Yuan Lv

A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin

OBJECTIVES The oxazolidinone-resistant Enterococcus faecalis E349 from a human patient tested negative for the cfr gene and 23S rRNA mutations. Here we report the identification of a novel oxazolidinone resistance gene, optrA, and a first investigation of the extent to which this gene was present in E. faecalis and Enterococcus faecium from humans and food-producing animals. METHODS The resistance gene optrA was identified by whole-plasmid sequencing and subsequent cloning and expression in a susceptible Enterococcus host. Transformation and conjugation assays served to investigate the transferability of optrA. All optrA-positive E. faecalis and E. faecium isolates of human and animal origin were analysed for their MICs and their genotype, as well as the location of optrA. RESULTS The novel plasmid-borne ABC transporter gene optrA from E. faecalis E349 conferred combined resistance or elevated MICs (when no clinical breakpoints were available) to oxazolidinones (linezolid and tedizolid) and phenicols (chloramphenicol and florfenicol). The corresponding conjugative plasmid pE349, on which optrA was located, had a size of 36 331 bp and also carried the phenicol exporter gene fexA. The optrA gene was functionally expressed in E. faecalis, E. faecium and Staphylococcus aureus. It was detected more frequently in E. faecalis and E. faecium from food-producing animals (20.3% and 5.7%, respectively) than from humans (4.2% and 0.6%, respectively). CONCLUSIONS Enterococci with elevated MICs of linezolid and tedizolid should be tested not only for 23S rRNA mutations and the gene cfr, but also for the novel resistance gene optrA.

Molecular Characterization and Antimicrobial Susceptibility Testing of Escherichia coli Isolates from Patients with Urinary Tract Infections in 20 Chinese Hospitals

ABSTRACT A total of 222 urinary Escherichia coli isolates from 20 tertiary hospitals in 15 different provinces and 4 municipalities in mainland China were characterized by antimicrobial susceptibility, phylogrouping, and the presence of plasmid-mediated quinolone resistance genes. A subset of 138 suspected extended-spectrum cephalosporinase (ESC) producers were examined for genes encoding cephalosporin resistance. Forty-three isolates harboring bla CTX-M-14 or bla CTX-M-15 were analyzed by pulsed-field gel electrophoresis (PFGE), and plasmids containing these genes were typed using PCR-based replicon typing (PBRT). Thirteen phylogroup B2 bla CTX-M-14- and bla CTX-M-15-positive isolates were analyzed by multilocus sequence typing (MLST). A frequent occurrence of resistance (>46%) was observed toward cephalosporins, gentamicin, and fluoroquinolones. Among the 222 isolates, 4 qnrS1, 4 qepA, and 16 aac(6′)-Ib-cr genes were confirmed. Four major phylogroups (A, B1, B2, and D) and nontypeable isolates (NTs) were found among the isolates, with phylogroup D (54%) being the most common phylogroup. A total of 110 (80%) of the 138 screened isolates harbored bla CTX-M genes, with bla CTX-M-14 (71%) and bla CTX-M-15 (24%) being the most prevalent of these genes. Nine of the 13 CTX-M-15- or CTX-M-14-containing B2 isolates belonged to ST131. PFGE typing showed a high level of diversity, and plasmid analysis indicated a very large pool of different resistance plasmids mediating the spread of bla CTX-M genes in mainland China. An equally very high frequency of resistance and equally high levels of diversity in phylogroups, PFGE types, and plasmids were observed among community- and hospital-acquired E. coli isolates, indicating the presence of a large reservoir in the community and a long-term spread of cephalosporin resistance in China.

Cfr-Mediated Linezolid-Resistance among Methicillin-Resistant Coagulase-Negative Staphylococci from Infections of Humans

Four methicillin-resistant coagulase-negative staphylococci (MRCoNS), one Staphylococcus haemolyticus and three Staphylococcus cohnii, from infections of humans collected via the Ministry of Health National Antimicrobial Resistance Surveillance Net (Mohnarin) program in China were identified as linezolid-resistant. These four isolates were negative for the 23S rRNA mutations, but positive for the gene cfr. Mutations in the gene for the ribosomal protein L3, which resulted in the amino acid exchanges Gly152Asp and Tyr158Phe, were identified in S. haemolyticus 09D279 and S. cohnii NDM113, respectively. In each isolate, the cfr gene was located on a plasmid of ca. 35.4 kb, as shown by S1 nuclease pulsed-field gel electrophoresis and Southern blotting experiments. This plasmid was indistinguishable from the previously described plasmid pSS-02 by its size, restriction pattern, and a sequenced 14-kb cfr-carrying segment. Plasmid pSS-02 was originally identified in staphylococci isolated from pigs. This is the first time that a cfr-carrying plasmid has been detected in MRCoNS obtained from intensive care patients in China. Based on the similarities to the cfr-carrying plasmid pSS-02 from porcine coagulase-negative staphylococci, a transmission of this cfr-carrying plasmid between staphylococci from pigs and humans appears to be likely.

Genetic environment of the transferable oxazolidinone/phenicol resistance gene optrA in Enterococcus faecalis isolates of human and animal origin

OBJECTIVES Aim of this study was to analyse 17 non-related Enterococcus faecalis isolates of human and animal origin for the genetic environment of the novel oxazolidinone/phenicol resistance gene optrA. METHODS WGS and de novo assembly were conducted to analyse the flanking sequences of the optrA gene in the 17 E. faecalis isolates. When optrA was located on a plasmid, conjugation assays were performed to check whether the plasmids are conjugative and to confirm the resistance phenotype associated with these plasmids. RESULTS All nine optrA-carrying plasmids were conjugated into E. faecalis JH2-2 and the transconjugants exhibited the optrA-associated phenotype. In these plasmids, an IS1216E element was detected either upstream and/or downstream of the optrA gene. In eight plasmids, the phenicol exporter gene fexA was found upstream of optrA and in six plasmids, a novel erm(A)-related gene for macrolide-lincosamide-streptogramin B resistance was detected downstream of optrA. When located in the chromosomal DNA, the optrA gene was found downstream of the transcriptional regulator gene araC in four isolates, or downstream of the fexA gene in another four isolates. Integration of the optrA region into a Tn558-Tn554 hybrid, located in the chromosomal radC gene, was seen in two isolates. CONCLUSIONS The findings of the present study extend the current knowledge about the genetic environment of optrA and suggest that IS1216E elements play an important role in the dissemination of optrA among different types of enterococcal plasmids. The mechanism underlying the integration of optrA into the chromosomal DNA requires further investigation.

Molecular characterization of erm(B)- and mef(E)-mediated erythromycin-resistant Streptococcus pneumoniae in China and complete DNA sequence of Tn2010

Aims:  To characterize the erm(B)‐ and mef(E)‐mediated erythromycin‐resistant Streptococcus pneumoniae clinical isolates obtained from ten hospitals located different cities in China.

Nationwide Surveillance of Novel Oxazolidinone Resistance Gene optrA in Enterococcus Isolates in China from 2004 to 2014.

ABSTRACT A total of 2,201 nonduplicate enterococcal isolates collected from 29 hospitals in 23 cities in China between 2004 and 2014 were screened for the oxazolidinone resistance gene optrA; 45 isolates (2.0%) were optrA positive with 11 OptrA variants identified. The positive rate of optrA increased from 0.4% to 3.9% during the 10-year surveillance period. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence type (MLST) analysis revealed that 37 optrA-positive Enterococcus faecalis isolates clustered into 25 PFGE patterns and 21 sequence types, while 6 Enterococcus faecium isolates represented 6 PFGE patterns and 6 sequence types. The present study underscores the importance of routine and persistent monitoring of oxazolidinone resistance and optrA gene.

LMP2/LMP7 gene variant: a risk factor for intestinal Mycobacterium tuberculosis infection in the Chinese population.

Background and Aims:  Low molecular mass protein‐2 (LMP2) and low molecular mass protein‐7 (LMP7) genes play a critical role in foreign antigen processing on the major histocompatibility complex‐I CD8+ cytotoxic T‐lymphocyte pathway. This study was designed to investigate whether the sequence variants in the LMP2/LMP7 coding region were associated with intestinal Mycobacterium tuberculosis (M. tuberculosis) infection or with the co‐infection of pulmonary tuberculosis.

Antimicrobial resistance surveillance of doripenem in China

To investigate the antibacterial resistance to doripenem in China and to understand the distribution trends of resistant bacteria. All the clinical isolates were collected from hospitals and the susceptibility tests were performed using the agar dilution method recommended by the Clinical Laboratory Standards Institute (CLSI) central laboratory. The susceptibility of the isolates to antimicrobial agents was determined using the CLSI (2014) or European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2013) guidelines. A total of 4047 pathogenic strains were isolated from 18 tertiary hospitals in 18 cities across China between July 2011 and June 2012. MIC results indicated that the vast majority of Enterobacteriaceae maintained high susceptibility to doripenem, with a lower resistance rate (1.9%) than that observed for other drugs tested. In the case of non-fermenting Gram-negative isolates, the resistance rate of Pseudomonas aeruginosa was 16.2%, which was less than that of imipenem and meropenem, and the Acinetobacter baumannii doripenem resistance rate was 67.4%. Doripenem also showed good in vitro activity against other the bacteria tested. This study suggests that the gradual increase in carbapenem nonsusceptible Enterobacteriaceae should be monitored carefully alongside the increasing multidrug-resistant and extensively drug-resistant A. baumannii.

Mechanism for transfer of transposon Tn2010 carrying macrolide resistance genes in Streptococcus pneumoniae and its effects on genome evolution

OBJECTIVES The objective of this study was to identify the mechanism responsible for the horizontal transfer of transposon Tn2010 in Streptococcus pneumoniae, and the genomic alterations introduced by the transfer process. METHODS Tn2010 was identified using PCR in 15 clinical isolates of S. pneumoniae with erythromycin resistance. S. pneumoniae and Enterococcus faecalis isolates were used as recipient cells in mating and transformation experiments to test the conjugative transferability and transformability of Tn2010. Whole-genome sequencing was used to assess the effects of the Tn2010 transfer on recipient genomes. The biological cost of the horizontal acquisition of Tn2010 and additional genomic changes was investigated by growth competition experiments. RESULTS Tn2010 was transformed at a frequency of 3 × 10(-7) transformants per cfu, whereas no transconjugants were detected using S. pneumoniae or E. faecalis as recipient cells. Genome analysis showed that many other recombinations were scattered throughout the genome of the transformants in addition to transposon Tn2010. The transformants demonstrated a negligible fitness cost compared with the wild-type strain. CONCLUSIONS Tn2010 tended to be transferred by transformation rather than conjugation in S. pneumoniae, and the spread of Tn2010 could have a profound effect on the evolution of the genome. The acquisition of Tn2010 with negligible fitness cost may facilitate spread of the transposon.

Underlying heart disease and microbiological spectrum of adult infective endocarditis in one Chinese university hospital: a 10-year retrospective study.

To identify the underlying heart disease and microbiological pathogen associated with infective endocarditis (IE) in Chinese patients in one university hospital over a 10‐year period.