Tingting Qu

Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Chinese hospitals

We investigated the molecular epidemiology and carbapenem resistance mechanisms of 258 non-duplicate carbapenem-resistant clinical isolates of Pseudomonas aeruginosa collected from 2006 to 2007 at 28 hospitals in China. Up to 88% of the carbapenem-resistant isolates were multidrug-resistant. Pulsed-field gel electrophoresis (PFGE) revealed that levels of intrahospital and interhospital dissemination of clones were low. To assess the mechanisms leading to resistance, all 258 carbapenem-resistant isolates were analysed for expression of the chromosomal beta-lactamase (AmpC), the porin important for entry of carbapenems (OprD) and an efflux system (MexAB-OprM) known to extrude some beta-lactams. Carbapenem resistance was driven mainly by mutational inactivation of OprD, accompanied or not by hyperexpression of AmpC or MexAB-OprM. Metallo-beta-lactamase genes were detected in 22 carbapenem-resistant isolates in China, belonging to eight pulsotypes. The bla(OXA-50) gene was detected among all of the carbapenem-resistant isolates, whereas the bla(GES-5) gene was detected in only one carbapenem-resistant isolate.

Evaluation of Phenotypic Tests for Detection of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Strains in China

ABSTRACT A total of 264 nonduplicate strains of imipenem (IPM)-nonsusceptible Pseudomonas aeruginosa were isolated from hospitals in 16 different regions throughout China. These 264 IPM-nonsusceptible clinical isolates of P. aeruginosa were examined by PCR, a metallo-β-lactamase (MBL) Etest, a double-disk synergy test (DDST), and a test using combined IPM disks supplemented with various amounts of EDTA. A total of 24 strains positive for MBLs were confirmed by PCR and DNA sequence analysis: 10 strains positive for the blaVIM-2 gene, 13 strains positive for the blaIMP-9 gene, and 1 strain positive for the blaIMP-1 gene. Real-time reverse transcriptase PCR (RT-PCR) was used to verify whether the isolates harboring MBL genes produced the enzyme and was considered the standard for evaluation of the methodology in this study. Of these 24 MBL-positive stains, 21 were confirmed as MBL-producing strains by real time RT-PCR for MBL expression and the other 3 had no expression of MBLs. The sensitivities, specificities, and positive and negative predictive values for the MBL Etest, the DDST, and the combined disk (CD) assay were evaluated. The best method for screening for MBL production in P. aeruginosa strains from China was the CD assay (IMP-EDTA) using 750 μg of EDTA/disk with a breakpoint of ≥6 mm. In the CD assay (IPM-EDTA) with 290 μg and 750 μg EDTA, the zone diameter increases for VIM-2-producing P. aeruginosa isolates were greater than those for IMP-9-producing P. aeruginosa isolates (P = 0.00).

Integrons Containing the VIM-2 Metallo-β-Lactamase Gene among Imipenem-Resistant Pseudomonas aeruginosa Strains from Different Chinese Hospitals

ABSTRACT A total of 140 nonrepetitive strains of imipenem-resistant Pseudomonas aeruginosa were isolated from five different Chinese hospitals. Fourteen isolates were confirmed to contain the VIM-2 metallo-β-lactamase gene. Twelve isolates harbored two kinds of class 1 integron, containing both VIM-2- and aminoglycoside-resistant genes.

Clinical and microbiological characteristics of Klebsiella pneumoniae liver abscess in East China

BackgroundKlebsiella pneumoniae has been the dominant pathogen for liver abscesses in several Asian countries. Although the prevalence of K. pneumoniae liver abscess (KLA) in mainland China is increasing recently, the clinical and microbiological characteristics of KLA in China have not been elucidated.MethodsClinical and microbiology characteristics of 45 consecutive patients with KLA from a tertiary teaching hospital in China between June 2008 and June 2012 were retrospectively evaluated.ResultsVast majority of the strains were susceptible to main antimicrobial agents. Most of K. pneumoniae strains from pyogenic liver abscess patients belonged to K1/K2 serotype (68.9% for K1 serotype and 20% for K2 serotype). All K. pneumoniae strains were rmpA positive, and 68.9% of these strains were magA positive. Overall, 57.8% (26/45) of K. pneumoniae strains belonged to ST23. Twenty-five of 26 ST23 K. pneumoniae isolates (96.2%) from KLA patients were magA-positive and K1 serotype. Only 28.9% (13/45) of KLA isolates exhibited hypermucoviscous phenotype, which is clinically used as the characteristic of hypervirulent K. pneumoniae (hvKP). Liver abscess sizes in patients infected with hvKP were tend to be larger than those in patients infected with cKP. There was no significant association between the microbiological and clinical characteristics including serotypes, magA and rmpA genotypes, and STs with the metastatic infection and prognosis of KLA.ConclusionsNeither the serotypes, magA and rmpA genotypes, nor the STs of K. pneumoniae were associated with the metastatic infection and prognosis of KLA. However, further studies with larger sample are needed in the future.

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple β-lactamases

Four clinical strains of extended-spectrum beta-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C(1) and C(2) were isolated prior to carbapenem therapy, whilst isolates C(3) and C(4) were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). beta-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C(1) in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple beta-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C(1) and C(2), isolates C(3) and C(4) failed to express OmpE36 owing to insertional inactivation by an IS903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene (kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C(3) and C(4) were apparently derived from the previously imipenem-susceptible isolates C(1) and C(2). Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.

Emergence of cfr-harbouring coagulase-negative staphylococci among patients receiving linezolid therapy in two hospitals in China

This study reports on the emergence of cfr-harbouring coagulase-negative staphylococci (CoNS) among patients who received linezolid therapy in two hospitals in Hangzhou, China. The mechanisms of resistance and transmission were analysed for these resistant isolates. Eight Staphylococcus capitis isolates, one Staphylococcus epidermidis isolate and one Staphylococcus hominis isolate, obtained from patients who had received linezolid therapy in two hospitals in Hangzhou, China, were confirmed as linezolid resistant, with MICs ranging from 8 to >256 mg l(-1). The linezolid usage data of the ten patients before isolation of the linezolid-resistant CoNS were collected. PFGE analysis showed that the eight S. capitis isolates from the two hospitals belonged to the same clone. Nine of the linezolid-resistant CoNS isolates carried the cfr gene, which was located on plasmids of a similar size. A 5.3 kb fragment containing the cfr gene, revealing 99 % identity to the sequence of the cfr-harbouring plasmid pSS-01 reported previously, was determined by PCR mapping for all cfr-positive isolates, and the cfr gene was flanked by two copies of IS256-like elements. Thus, these results document the emergence of linezolid-resistant CoNS isolates carrying the cfr gene in Hangzhou, China. Effective nosocomial infection control strategies and the judicious use of antibiotics will be required to prevent further spread of this resistance mechanism.

Novel Vancomycin-Resistance Transposon, Plasmid Replicon Types, and Virulence Factors of Vancomycin-Resistant Enterococci in Zhejiang, China

Forty-seven vancomycin-resistant Enterococcus (VRE) strains were isolated from clinical samples in 13 Zhejiang hospitals and fecal samples from ICU patients in a large teaching hospital in China. No VRE isolates were detected in healthy human subjects. CC17 was the main clonal complex in clinical Enterococcus faecium isolates but not in isolates from healthy human subjects. Novel vancomycin-resistance transposons were detected among VRE strains. This is the first report demonstrating insertion of tnpA and fosB genes in the vanRS-vanH intergenic region of Tn1546 leading to coresistance to vancomycin and fosfomycin. The four plasmid replicon types (pRUM, pRE25, pEF418, and pB82) were more common in VRE isolates, suggesting their association with vancomycin resistance and nosocomial transmission. The prevalence rate of vancomycin-resistant Staphylococcus aureus-related Inc18-like plasmid, pIP501, in VRE was 21.3%. The prevalence of the esp gene among VRE isolates was high (76.6%). In several VRE strains, the esp and hyl genes were cotransferred with the vanA gene by conjugation. Although the frequency of VRE is low in Chinese hospitals, its association with virulence determinants, the vancomycin-resistance transposon with other resistance gene insertions or plasmids may lead to multidrug resistance and the evolution of pathogenic VRE.

Whole Genome Analysis of a Community-Associated Methicillin-Resistant Staphylococcus aureus ST59 Isolate from a Case of Human Sepsis and Severe Pneumonia in China

We report a case of necrotizing pneumonia in a young patient caused by community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) in a teaching hospital in the People’s Republic of China. The patient had a typical clinical presentation and was successfully treated with antibiotics and intravenous immunoglobulin. A CA-MRSA strain, named SA268, was isolated from the blood of the patient. The isolate was susceptible to most antimicrobial agents, except cephalosporins, penicillins, and β-lactamase inhibitor combinations. Multi-locus sequence typing (MLST) assigned SA268 to ST59, a clone widely spread in eastern Asia. The strain was positive for Panton Valentine Leukocidin (PVL)-encoding genes and SCCmec type V. We sequenced the complete genome of the SA268 isolate. The genome of SA268 was almost identical to that of the Taiwanese ST59 CA-MRSA strains M013 and SA957. However, we observed several differences in gene composition, which included differences in the SCCmec element and several lipoprotein genes that were present in the Taiwanese strains but absent from SA268.

Prevalence of the fosfomycin-resistance determinant, fosB3, in Enterococcus faecium clinical isolates from China.

In order to investigate the prevalence of fosfomycin-resistance (fos) determinants in Enterococcus faecium, clinical strains were collected from hospitals throughout China between January 2008 and December 2009. Antimicrobial susceptibility testing was performed, after which the fos genes in all isolates and van genes in vancomycin-resistant isolates were characterized by PCR and sequencing. Conjugation experiments were carried out with fosB-positive E. faecium, DNA fragments flanking the fosB3 gene were sequenced and the genetic environment of fosB3 was analysed. Fosfomycin-resistant E. faecium (FREF) strains were characterized further by multilocus sequence typing (MLST) and PFGE. Among 145 E. faecium clinical isolates, 10 were resistant to fosfomycin with MICs>1024 mg l(-1) including six vancomycin-resistant strains of which five were vanA-positive and the sixth vanM-positive. All ten FREF strains harboured the fosB3 gene. Fosfomycin resistance and fosB3 could be transferred by conjugation from nine isolates. The fosB3 and tnpA genes were located in a circular DNA intermediate in all FREF strains and reversely inserted into vanA transposon Tn1546 in four vanA-positive FREF isolates. Ten different PFGE types and seven MLST types were found among the ten fosB3-positive isolates, while all strains belonged to the common clonal complex CC17. In conclusion, the transferable fosfomycin-resistance determinant fosB3 plays an important role in E. faecium resistance to fosfomycin in China.

Heteroresistance to Teicoplanin in Enterococcus faecium Harboring the vanA Gene

Hospital-acquired infections caused by vancomycin-resistant enterococci (VRE) are increasing. The resistance of VRE to multiple antibiotics makes them clinically challenging. There are at least six phenotypes of VRE, including VanA, VanB, VanC, VanD, VanE, and VanG ([2][1], [8][2]). These phenotypes