Cfr-carrying Plasmids Research Articles

Two novel plasmids harbouring the multiresistance gene cfr in porcine Staphylococcus equorum

BackgroundThe emergence and transmission of the multidrug resistance gene cfr have raised public health concerns worldwide. ObjectivesMultidrug-resistant Staphylococcus equorum isolates can pose a threat to public health. In this study, we have characterised the whole-genome of one Staphylococcus equorum isolate harbouring two distinct cfr-carrying plasmids. MethodsAntimicrobial susceptibility testing was performed by broth microdilution. Genomic DNA was sequenced using both the Illumina HiSeq X Ten and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. ResultsStaphylococcus equorum isolate SN42 exhibited resistance or high MICs to linezolid, erythromycin, tetracycline, oxacillin, clindamycin, virginiamycin, tiamulin, chloramphenicol and florfenicol. It carried two cfr-harbouring plasmids: the RepA N-family plasmid pSN42–51 K and the Inc18-family plasmid pSN42–50 K. These two plasmids exhibited low structural similarities to the so far reported cfr-carrying plasmids. Both plasmids harboured an arsenic resistance operon, copper and cadmium resistance genes as well as the lincosamide-pleuromutilin-streptogramin A resistance gene lsa(B). In addition, plasmid pSN42–51 K carried two erm(B) genes for macrolide-lincosamide-streptogramin B resistance, the streptomycin resistance gene ant(6)-Ia as well as mercury resistance genes while pSN42–50 K was associated with the heavy metal translocating P-type ATPase gene hmtp. The co-carriage and co-existence of these antimicrobial resistance and heavy metal resistance genes increases the likelihood of co-selection of the cfr-carrying plasmids. ConclusionThis is the first report of S. equorum carrying two distinct cfr-carrying plasmids, underscoring the need for ongoing surveillance to address the potential dissemination of multi-drug resistance in bacteria from food-producing animals to ensure food safety and public health.

Linezolid-Resistant Enterococcus faecalis of Chicken Origin Harbored Chromosome-Borne optrA and Plasmid-Borne cfr, cfr(D), and poxtA2 Genes.

The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an Enterococcus faecalis isolate QZ076, cocarrying optrA, cfr, cfr(D), and poxtA2 genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using E. faecalis JH2-2 and clinical methicillin-resistant Staphylococcus aureus (MRSA) 109 as recipients. E. faecalis QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with optrA located in the chromosomal DNA. The gene cfr was located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes cfr(D) and poxtA2 were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The cfr-carrying plasmid pQZ076-1 could transfer from E. faecalis QZ076 to E. faecalis JH2-2, along with the cfr(D)- and poxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA, cfr, cfr(D), and poxtA2] being simultaneously present in the same E. faecalis isolate. The location of the cfr gene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the cfr-carrying pheromone-responsive conjugative plasmid in E. faecalis was also able to mobilize the interspecies transfer of the cfr(D)- and poxtA2-cocarrying plasmid between enterococci and staphylococci. IMPORTANCE In this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA, cfr, cfr(D), and poxtA2] was identified in an E. faecalis isolate of chicken origin. The association of the cfr gene with a novel pseudocompound transposon Tn7515 integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genes cfr(D) and poxtA2 on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such as cfr, cfr(D), and poxtA2, among Gram-positive pathogens.

An 11-year linezolid-resistant Staphylococcus capitis clone dissemination with a similar cfr-carrying plasmid in China.

Linezolid resistance has represented a global concern with its wide dissemination among nosocomial pathogens in recent years. One hundred and two linezolid-resistant Staphylococcus capitis (LRSC) were constantly isolated from 2011 to 2021, which demonstrated single clonal dissemination in a Chinese tertiary hospital. A structurally similar cfr-carrying plasmid was identified among 90 isolates. A chromosomal cfr was located beside a Tn4001-like transposon and ISEnfa4 in one strain (LR95). The loss of cfr-carrying plasmid was observed in 11 isolates and the invitro passage experiments. Conjugation experiments demonstrated the horizontal transferability of the cfr-carrying plasmid into Staphylococcus aureus RN4220. Both cfr-positive LRSC and S.aureus showed no significant differences in growth rates, while only the former displayed competition defect, suggesting this plasmid imposed a certain fitness cost on LRSC. Hence, ongoing measurements are supposed to be adopted to control the spread of these antimicrobial-resistant bacteria.

Detection of linezolid resistance cfr gene among MRSA isolates

BackgroundLinezolid (Oxazolidinones) is commonly used against a variety of Gram-positive infections, especially methicillin-resistant Staphylococcus aureus (MRSA). The emerging resistance to linezolid curtail the treatment of infections caused by MRSA and other Gram-positive bacteria. Presence of cfr gene plays a crucial role in Linezolid resistance. ObjectivePresent study was aimed to detect cfr gene among clinical MRSA isolates. Materials and methodsThe suspected Staphylococcus aureus isolates were processed through Kirby Bauer disc diffusion methods for the confirmation of MRSA strains. Phenotypic Linezolid resistance was determined through broth micro-dilution method. The plasmid and DNA of Linezolid resistant isolates were subjected to molecular characterization for the presence of cfr gene. ResultsAmong 100 Staphylococcus aureus isolates, 85 of them were confirmed as MRSA isolates. Categorically, 65% MRSA isolates were sensitive to linezolid with MIC lower than 8 µg/ml, whereas, 35% of them were resistant to linezolid having MIC greater than 8 µg/ml. MIC level of 128 µg/ml was observed among 3.5% of the resistant isolates. Similarly, MIC level of 64 µg/ml, 32 µg/ml, 16 µg/ml and 8 µg/ml were noted for 3.5%, 4.7%, 8.2% and 15.3% isolates respectively. Linezolid resistance cfr gene was detected only in 9.4% of the resistant isolates. ConclusionMulti drug resistance among MRSA isolates is keenly attributed to the presence of cfr gene as evident in the present study, and horizontal dissemination of cfr gene among MRSA strains is accredited to cfr-carrying transposons and plasmids.

Persistence of a multidrug-resistant worldwide-disseminated methicillin-resistant Staphylococcus epidermidis clone harbouring the cfr linezolid resistance gene in a French hospital with evidence of interspecies transfer to several Staphylococcus aureus lineages.

Resistance to linezolid has become a worldwide concern since it is one of the last-resort antibiotics to treat multidrug-resistant staphylococcal and enterococcal infections. We investigated staphylococcal infections caused by 16 cfr-positive linezolid-resistant Staphylococcus epidermidis and Staphylococcus aureus isolates in a French university hospital from 2015 to 2018. Antimicrobial susceptibility of isolates was tested by broth microdilution and gradient strips. Genetic determinants of linezolid resistance (including cfr gene and 23S rRNA mutations) were assessed by PCR and WGS; the latter was also used to characterize the cfr-carrying plasmids in S. epidermidis and S. aureus, and to explore the clonal relationship of isolates. All linezolid-resistant staphylococcal isolates harboured the same cfr-carrying plasmid, sharing 99% identity with the previously described pSA737. The three S. aureus isolates belonged to different STs (ST8, ST72, ST2416); the 13 methicillin-resistant S. epidermidis (MRSE) belonged to ST2 and harboured both cfr and mutations in genes encoding 23S rRNA and ribosomal proteins. Phylogenetic analysis grouped the MRSE isolates into two clusters, one of which (n = 12 isolates) belonged to the recently reported multidrug-resistant worldwide-disseminated S. epidermidis lineages. The results presented herein highlight the persistence and efficient spread of a cfr-carrying plasmid in a hospital related both to the dissemination of a multidrug-resistant S. epidermidis clone and the in vivo interspecies transfer of cfr between S. epidermidis and S. aureus. The emergence of linezolid-resistant strains should be closely monitored, and the mechanisms involved systematically explored in order to limit the spread of plasmid-mediated resistance.

Phenotypic and genotypic characterisation of linezolid-resistant coagulase-negative staphylococci possessing cfr-carrying plasmid

Linezolid-dependent growth has contributed to wide dissemination of Staphylococcus epidermidis in hospitals. This study aimed to characterise linezolid-resistant coagulase-negative staphylococci (CoNS) isolates and the possibility of linezolid dependence in China. Phenotypic and genotypic resistance of 13 CoNS isolates were investigated by antimicrobial susceptibility testing and PCR. Similarity of isolates was determined by pulsed-field gel electrophoresis (PFGE). Characterisation of the cfr-carrying plasmid was performed by S1 nuclease PFGE, Southern blotting and whole-genome sequencing (WGS). A maximum likelihood phylogenetic tree was constructed for phylogenetic analysis. Growth curve analysis was performed with and without linezolid to determinate the possible contribution of linezolid dependence to linezolid-resistant CoNS dissemination. Thirteen CoNS isolates showed linezolid MICs of 8 mg/L to >256 mg/L, typed into three PFGE profiles. Southern blotting and WGS indicated that the cfr gene was located on a 39.5-kb plasmid with 99% identity to cfr-harbouring plasmids pSR01, pLRSA417 and pH29-46. The cfr gene was flanked by two copies of an IS256-like element ISEnfa4 family transposase, indicating the transferability of linezolid resistance conferred by the cfr gene. Comparative phylogenetic analysis revealed that Staphylococcus capitis XZ03 shared high similarity with linezolid-resistant S. capitis isolates in Huashan Hospital, Shanghai. Thirteen CoNS isolates did not exhibit linezolid dependence on exposure from 8-32 mg/L. The endemic CoNS clone carrying the cfr gene in our hospital showed high-level linezolid resistance, threatening linezolid use. Linezolid-dependent growth under linezolid selective pressure was not observed, indicating that it may not yet be a common phenotype in Staphylococcus spp.

Co-occurrence of cfr-mediated linezolid-resistance in ST398 LA-MRSA and non-aureus staphylococci isolated from a pig farm

Linezolid resistance, mediated by the cfr gene, which confers resistant phenotypes to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antimicrobials, has emerged in S. aureus and non-aureus staphylococci (NAS). Moreover, due to the transferable potential via plasmids, the spread of cfr among staphylococci is of great concern. In the present study, we investigated the prevalence of cfr-mediated linezolid resistance in ST398 methicillin-resistant S. aureus (MRSA) and NAS strains isolated from a pig farm. Among the 26 staphylococci isolates collected from a pig farm, 14 cfr-harboring ST398 MRSA and NAS (S. epidermidis, S. pasteuri, S. cohnii, and S. rostri) strains were resistant to linezolid and also carried the fexA gene. Comparative genome analysis of cfr-carrying linezolid-resistant ST398 MRSA and NAS (S. pasteuri, S. cohnii, and S. epidermidis) strains revealed that the segments harboring cfr in different staphylococcal strains showed ≥ 99 % sequence identity and the corresponding region containing the cfr, fexA, and Tn558 elements were located in a 38-kb plasmid, designated pSA12 of ST398 MRSA. These observations indicate that the cfr-carrying plasmids and/or fragments may be disseminated among staphylococci in a pig farm and possibly transmitted to staphylococci of human origin, subsequently posing a threat to public health. This is the first report of the co-existence of cfr in linezolid-resistant ST398 MRSA and NAS isolated from a pig farm in South Korea.

Characterization of the Multi-Drug Resistance Gene cfr in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated From Animals and Humans in China.

We investigated cfr-positive and -negative MRSA strains isolated from animals and humans in different geographical areas of China, from 2011 to 2016. Twenty cfr-positive strains (15.6%) were identified from 128 MRSA strains including 17 from food animals and three from humans. The resistance rates and prevalence of the tested antibiotic resistance genes (ARGs) in the cfr-positive MRSA isolates were higher than that in the cfr-negative MRSA isolates. All cfr-positive MRSA isolates were co-carrying fexA and ermC, and had significantly higher optrA incidence rate vs. the cfr-negative isolates (P < 0.05). In addition, multilocus sequence typing (MLST) assays showed that ST9 and spa-type t899 were the most prevalent ST and spa types in the study strains. However, all of the 20 cfr-positive and 10 randomly selected cfr-negative MRSA isolates were clonally unrelated as determined by pulsed-field gel electrophoresis (PFGE) analyses. Importantly, the cfr gene was successfully transferred to a recipient Staphylococcus aureus strain RN4220 from 13 of the 20 cfr-positive MRSA isolates by electroporation. Among these 13 cfr-positive MRSA isolates, two different genetic contexts surrounding cfr were determined and each was associated with one type of cfr-carrying plasmids. Of note, the predominant genetic context of cfr was found to be a Tn558 variant and locate on large plasmids (∼50 kb) co-harboring fexA in 11 of the 13 MRSA isolates. Furthermore, the cfr gene was also identified on small plasmids (∼ 7.1 kb) that co-carried ermC in two of the 13 MRSA isolates. Our results demonstrated a high occurrence of multi-drug resistance in cfr-positive MRSA isolates, and the spread of cfr might be attributed to horizontal dissemination of similar cfr-carrying transposons and plasmids.

Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate.

Enterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.

Prevalence of cfr in Enterococcus faecalis strains isolated from swine farms in China: Predominated cfr-carrying pCPPF5-like plasmids conferring “non-linezolid resistance” phenotype

The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr-positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr-positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1-PFGE and Southern blotting. A 12 kb cfr-positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr-carrying ISEnfa5-composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr.

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

To investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE) in an interdisciplinary ICU, linezolid consumption and infection control measures taken. 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. 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). 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.

F14:A-:B- and IncX4 Inc group cfr-positive plasmids circulating in Escherichia coli of animal origin in Northeast China

The objective of this study was to investigate the prevalence of the cfr gene in Escherichia coli isolates from domestic animals in Northeast China and to characterize the cfr-containing plasmids. Between June 2015 and April 2016, 370 E. coli isolates were collected from pigs, chickens, and dairy cows in Northeast China. Among these, 111 were florfenicol resistant, including 109 isolates carrying the floR gene and 6 positives for cfr. The prevalence of cfr in E. coli isolates from the four northeast provinces in China was 1.6% (6/370), which was higher than that previously reported (0.08% and 0.5%). All six cfr-containing E. coli isolates were highly resistant to florfenicol (100%), cefotaxime (100%), and fosfomycin (100%). Complete sequence analysis of two cfr-carrying plasmids revealed high homology of the IncX4-type pEC14cfr plasmid with two other cfr-harboring plasmids, pSD11 and pGXEC6, found in swine E. coli isolates from southern China. pEC14cfr-like plasmids have been isolated in five provinces in southern and northern China. The isolation sites were up to 2700 kilometers apart, implying that pEC14cfr-like plasmids are likely to be national epidemic cfr-carrying plasmids that mediate the dissemination of cfr in China. Moreover, the genetic structure (IS26-IS26-cfr-rec-pre/mob-ramA-IS26) of the second cfr-carrying plasmid, IncF14:A-:B- pEC295cfr, represents a novel genetic environment for cfr identified for the first time in the present study. Sequence homology analysis indicated that the cfr-carrying element was most likely introduced into a cfr-negative pEC12 plasmid backbone, which evolved into the cfr-carrying vector, pEC295cfr. Moreover, isolation of the IncF14:A-:B- pEC295cfr plasmid harboring cfr suggests that IncFII plasmids maybe have become additional effective vehicles for cfr dissemination. These results highlight the importance of surveying the prevalence of IncX4 and IncFII plasmids in gram-negative bacteria, especially in swine E. coli isolates.

Long-lasting successful dissemination of resistance to oxazolidinones in MDR Staphylococcus epidermidis clinical isolates in a tertiary care hospital in France.

ObjectivesPatient- and procedure-related changes in modern medicine have turned CoNS into one of the major nosocomial pathogens. Treatments of CoNS infections are challenging owing to the large proportion of MDR strains and oxazolidinones often remain the last active antimicrobial molecules. Here, we have investigated a long-lasting outbreak (2010–13) due to methicillin- and linezolid-resistant (LR) CoNS (n = 168), involving 72 carriers and 49 infected patients.MethodsAntimicrobial susceptibilities were tested by the disc diffusion method and MICs were determined by broth microdilution or Etest. The clonal relationship of LR Staphylococcus epidermidis (LRSE) was first determined using a semi-automated repetitive element palindromic PCR (rep-PCR) method. Then, WGS was performed on all cfr-positive LRSE (n = 30) and LRSE isolates representative of each rep-PCR-defined clone (n = 17). Self-transferability of cfr-carrying plasmids was analysed by filter-mating experiments.ResultsThis outbreak was caused by the dissemination of three clones (ST2, ST5 and ST22) of LRSE. In these clones, linezolid resistance was caused by (i) mutations in the chromosome-located genes encoding the 23S RNA and L3 and L4 ribosomal proteins, but also by (ii) the dissemination of two different self-conjugative plasmids carrying the cfr gene encoding a 23S RNA methylase. By monitoring linezolid prescriptions in two neighbouring hospitals, we highlighted that the spread of LR-CoNS was strongly associated with linezolid use.ConclusionsPhysicians should be aware that plasmid-encoded linezolid resistance has started to disseminate among CoNS and that rational use of oxazolidinones is critical to preserve these molecules as efficient treatment options for MDR Gram-positive pathogens.

First Report of cfr-Carrying Plasmids in the Pandemic Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Staphylococcal Cassette Chromosome mec Type IV Clone.

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins; cfr, encoding phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance; its homologue cfr(B); or optrA, conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr is even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPSA susceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel electrophoresis (PFGE), plasmid curing, and conjugative transfer. Whole-genome sequencing was used for single-nucleotide variant (SNV) analysis, multilocus sequence typing, L protein mutation identification, cfr plasmid sequence analysis, and optrA and cfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles, compared to 1/5 in M13/0401. Both isolates were sequence type 22 MRSA staphylococcal cassette chromosome mec type IV (ST22-MRSA-IV)/spa type t032 isolates, harbored cfr, exhibited the PhLOPSA phenotype, and lacked optrA and cfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid. This is the first report of cfr in the pandemic ST22-MRSA-IV clone. Different cfr plasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlight that prudent management of linezolid use is essential.

Characterization of novel conjugative multiresistance plasmids carryingcfrfrom linezolid-resistantStaphylococcus epidermidisclinical isolates from Italy

The objective of this study was to investigate the genetic environment of the cfr gene from two linezolid-resistant clinical isolates of Staphylococcus epidermidis from Italy. The two strains (SP1 and SP2) were phenotypically and genotypically characterized. Transferability of cfr was assessed by electrotransformation and conjugation. The genetic contexts of cfr were investigated by PCR mapping, sequencing and comparative sequence analyses. SP1 and SP2 belonged to ST23 and ST83, respectively. In both strains, the cfr gene was located on a plasmid, which could be transferred to Staphylococcus aureus by transformation and conjugation. In isolate SP1, linezolid resistance mediated by mutations in 23S rRNA and the L3 ribosomal protein was also detected. pSP01, the cfr-carrying plasmid from strain SP1, had a larger number of additional resistance genes and was sequenced (76 991 bp). It disclosed a distinctive mosaic structure, with four cargo regions interpolated into a backbone 95% identical to that of S. aureus plasmid pPR9. Besides cfr, resistance genes distributed in the cargo regions included blaZ, lsa(B), msr(A) and aad, and a gene cluster for resistance to heavy metals. A closely related cfr plasmid (pSP01.1, ∼ 49 kb), differing from pSP01 by the lack of a large cargo region with some resistance genes, was detected in strain SP2. The conjugative multiresistance plasmid pSP01 is the first cfr-carrying plasmid to be sequenced in Italy. This is the first time cfr has been found: (i) in association with blaZ, msr(A) and heavy metal resistance genes; and (ii) in an S. epidermidis strain (SP2) belonging to ST83.

Identification and characterization of cfr-positive Staphylococcus aureus isolates from community-onset infectious patients in a county hospital in China.

The cfr gene was detected in 14 meticillin-susceptible Staphylococcus aureus isolates recovered from outpatients with community-onset infections in a county hospital in China. The MIC of linezolid was 4 μg ml- 1 in eight isolates and 2 μg ml- 1 in six isolates. All isolates were susceptible to vancomycin and teicoplanin, but had elevated MICs for penicillin (0.5-128 μg ml- 1), chloramphenicol (2-32 μg ml- 1), clindamycin (0.5-128 μg ml- 1) and erythromycin (4-128 μg ml- 1). Nine isolates had mutations on domain V of 23S rRNA and/or the ribosomal L proteins that were not located close to the linezolid-binding pocket. Southern blotting experiments demonstrated that the cfr genes in all 14 isolates resided on plasmids. Sequence analysis of the 5.6 kb cfr-carrying plasmid segment revealed 99 % identity to the corresponding sequences in plasmid pSS-01 from animal staphylococci and plasmid pRM-01 from human staphylococci. Five isolates belonged to sequence type (ST)188 and three to ST965; the two ST types were previously reported in isolates of animal origin in some areas of China. These results indicate that the cfr-carrying plasmids in this study are likely of animal origin. The present study shows that cfr-harbouring S. aureus isolates have emerged in some areas of China and that cfr-carrying isolates may be transmitted between animals and humans.

Dissemination of the same cfr-carrying plasmid among methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococcal isolates in China.

Six cfr-harboring methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to the same clone of sequence type 5 (ST5)-staphylococcal cassette chromosome mec element II (SCCmec II)-spa t311, were investigated in this study. Complete sequencing of a cfr-carrying plasmid, pLRSA417, revealed an 8,487-bp fragment containing a Tn4001-like transposon, cfr, orf1, and ISEnfa4. This segment, first identified in an animal plasmid, pSS-01, was observed in several plasmids from clinical coagulase-negative staphylococci in China, suggesting that the cfr gene, which might originate from livestock, was located in the same mobile element and disseminated among different clinical staphylococcal species.

Linezolid resistance in clinical isolates of Staphylococcus epidermidis from German hospitals and characterization of two cfr-carrying plasmids.

This study was a detailed investigation of Staphylococcus epidermidis clinical isolates exhibiting linezolid resistance. Thirty-six linezolid-resistant S. epidermidis from eight German hospitals, including isolates from suspected hospital-associated outbreaks between January 2012 and April 2013, were analysed with respect to their antimicrobial susceptibility and the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. Relatedness of isolates was estimated by MLST and SmaI macrorestriction analysis. Characterization of cfr plasmids was carried out by means of Illumina sequencing. The MICs of linezolid varied substantially between the isolates. No apparent correlation was detected between the level of resistance, the presence of cfr and ribosomal target site mutations. S. epidermidis isolates from two hospitals were confirmed as clonally related, indicating the spread of the respective clone over a period of 1 year. Next-generation sequencing revealed two different categories of cfr-expressing plasmids, both of them varying in genetic arrangement and composition from previously published cfr plasmids: p12-00322-like plasmids showed incorporation of cfr into a pGO1-like backbone and displayed capabilities for intra- and inter-species conjugational transfer. To date, linezolid-resistant S. epidermidis have rarely been isolated from human clinical sources in Germany. Here, we describe the emergence and outbreaks of these strains. We detected previously described and novel point mutations in the 23S ribosomal genes. The cfr gene was only present in six isolates. However, this is the first known description of cfr incorporation into conjugative vectors; under selective pressure, these vectors could give reasonable cause for concern.

Ribosomal protein L3 mutations are associated with cfr-mediated linezolid resistance in clinical isolates of Staphylococcus cohnii.

From June, 2012 to November, 2013 five linezolid-resistant Staphylococcus cohnii isolates were identified in our hospital in Beijing, China. The investigation of the resistance mechanisms confirmed that the cfr-carrying plasmids were the main cause of linezolid resistance in those clinical isolates. Moreover, all the five isolates had ribosomal protein L3 mutations, which had different coordinate effect on cfr-mediated linezolid resistance directly through the substitution of serine 158 by phenylalanine or tyrosine in L3 protein. In this study, two types of plasmids (p432, p438) (Accession No. KM114207) were found, which share high sequence identity with previously reported cfr-carrying pRM01 and pMHZ plasmids originated from northern and southern China, showing wide regional dissemination in China. The stability of linezolid resistance was studied by passaging single colonies serially on antibiotic-free blood medium, which showed that the susceptible derivatives emerged until the passages 39-42 with the elimination of cfr-carrying plasmid. Thus the high stability of this plasmid may pose a risk for the transmission among patients or even cause an outbreak in clinical settings.

Investigation of a multiresistance gene cfr that fails to mediate resistance to phenicols and oxazolidinones in Enterococcus faecalis

To investigate the basis of susceptibility to phenicols and oxazolidinones of the porcine Enterococcus faecalis CPPF5 despite the presence of the multiresistance gene cfr. Southern blotting, conjugation and transformation analyses were conducted to confirm the plasmid location and transferability of cfr in CPPF5. The genetic environment of cfr was determined by sequence analysis. Transcription and translation of cfr were examined by RT-PCR and western blotting, respectively, and modifications at A2503 within the 23S rRNA sequence were identified by primer extension. Electrotransformation and Southern blotting indicated that CPPF5 and its transformant 5B2-3 contained two cfr-carrying plasmids ∼ 50 and ∼ 12 kb in size. The complete 12,270 bp sequence of the smaller plasmid, pCPPF5, was determined and shared 99.9% (12,269/12,270 bp) identity with the corresponding region of the cfr-carrying plasmid pEF-01 in E. faecalis of cattle origin. Moreover, the genetic environment of cfr in the ∼ 50 kb plasmid was the same as that in pCPPF5 according to sequencing results. Although cfr mRNA, Cfr protein and a modification at the A2503 site were detected, the cfr-carrying transformant 5B2-3 did not have elevated MICs of chloramphenicol, florfenicol and linezolid, indicating that cfr fails to mediate resistance to the respective antibiotics in E. faecalis. This is the first report of the cfr gene failing to elevate MICs of the corresponding antibiotics. Although the genetic basis for the apparent 'no resistance' phenotype remains to be determined, this finding may have implications for surveillance studies that target the cfr gene.