Yingyuan Zhang

Plasmid-Mediated Quinolone Resistance in Clinical Isolates of Escherichia coli from Shanghai, China

ABSTRACT Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16- to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA→CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinolone-resistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.

vanM, a New Glycopeptide Resistance Gene Cluster Found in Enterococcus faecium

ABSTRACT Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcusfaecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-d-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in d-Ala-d-Lac.

Clostridium difficile infections in a Shanghai hospital: antimicrobial resistance, toxin profiles and ribotypes

The incidence of Clostridium difficile infection (CDI) has risen markedly since 2003, however data from China are limited. A 1-year study was conducted at the University Hospital Huashan to characterise clinical isolates of C. difficile. Of 74 isolates, 56 were from the first episode of CDI (43 A(+)B(+) and 13 A(-)B(+)), 5 were from recurrences and 13 were toxin-negative. No binary toxin or TcdC deletion was detected. All strains were susceptible to metronidazole, vancomycin, meropenem and piperacillin/tazobactam. Resistance to moxifloxacin, ciprofloxacin, levofloxacin, erythromycin, clindamycin, tetracycline, rifampicin and fusidic acid was found in 46.4%, 100%, 60.7%, 71.4%, 71.4%, 35.7%, 25.0% and 17.9% of the isolates, respectively. All moxifloxacin-resistant isolates carried a mutation in either gyrA, gyrB or both. Fourteen different polymerase chain reaction ribotypes were identified, with a specific clone (SH II) accounting for 25% of isolates. No isolates belonged to ribotype 027. The present study is the first systematic survey of clinical C. difficile isolates in China. Further surveillance is required to detect clustering of cases and to monitor the emergence of specific highly virulent clones and resistance.

Antimicrobial susceptibility and heteroresistance in Chinese Clostridium difficile strains

One hundred and ten toxigenic Clostridium difficile isolates collected between December 2008 and May 2009 at Fudan University Hospital Huashan were analyzed for their antibiotic susceptibility patterns and resistance molecular basis. The heteroresistance to metronidazole in fresh isolates were detected as well. Sixteen different PCR ribotypes were identified with a dominant clone 017 accounting for 37.3% of the isolates, followed by 001 and H. Ribotype 027 was not found but one isolate belonged to ribotype 078. All the isolates were susceptible to vancomycin and piperacillin/tazobactam. Seventy-eight fresh isolates were tested for heteroresistance to metronidazole, 18 (23.1%) of them were found to be positive. Resistance to moxifloxacin, ciprofloxacin, levofloxacin, erythromycin, clindamycin, tetracycline, rifampin, rifaximin and fusidic acid was found in 61.8%, 100%, 66.4%, 85.3%, 88.1%, 62.7%, 29.1%, 29.1% and 8.2% of the isolates, respectively. The isolates of common PCR ribotypes were more frequently resistant than the uncommon ribotypes. The prevalence of resistance genes and mutations among the resistant isolates was as follows: ermB, 69.1%; tetM, 97.1%; gyrA mutation, 63.2%; gyrB mutation, 4.4%; gyrA and gyrB mutation, 32.4%; rpoB mutation, 100%, respectively. The resistance related fusA mutation was only found in one isolate with minimum inhibitory concentration of 4 mg/L.

Community-acquired pneumonia in Shanghai, China: microbial etiology and implications for empirical therapy in a prospective study of 389 patients.

The aim of this multicenter study was to identify the causative pathogens of community-acquired pneumonia (CAP) in Shanghai, China, and to determine their susceptibility to antimicrobial agents. Pathogens obtained from 389 patients with documented CAP during 2001–2003 were identified by multiple diagnostic tools that included bacterial culture, polymerase chain reaction (PCR), and specific immunological assays. Susceptibility of the bacterial isolates was tested by the broth microdilution method. A specific pathogen was identified in 39.8% (155/389) of the patients: Haemophilus influenzae (n=80), Klebsiella spp. (n=15), Streptococcus pneumoniae (n=12), Staphylococcus aureus (n=6), Moraxella catarrhalis (n=1), other gram-negative organisms (n=9), and atypical pathogens that comprised Mycoplasma pneumoniae (n=42), Chlamydia pneumoniae (n=17), and Legionella pneumophila (n=2). Most H. influenzae isolates were susceptible to ampicillin (88.3%), and all were susceptible to macrolides. Of the S. pneumoniae isolates, 75% (9/12) were susceptible to penicillin, while 25% (3/12) were intermediately susceptible. H. influenzae and atypical pathogens are among the most important pathogens of CAP. Ampicillin, cephalosporins, and the newer fluoroquinolones can be used as empirical therapy for CAP in the Shanghai area. The efficacy of monotherapy with newer macrolides for CAP caused by S. pneumoniae requires further evaluation.

Emergence of carbapenem-resistant clinical Enterobacteriaceae isolates from a teaching hospital in Shanghai, China

Carbapenems such as imipenem and meropenem are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant clinical isolates of bacteria belonging to the family Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among members of the family Enterobacteriaceae isolated from a teaching hospital in Shanghai, China. In the present study, we investigated the prevalence and molecular characteristics of carbapenem-resistant clinical isolates of Enterobacteriaceae. None of the 77 clinical isolates collected from 2002 to 2009 were susceptible to ertapenem and only 6.5 % and 1.3 % of isolates were susceptible to imipenem and meropenem, respectively. Colistin and tigecycline were found to be the most active agents against carbapenem-resistant Enterobacteriaceae isolates, inhibiting 90 % of isolates at a concentration of 1 µg ml(-1) and 4 µg ml(-1), respectively. The results of PFGE analysis suggested that many of the KPC-2-producing isolates of Citrobacter freundii and Klebsiella pneumoniae were clonally related. Most of these isolates were isolated from the same ward, namely the neurosurgical ward, suggesting horizontal transfer of the KPC-2-encoding gene in these isolates. Of the 77 isolates, 84.4 % were found, by PCR, to be capable of carbapenemase production. SDS-PAGE analysis revealed that 75.3 % (58/77) of the isolates had lost at least one porin protein. Our results suggested that the prompt detection of carbapenemase-producing strains is critical for the containment of nosocomial transmission. As no novel antimicrobials have been identified for use in the treatment of these pan-drug-resistant isolates, further studies should focus on the rational use of available antibiotics, the implementation of active antibiotic resistance surveillance and the strict implementation of infection control measures to avoid the rapid spread or outbreak of carbapenemase-producing Enterobacteriaceae in health-care facilities.

Activities of Newer Quinolones against Escherichia coli and Klebsiella pneumoniae Containing the Plasmid-Mediated Quinolone Resistance Determinant qnr

ABSTRACT Seventeen qnr-containing transconjugants were constructed with azide-resistant Escherichia coli J53 as the recipient, and the MICs of 12 quinolones were tested by agar dilution methods. Sitafloxacin, BAYy3118, and premafloxacin had higher activity in vitro than ciprofloxacin against transconjugants and donors containing qnr. The donors had higher quinolone MICs than the transconjugants.

Prevalence and Expression of the Plasmid-Mediated Quinolone Resistance Determinant qnrA1

ABSTRACT Since its discovery, qnrA has been found in most common Enterobacteriaceae. Ciprofloxacin MICs conferred by different qnrA-positive plasmids could range from 0.1 μg/ml to 2 μg/ml in Escherichia coli J53. The reasons for different ciprofloxacin MICs conferred by qnrA have not been fully clarified. Five hundred forty-one consecutive gram-negative clinical strains that were resistant or intermediate to ciprofloxacin and that were isolated in Shanghai in 2005 were screened for qnrA by PCR. For qnrA-positive isolates, the transferability of quinolone resistance was determined by conjugation and mutations within the quinolone resistance-determining region (QRDR) of gyrA and parC. aac(6′)-Ib-cr was detected and qnrA RNA expression was determined using real-time reverse transcription-PCR for transconjugants with different ciprofloxacin MICs. The qnrA gene was detected in 7 of the 541 clinical isolates. Quinolone resistance was transferred in four strains by conjugation. Mutations in the QRDR of gyrA and parC were detected in five qnrA-positive clinical strains with higher ciprofloxacin MICs. Of four qnrA-bearing plasmids in E. coli J53, pHS4 and pHS5 conferred ciprofloxacin MICs of 0.094 to 0.125 μg/ml; pHS3, which harbored the aac(6′)-Ib-cr gene as well, conferred a ciprofloxacin MIC of 0.25 μg/ml, and pHS6, which had both the aac(6′)-Ib-cr gene and a high expression level of qnrA, had a ciprofloxacin MIC of 1.0 μg/ml. The prevalence of qnrA appeared to be higher in Enterobacter cloacae than in other Enterobacteriaceae. The coexistence of qnrA and aac(6′)-Ib-cr in a single plasmid and increased qnrA expression can account for the different levels of ciprofloxacin resistance seen in transconjugants.

Pathogenic Implication of a Fibrinogen-Binding Protein of Staphylococcus epidermidis in a Rat Model of Intravascular-Catheter-Associated Infection

ABSTRACT The involvement of Fbe, a fibrinogen-binding protein of Staphylococcus epidermidis, in the pathogenesis of catheter-associated infection was investigated. An fbe (gene encoding Fbe protein) mutant was constructed by allelic replacement, wherein an erythromycin resistance gene replaced a portion of the A region of fbe. Meanwhile, a rat central venous catheter (CVC) infection model was established to assess the importance of Fbe in the pathogenesis of CVC-associated infection due to S. epidermidis. Fbe-positive S. epidermidis strain HB was significantly more likely to cause a CVC-associated infection resulting in bacteremia and metastatic disease than its isogenic Fbe-deficient mutant (100% versus 20%, P < 0.01). These results confirm the importance of adherence associated with Fbe in the pathogenesis of CVC-associated infection caused by S. epidermidis.

Pharmacokinetics and Pharmacodynamics of Nemonoxacin against Streptococcus pneumoniae in an In Vitro Infection Model

ABSTRACT The aim of this paper was to investigate the pharmacokinetics (PK) and pharmacodynamics (PD) of nemonoxacin, a novel nonfluorinated quinolone, against Streptococcus pneumoniae in vitro. A modified infection model was used to simulate the pharmacokinetics of nemonoxacin following scaling of single oral doses and multiple oral dosing. Four S. pneumoniae strains with different penicillin sensitivities were selected, and the drug efficacy was quantified by the change in log colony counts within 24 h. A sigmoid maximum-effect (Emax) model was used to analyze the relationship between PK/PD parameters and drug effect. Analysis indicated that the killing pattern of nemonoxacin shows a dualism which is mainly concentration dependent when the MIC is low and that the better PK/PD index should be the area under the concentration-time curve for the free, unbound fraction of the drug divided by the MIC (fAUC0–24/MIC), which means that giving the total daily amount of drug as one dose is appropriate under those conditions. When the MIC is high, the time (T) dependency is important and the valid PK/PD index should be the cumulative percentage of a 24-h period in which the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (f%T>MIC), which means that to split the maximum daily dose into several separate doses will benefit the eradication of the bacteria. To obtain a 3-log10-unit decrease, the target values of fAUC0–24/MIC and f%T>MIC are 47.05 and 53.4%, respectively.