Characterization of linezolid- and methicillin-resistant coagulase-negative staphylococci in a tertiary hospital in China.

Recently, linezolid-resistant staphylococci have become an emerging problem worldwide. Understanding the mechanisms of resistance, molecular epidemiology and transmission of linezolid-resistant CoNS in hospitals is very important. The antimicrobial susceptibilities of all isolates were determined by the microdilution method. The resistance mechanisms and molecular characteristics of the strains were determined using whole-genome sequencing and PCR. All the strains were resistant to oxacillin and carried the mecA gene; 13 patients (36.1%) had prior linezolid exposure. Most S. epidermidis and S. hominis isolates were ST22 and ST1, respectively. MLST typing and evolutionary analysis indicated most linezolid-resistant CoNS strains were genetically related. In this study, we revealed that distinct CoNS strains have different mechanisms of linezolid resistance. Among ST22-type S. epidermidis, acquisition of the T2504A and C2534T mutations in the V domain of the 23 S rRNA gene, as well as mutations in the ribosomal proteins L3 (L101V, G152D, and D159Y) and L4 (N158S), were linked to the development of linezolid resistance. In S. cohnii isolates, cfr, S158Y and D159Y mutations in the ribosomal protein L3 were detected. Additionally, emergence of the G2576T mutation and the cfr gene were major causes of linezolid resistance in S. hominis isolates. The cfr gene, G2576T and C2104T mutations, M156T change in L3 protein, and I188S change in L4 protein were found in S. capitis isolates. The emergence of linezolid-resistant CoNS in the environment is concerning because it involves clonal dissemination and frequently coexists with various drug resistance mechanisms.

Liu C ，Yu J ，Chen C ，Li X ，Ye Y ，Dong Y ，Ying X ，Li H ，Wang W ... -  《BMC INFECTIOUS DISEASES》

cfr-mediated linezolid-resistant clinical isolates of methicillin-resistant coagulase-negative staphylococci from China.

Three linezolid-resistant coagulase-negative staphylococci (LR-CoNS), including two Staphylococcus cohnii and one Staphylococcus capitis, were isolated from 1104 clinical staphylococcal isolates across China in 2013-2014. Antibiotic susceptibilities of the bacteria were determined by the agar dilution method. PCR and DNA sequencing were performed to determine the potential molecular mechanism of linezolid resistance. The two linezolid-resistant S. cohnii isolates were subjected to pulsed-field gel electrophoresis (PFGE) to investigate their genetic relatedness. Primer walking, S1 nuclease PFGE and Southern blot hybridisation were conducted to ascertain the location and environment of the cfr gene. All three isolates were positive for the cfr gene. Amino acid mutations S158F and S158Y in the ribosomal protein L3 were identified in S. cohnii 13B289 and 13L105, respectively, both of which also had an additional substitution (D159Y) in L3. PFGE indicated that the two S. cohnii isolates belonged to diverse clonal strains. S1 nuclease PFGE and Southern blotting experiments indicated that the cfr gene of the three isolates resided on plasmids of similar size (ca. 35.4kb). The cfr-harbouring segments of S. capitis 13G350 and S. cohnii 13L105 were identical to plasmid pSS-01 reported previously. The cfr-carrying fragment of S. cohnii 13B289 was indistinguishable from the formerly described plasmid pSS-02. In conclusion, the presence of the cfr gene located on a plasmid was the main mechanism contributing to resistance to linezolid in the three staphylococcal isolates. Hence, timely detection and judicious use of antibiotics are essential to prevent further transmission of this resistance mechanism.

Song Y ，Lv Y ，Cui L ，Li Y ，Ke Q ，Zhao Y ... -  《-》

Mechanisms of Linezolid Resistance Among Clinical Staphylococcus spp. in Spain: Spread of Methicillin- and Linezolid-Resistant S. epidermidis ST2.

Ruiz-Ripa L ，Feßler AT ，Hanke D ，Eichhorn I ，Azcona-Gutiérrez JM ，Alonso CA ，Pérez-Moreno MO ，Aspiroz C ，Bellés A ，Schwarz S ，Torres C ... -  《-》

Mechanisms of linezolid resistance among coagulase-negative staphylococci determined by whole-genome sequencing.

Linezolid resistance is uncommon among staphylococci, but approximately 2% of clinical isolates of coagulase-negative staphylococci (CoNS) may exhibit resistance to linezolid (MIC, ≥8 µg/ml). We performed whole-genome sequencing (WGS) to characterize the resistance mechanisms and genetic backgrounds of 28 linezolid-resistant CoNS (21 Staphylococcus epidermidis isolates and 7 Staphylococcus haemolyticus isolates) obtained from blood cultures at a large teaching health system in California between 2007 and 2012. The following well-characterized mutations associated with linezolid resistance were identified in the 23S rRNA: G2576U, G2447U, and U2504A, along with the mutation C2534U. Mutations in the L3 and L4 riboproteins, at sites previously associated with linezolid resistance, were also identified in 20 isolates. The majority of isolates harbored more than one mutation in the 23S rRNA and L3 and L4 genes. In addition, the cfr methylase gene was found in almost half (48%) of S. epidermidis isolates. cfr had been only rarely identified in staphylococci in the United States prior to this study. Isolates of the same sequence type were identified with unique mutations associated with linezolid resistance, suggesting independent acquisition of linezolid resistance in each isolate. Linezolid is one of a limited number of antimicrobials available to treat drug-resistant Gram-positive bacteria, but resistance has begun to emerge. We evaluated the genomes of 28 linezolid-resistant staphylococci isolated from patients. Multiple mutations in the rRNA and associated proteins previously associated with linezolid resistance were found in the isolates investigated, underscoring the multifocal nature of resistance to linezolid in Staphylococcus. Importantly, almost half the S. epidermidis isolates studied harbored a plasmid-borne cfr RNA methylase gene, suggesting that the incidence of cfr may be higher in the United States than previously documented. This finding has important implications for infection control practices in the United States. Further, cfr is commonly detected in bacteria isolated from livestock, where the use of phenicols, lincosamides, and pleuromutilins in veterinary medicine may provide selective pressure and lead to maintenance of this gene in animal bacteria.

Tewhey R ，Gu B ，Kelesidis T ，Charlton C ，Bobenchik A ，Hindler J ，Schork NJ ，Humphries RM ... -  《mBio》

Linezolid-resistant clinical isolates of enterococci and Staphylococcus cohnii from a multicentre study in China: molecular epidemiology and resistance mechanisms.

Genetic characterisation of linezolid-resistant Gram-positive cocci in a multicentre study in China has not been reported previously. To study the mechanism underlying the resistance of linezolid-resistant isolates, nine Enterococcus faecalis, one Enterococcus faecium and three Staphylococcus cohnii isolates with various levels of resistance were collected from five hospitals across China in 2009-2012. The nine E. faecalis isolates were classified into seven sequence types, indicating that these linezolid-resistant E. faecalis isolates were polyclonal. Enterococci isolates had reduced susceptibility to linezolid (MICs of 4-8 mg/L) and had mutation of ribosomal protein L3, with three also having mutation of L4, but without the multidrug resistance gene cfr or the 23S rRNA mutation G2576T. The three S. cohnii isolates were highly resistant to linezolid (MICs of 64 mg/L to >256 mg/L), harboured the cfr gene and had the 23S rRNA mutation G2576T. Southern blotting indicated that the cfr gene of these three isolates resided on different plasmids (pHK01, pRM01 and pRA01). In plasmid pHK01, IS21-558 and the cfr gene were integrated into transposon Tn558. In plasmids pRM01 and pRA01, the cfr gene was flanked by two copies of an IS256-like insertion sequence, indicating that the transferable form of linezolid resistance is conferred by the cfr gene. In conclusion, the emergence of linezolid-resistant Gram-positive cocci in different regions of China is of concern. The cfr gene and the 23S rRNA mutation contribute to high-level linezolid resistance in S. cohnii, and the L3 and L4 mutations are associated with low-level linezolid resistance in enterococci.

Chen H ，Wu W ，Ni M ，Liu Y ，Zhang J ，Xia F ，He W ，Wang Q ，Wang Z ，Cao B ，Wang H ... -  《-》