Empirical therapy of severe infections in MRSA-positive and MRSA-negative patients with linezolid seemed to facilitate the concomitant selection and spread of LRSE on the ward. Treatment with linezolid can suppress the susceptible cutaneous flora of patients. Hence, resistant strains may become predominant. Even if colonization of patients with LRSE is microbiologically not yet detectable, therapy with linezolid could initiate selection of the earlier-transmitted resistant strain. Therefore, our infection control measures included the restriction of linezolid prescription, which has already been published by Mulanovich et al.19 and others.30–34 They proposed a threshold of ≥13 DDDs/100 patient-days being associated with the development of an outbreak of LRSE.19 In accordance with the present study, the increased use of linezolid seemed efficient in promoting the emergence and persistence of linezolid-resistant clones in ICU patients.20,35 In conclusion, prospective surveillance, screening procedures, isolation precautions, education of ICU staff and providing them with information, control of MRSA and the restriction of linezolid use were efficient in controlling the emergence and transmission of LRSE. Molecular epidemiology of LRSE Worldwide, the nosocomial S. epidermidis population is dominated by strains that belong to the MLST clonal complex 5 (CC5). These epidemic clones are multiresistant, can persist in hospitals over a long period of time and seem to adapt quickly to different environments through recombination and frequent exchange of genetic mobile elements.36–39 In the present study three different STs were identified: ST2, ST5 and ST168 (a single-locus variant of ST2), all of them belonging to CC5. SmaI macrorestriction analysis confirmed the presence of three distinct clusters; no further differentiation was possible. Ribosomal mutations were analysed, thereby confirming an identical pattern of ribosomal protein alterations within the respective clade and indicating the spread of LRSE ST5 and ST168 on the ward. LRSE ST2 was only detected once, in January 2015, from the blood culture of the last defined case. LRSE ST168 and ST2 and 4/10 LRSE ST5 carried a cfr plasmid. Due to the sequencing of one plasmid from each clonal lineage, we confirmed the presence of a highly similar cfr plasmid in all three genetic backgrounds. The acquisition of this plasmid via horizontal gene transfer is possible, as similar scenarios with the respective conjugative cfr vectors are described elsewhere.15,40 There are only a few reports dealing with outbreaks caused by LRSE in Europe.34 In 2015 we published data concerning clusters of LRSE ST2 and ST22 in German hospitals, where we showed evidence for the acquisition of cfr plasmids via horizontal gene transfer during spread of the respective strains.15 The same year, O’Connor et al.17 described the first cfr-mediated LRSE outbreak in the Republic of Ireland. Several European studies present evidence for the presence of endemic LRSE clones that circulate in hospital settings.18,41,42 Similar to our study, these strains differ from commensal S. epidermidis isolates by exhibiting MDR and biofilm-forming capabilities, therefore being probably more successful in the hospital environment.39,43,44 Linezolid-resistance mechanisms We examined the isolates for molecular mechanisms that may contribute to linezolid resistance in CoNS. All strains showed a C2190T modification of the 23S rRNA that is occasionally described in linezolid-resistant CoNS.34 In contrast to G2576T, its association with increased linezolid MICs has not been functionally verified so far.1,7,34 Mutations in rplC and rplD genes, encoding 50S ribosomal proteins of the PTC of the ribosome, revealed non-synonymous mutations in the 50S ribosomal proteins L3 and L4. The impact of these alterations on the structure of the PTC and linezolid binding remains to be determined, as the influence of L3 mutations on its tertiary folding was shown in silico.45 An earlier study using laboratory-derived linezolid-resistant strains confirmed a correlation for selected L3 and L4 mutations.46 Although many articles report multiple ribosomal mutations in clinical LRSE, data concerning the functional proof of the putative resistance mechanisms are rare.1,8,15,47 However, a combination of mutations leads to functional and structural adaptation to linezolid and promotes a faster growth rate of linezolid-dependent strains.20,35 Ten (62.5%) of our isolates additionally carried a cfr-positive plasmid. In all cases cfr was detected in combination with alterations in ribosomal genes. WGS showed that all cfr plasmids were highly similar to the recently described cfr plasmid p12-02300 from a clinical S. epidermidis isolate in Germany15 and differed only by varying numbers of tandem repeats. The function of these sequences requires further investigation. As a result of highly similar plasmid content, the spread of endemic clones in addition to dissemination of the cfr plasmid(s) within the hospital environment can be suspected. Acknowledgements We acknowledge Dr Helmers and Dr Téllez-Castillo from MVZ Synlab Leverkusen for microbial routine diagnostics and for providing LRSE isolates and the pharmacist Hartmut Paul for providing us with monthly data on antibiotic use at the investigated ICU. We thank the staff at the NRC for excellent technical assistance. Funding The study used core funding from the hospitals of the city of Cologne. The German Reference Centre for Staphylococci and Enterococci is funded by the German Federal Ministry of Health. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 1, 2018

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