Shi Wu

New Plasmid-Mediated Quinolone Resistance Gene, qnrC, Found in a Clinical Isolate of Proteus mirabilis

ABSTRACT Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6′)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 μg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.

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.

Comparison of Adhesin Genes and Antimicrobial Susceptibilities between Uropathogenic and Intestinal Commensal Escherichia coli Strains

The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.

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.

Risk factors of Clostridium difficile infections among patients in a university hospital in Shanghai, China

Clostridium difficile infection (CDI) is an increasing concern in China. However, the risk factors of CDI are rarely reported in the Chinese population. A prospective observational study was therefore conducted among patients with hospital-acquired C. difficile diarrhoea and the risk factors of CDI in a retrospective case-control study. The CDI patients were compared with the non-CDI diarrhoeal patients and those without diarrhoea, respectively. The recurrent CDI patients were compared with the corresponding non-recurrent CDI patients and those without diarrhoea, respectively. Overall, of the 240 patients with hospital-acquired diarrhoea 90 (37.5%) were diagnosed as CDI, and 12 (13.3%) of the 90 CDI patients experienced recurrence. Multivariate analysis indicated that renal disease, malignancy, hypoalbuminemia, prior antibiotic treatment, chemotherapy, nasogastric tube use, length of stay>14 days and intra-abdominal surgery, defined daily dose of antimicrobial agents≥19, prior use of more than three antimicrobial agents, and use of carbapenems were independent risk factors for the first episode of CDI. Use of laxatives, the first- and second-generation narrow-spectrum cephalosporins or metronidazole was identified as protective factors. It is necessary to make testing of C. difficile available as a routine practice and control these risk factors in Chinese hospitals to avoid CDI outbreaks.

Clonal dissemination of extensively drug-resistant Acinetobacter baumannii producing an OXA-23 β-lactamase at a teaching hospital in Shanghai, China

BACKGROUND/PURPOSE Extensively drug-resistant (XDR) Acinetobacter baumannii presents a serious therapeutic and infection control challenge. This study aimed to explore the causes for the rapid increase of XDR A. baumannii at a teaching hospital in Shanghai. METHODS All consecutive clinical isolates of XDR A. baumannii were collected from January to December 2010 at Huashan Hospital in Shanghai. The prevalence of carbapenemase genes was investigated by polymerase chain reaction (PCR) amplification. Genetic relatedness of the isolates was determined by enterobacterial repetitive intergenic consensus-PCR and multilocus sequence typing. A retrospective case-control study was performed for the identification of risk factors of XDR A. baumannii infections. RESULTS All 106 XDR A. baumannii isolates carried the blaOxA-23 gene and were resistant to all antimicrobial agents tested, except colistin, tigecycline and cefoperazone-sulbactam. One hundred and five of the strains belonged to clonal complex 92 by multilocus sequence typing, and 78 were classified as clone A1 by enterobacterial repetitive intergenic consensus-PCR. Intensive care unit residency at the time of isolation, recent general anesthesia, the number of previous antibiotic classes administered and previous hospitalization were identified as risk factors by case-control study. Efficacy rates were 62.5% (5/8), 47.4% (9/19), and 42.9% (3/7) when the XDR patients were treated with cefoperazone-sulbactam, carbapenems, or both cefoperazone-sulbactam and carbapenem, alone or in combination with other agents, respectively. CONCLUSION XDR A. baumannii producing OXA-23 β-lactamase was clonally disseminated at a university hospital in Shanghai. Cefoperazone-sulbactam and carbapenems alone or combined with other antibiotics may benefit XDR A. baumannii infections in the absence of other effective antibiotics.

High ceftazidime hydrolysis activity and porin OmpK35 deficiency contribute to the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae

Objectives To investigate mechanisms for the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae (KPC-KP). Methods A total of 24 isolates, 8 each with ceftazidime/avibactam MICs of 4-8, 1-2 and ≤0.5 mg/L, were randomly selected from 214 clinical isolates of KPC-KP, and the β-lactamase hydrolysis activity and porin expression profiles were determined. Plasmid profile and relative expression and copy number of the bla KPC gene were also analysed. Results Ceftazidime/avibactam MIC 50 and MIC 90 were 2 and 4 mg/L, respectively, for the 214 KPC-KP isolates. The hydrolysis activities of nitrocefin and ceftazidime in both of the ceftazidime/avibactam MIC 4-8 and 1-2 mg/L groups were significantly higher than those of the MIC ≤0.5 mg/L group, while the hydrolysis activities were 4-4.6-fold higher in the MIC 4-8 mg/L group than in the other two groups when 4 mg/L avibactam was added. The relative expression and copy number of the bla KPC gene in the MIC 4-8 mg/L group were 4.2-4.8-fold higher than in the other two groups. Meanwhile, SDS-PAGE showed that all isolates in the two groups with MIC ≥1 mg/L lacked OmpK35, which had either an early frameshift with a premature stop codon ( n  =   15, ST11) or overexpression of the negative regulation genes, micF and ompR ( n  =   1, ST15), whereas OmpK35 and OmpK36 could both be observed in all isolates with MIC ≤0.5 mg/L. Conclusions Decreased ceftazidime/avibactam susceptibility in KPC-KP clinical isolates is caused by high ceftazidime hydrolysis activity and OmpK35 porin deficiency and the majority of isolates belong to ST11.

Toxin profiles, PCR ribotypes and resistance patterns of Clostridium difficile: a multicentre study in China, 2012–2013

A total of 178 clinical isolates of Clostridium difficile were collected from five major teaching hospitals representing northern, eastern and southern China from August 2012 to July 2013. Among the 178 isolates, 162 (91.0%) were toxigenic, including 66 (40.7%) toxin A-negative, toxin B-positive, 95 (58.6%) toxin A-positive, toxin B-positive and only 1 (0.6%) toxin A-, toxin B- and binary toxin-positive. Twenty-nine different PCR ribotypes were identified, of which 017 (21.0%), 012 (17.3%) and novel type H (16.7%) were the most prevalent. PCR ribotypes 027 and 078 were not found. All toxigenic strains were susceptible to metronidazole, vancomycin and piperacillin/tazobactam. Resistance to moxifloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, fusidic acid, imipenem, linezolid and rifampicin was observed in 45.1%, 79.6%, 75.3%, 46.9%, 3.7%, 29.6%, 4.9%, 2.5% and 12.3% of the isolates, respectively. Ribotype 017 showed resistance to more antimicrobial agents tested than other ribotype strains.