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

BACKGROUND: Klebsiella 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. METHODS: Clinical 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. RESULTS: Vast 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. CONCLUSIONS: Neither 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.

Risk factors, outcomes and epidemiology associated with Clostridium difficile infection in patients with haematological malignancies in a tertiary care hospital in China.

The purpose of this study was to evaluate the risk factors, outcomes and epidemiology associated with Clostridium difficile infection (CDI) in patients with haematological malignancies in a tertiary care hospital in China. C. difficile screening was performed on patients admitted for chemotherapy or haematopoietic stem cell transplantation between 2009 and 2013. C. difficile isolates were analysed by multilocus sequence typing, and a retrospective chart review was performed on all patients with a positive toxin assay. CDI was diagnosed in 21 haematology-oncology ward patients and 14 marrow transplantation service patients for a cumulative incidence of 1.89/1000 and 3.69/1000 patient-days, respectively. Univariate analyses showed that patients who received etoposide had an increased risk of CDI (odds ratio 4.25, 95 % confidence interval 1.32-13.64). There was only one patient death, for which CDI was not the primary cause. Ten sequence types (STs) were identified, of which ST-3 and ST-54 were the most common; the hypervirulent ST-1 (ribotype 027) and ST-11 (ribotype 078) C. difficile strains were not detected in the patients in this study. The incidence of CDI did not differ between patients receiving chemotherapy and those receiving haematopoietic stem cell transplantation. The only risk factor for chemotherapy patients was treatment with etoposide.

Fosfomycin resistance among vancomycin-resistant enterococci owing to transfer of a plasmid harbouring the fosB gene.

The presence and characterisation of plasmid-mediated fosfomycin resistance determinants were investigated among 45 clinical vancomycin-resistant enterococci (VRE) isolated in Zhejiang Province, China. In total, 19 VRE were resistant to fosfomycin, of which 18 isolates had conjugative fosfomycin resistance and were positive for fosB. No reported fos genes were detected in the remaining isolate. Among the 18 fosB-carrying isolates, the fosB gene was always flanked by tnpA, suggesting the same novel fosB transposon. In 10 of the 18 fosB-carrying isolates, the fosB and tnpA genes were found reversely inserted in the vanA transposon Tn1546. In the remaining eight isolates the fosB and vanA genes were located on different plasmids. These findings indicate that acquisition of the conjugative plasmid harbouring the novel fosB transposon (ISL3-like transposon) and the Tn1546-like transposon (containing vanA and fosB) may explain, at least in part, the recent increase in fosfomycin-resistant Enterococcus faecium in China.

Molecular epidemiology of Clostridium difficile in a tertiary hospital of China.

Clostridium difficile infection (CDI) is caused by toxin-producing strains. It accounts for 20-30 % of antibiotic-associated diarrhoea and particularly accounts for 90 % of pseudomembranous colitis. The epidemiological study of C. difficile is thus important. In this study, we report the molecular epidemiology and ward distribution of C. difficile in a tertiary hospital of China. A total of 161 toxigenic strains were isolated from 1845 patients originating from different wards and the strains were characterized based on toxin profile and multilocus sequence typing. Variable isolation rates were observed in different wards and the occurrence was higher in intensive care unit and geriatric wards. Toxin gene profiling revealed that, out of the 161 isolates, 134 (83.2)% were positive for both toxin A (tcdA) and toxin B (tcdB) (A+B+) followed by toxin A-negative and B-positive (A-B+) (16.8 %) isolates. However, only three of the toxigenic strains (1.9 %) were positive for both the cdtA and cdtB genes. Based on the molecular epidemiology study, a total of 30 different sequence types (STs), including one new ST (ST-220), were distinguishable. ST-54 was the most prevalent (23.0 %), followed by ST-35 (19.3 %) and ST-37 (10.0 %). None of the isolates belonged to ST-1 (ribotype 027) or ST-11 (ribotype 078). Taken together, the toxin profile and the molecular epidemiological data showed that all the ST-37 clades were of toxin type A-B+, which accounted for 59.3 % of all type A-B+ isolates. Meanwhile the clade 1 genotype, ST-54, was widely distributed among the geriatric, infection and haematology wards. There was no outbreak of C. difficile infection during our study; however the possibility of prolonged outbreaks cannot be completely ignored.

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.

Epidemiology and characteristics of antimicrobial resistance in China.

A comprehensive surveillance system for bacterial resistance in tertiary hospitals has been established in China that involves tertiary hospitals in distinct regions nationwide, enabling the collection of a large amount of antimicrobial surveillance data. Antimicrobial resistance in China has become a serious healthcare problem, with high resistance rates of most common bacteria to clinically important antimicrobial agents. Methicillin-resistant S. aureus, ESBL-producing Enterobacteriaceae and carbapenem-resistant Acinetobacter baumannii represent more than 50% of microbial isolates. Additionally, bacterial resistance to fluoroquinolones, macrolides and third-generation cephalosporins is of serious concern. The molecular epidemiology and resistance mechanisms of the antimicrobial strains in China exhibited regional specificity, as well as the influence of dissemination of international clonal complexes. The molecular characteristics of MRSA, ESBL- and carbapenemase-producing Enterobacteriaceae, and macrolide-resistant gram-positive Streptococci in China were significantly different from those in other countries and regions, while S. pneumoniae serotypes appear to have been affected by the global spread of prevalent clones in other parts of the world. Moreover, important antimicrobial resistant bacteria such as community-acquired-MRSA, multidrug-resistant P. aeruginosa and extensive-resistant A. baumannii, and the antimicrobial resistance in primary healthcare and outpatient setting should be intensely monitored and investigated in the future.

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.

In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple beta-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.

Clonal spread of imipenem-resistant Acinetobacter baumannii among different cities of China.

A total of 342 imipenem-resistant Acinetobacter baumannii isolates (IRABs) were collected from 16 Chinese cities. Six predominant clones had spread widely, and four clones were detected in distant hospitals. The majority of the IRABs contain bla(OXA-23), with ISAba1 upstream of the gene. These results suggested that clonal spread played an important role in the outbreak of IRABs in China.

[Study on the carbapenemase genotype and molecular epidemiology of Acinetobacter baumannii].

OBJECTIVE: To investigate antibiotic resistance, clonal relatedness and carbapenemase genotype among carbapenem-resistant Acinetobacter baumannii collected from 3 comprehensive hospitals in Ningbo city, Zhejiang province. METHODS: 28 strains of carbapenem resistant Acinetobacter baumannii were collected from Ningbo Li Hui-li Hospital, Ningbo Li Hui-li Hospital, Ningbo First Hospital, and N ingbo Second Hospital. The minimum inhibitory concentrations (MIC) of these strains were examined by agar dilution and E-test method. Homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE) and Genotype of carbapenemases were analyzed by PCR and verified by DNA sequencing. RESULTS: 28 strains of Acinetobacter baumanii were highly resistant to all of the antibiotics except polymyxin E. They were classified into 4 clones based on PFGE pattern. Clone A and B had been spreading widely. All of the 28 strains produced carbapenemases which were confirmed as OXA-23 by PCR and sequencing. Metallo-beta-lactamase was not detected in any of the isolates. CONCLUSION: All of t hecarbapenem-resistant Acinetobacter baumannii collected from Ningbo were producing OXA-23 carbapenemase, suggesting that the transmission of clones had occurred in the 3 hospitals.

Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China.

A carbapenem-resistant isolate of Klebsiella pneumoniae producing class A carbapenemase KPC-2 was identified in Zhejiang, China. The KPC-2 gene was located on an approximately 60-kb plasmid in a genetic environment partially different from that of blaKPC-2 in the isolates from the United States and Colombia.

[A novel plasmid-mediated aminoglycosides-modifying enzyme gene, aph (2'')-Ie, in a strain of high-level gentamicin resistant Enterococcus casseliflavus].

OBJECTIVE: To identify the aminoglycoside resistance gene in the high-level gentamicin resistant (HLGR) enterococcus and its transmission mechanism. METHODS: A HLGR strain, HZ95, of Enterococcus casseliflavus was screened by agar dilution method. The aminoglycoside activation enzyme genes were screened by PCR. The PCR products underwent purification, cloned, and sequenced. Plasmids were extracted and underwent Southern hybridization. Plasmid-mediated aminoglycoside modifying enzymes were cloned. The resistance of the HZ05 strain to different antimicrobial agents was determined by K-B method or agar dilution method. RESULTS: A new aminoglycosides-modifying enzyme, APH (2'')-Ie, leading to HLGR, was found in the plasmid of the HZ95 bacteria. The aph (2'')-Ie gene and partial sequence of a streptomycin adenylyltransferase gene (str) were contained in the 8.7-kb cloned fragment, and the transposase gene (tnpA) was on the upper stream. The aph (2'')-Ie gene was located on the 16-kb plasmid with the amino acid sequence 96.1% homologous with chromosome-mediated APH (2'')-Id aminoglycoside-modifying enzyme. CONCLUSION: HLGR can be caused by a new plasmid-mediated aminoglycosides-modifying enzyme, APH (2'')-Ie, which can be transmitted among plasmids or chromosome with other resistant genes through transposase.

[A multicentre study on the pathogenic agents in 665 adult patients with community-acquired pneumonia in cities of China].

OBJECTIVE: To investigate the pathogenic causes of community-acquired pneumonia (CAP) in adult patients in China, the relation of previous antibiotic use and the Pneumonia Patient Outcome Research Team (PORT) classification to microbial etiology, and the prevalence of drug resistance of common CAP bacteria. METHODS: A prospective study was performed on 665 consecutive adult patients with CAP at 12 centers in 7 Chinese cities during one year. The etiology of pneumonia was considered if one of the following criteria was met: (1) valid sputum sample yielding one or more predominant strains; (2) blood cultures yielding a bacterial pathogen; (3) seroconversion, a > or = 4-fold increase or decrease titers of antibodies to Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila. Minimum inhibitory concentration (MIC) of respiratory tract isolates was determined using the agar dilution method. RESULTS: Pathogens were identified in 324/610 patients (53.1%) with valid serum samples and sputum cultures as follows: Mycoplasma pneumoniae (126, 20.7%), Streptococcus pneumoniae (63, 10.3%), Haemophilus influenzae (56, 9.2%), Chlamydia pneumoniae (40, 6.6%), Klebsiella pneumoniae (37, 6.1%), Legionella pneumophila (31, 5.1%), Staphylococcus aureus (23, 3.8%), Escherichia coli (10, 1.6%), Moraxella catarrhalis (8, 1.3%), Pseudomonas aeruginosa (6, 1.0%). Of 195 patients with a bacterial pathogen, an atypical pathogen was identified in 62 (10.2%) cases. The non-susceptibility rate of Streptococcus pneumoniae to penicillin, azithromycin, and moxifloxacin was 20.3%, 75.4% and 4.3% respectively. CONCLUSIONS: Atypical pathogens have important role in CAP, with Mycoplasma pneumoniae being the most common pathogen, and mixed infection of atypical pathogens with bacteria was found in 10.2% of the cases. Streptococcus pneumoniae and Haemophilus influenzae remain the most important bacteria for CAP. More than 75.0% of Streptococcus pneumoniae was resistant to macrolides and 20.3% was resistant to penicillin.

[Genotypes of aminoglycoside-modifying enzyme and clinical study of high-level gentamycin resistant enterococcus].

OBJECTIVE: To determine the antibiotics resistance, aminoglycoside-modifying enzymes and homology of high-level gentamycin resistant enterococcus in clinical specimens. METHODS: The high-level gentamicin resistant (HLGR) isolates were screened by the agar method and the resistance of 14 antimicrobial agents was determined by K-B method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates. RESULTS: The ratio of HLGR was 64.2% (68/106). Among the HLGR,there were no isolates resistant to linezolid, vancomycin and tecoplanin, and Enterococcus faecium was more resistant to beta-lactam antibiotics and quinolone than Enterococcus faecalis. The positive rate of aac(6')-Ie-aph(2')-Ia was 92.6% and 3 isolates had the resistance gene mostly similar to aph(2')-Id. And among 51 HLGR isolates from the hospitalized patients, PFGE grouped 17 E. faecalis isolates into 4 clusters (A-D), and 33 E. faecium isolates into 8 clusters (A-H) with A cluster as predominant. CONCLUSION: HLGR has become the important antibiotic resistance bacteria which results in nosocomial infection; and aac(6')-Ie-aph(2')-Ia is the main aminoglycoside-modifying enzyme gene which causes HLGR.

Genotypic diversity and epidemiology of high-level gentamicin resistant Enterococcus in a Chinese hospital.

OBJECTIVE: To investigate the antibiotics resistance of Enterococcus, the aminoglycoside-modifying enzymes (AME) and homology of high-level gentamicin resistant (HLGR) Enterococcus in clinical specimens for the implementation of effective infection control measures. METHODS: The resistance of 13 antimicrobial agents was determined by Kirby-Bauer (K-B) or agar dilution method. And the HLGR and high-level streptomycin resistant (HLSR) isolates were screened by agar screen. Production of beta-lactamases was tested by the nitrocefin disc method. The aminoglycoside-modifying enzyme genes were detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis (PFGE) was used to analyze the homology of HLGR isolates from in-patients. RESULTS: No isolates resistant to linezolid, vancomycin and teicoplanin were found. Ampicillin-resistant isolates did not produce beta-lactamases and 68 HLGR isolates were screened at the rate of 64.2%. The positive rate of aac(6')-Ie-aph(2'')-Ia was 86.8% and 3 isolates had the new AME gene designated aph(2'')-Ie mostly similar to aph(2'')-Id. Among 51 HLGR isolates from in-patients, PFGE grouped 17 Enterococcus faecalis (E. faecalis) isolates into 4 clusters (A-D), and 33 Enterococcus faecium (E. faecium) isolates into 8 clusters (A-H), of which the A cluster is the main. CONCLUSIONS: HLGR has become the important antibiotic resistance pathogen causing nocosomial infection. And the aac(6')-Ie-aph(2'')-Ia gene was the main aminoglycoside-modifying enzyme gene leading to HLGR.

[Study on the genetic structure and transmission mechanism of a plasmid-mediated AmpC beta-lactamase].

OBJECTIVE: To clone the gene of plasmid-mediated AmpC beta-lactamase from the plasmid of multiple-drug resistance Klebsiella pneumoniae producing plasmid-mediated AmpC beta-lactamase and demonstrate its mechanism of transmission. METHODS: Plasmids of the transconjugant were extracted and digested with restriction endonuclease HindIII. Taq DNA polymerase was applied to fill the recessed 3' termini, and a single deoxyadenosine was added to the 3' termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5alpha containing recombinant plasmid was selected on MacConkey agar plates containing ampicillin and cefoxitin. Insert fragments were sequenced by primer walking. MIC determinations and isoelectric focusing electrophoresis (IFE) were utilized to analyze recombinant. RESULTS: The recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The inserted fragment contained a bla(DHA-1) and a regulatory gene ampR. The insertion sequence (IS26), qacEDelta1 and sulI genes of the I type integron were obtained near the bla(DHA-1) gene. Recombinant expressed a beta-lactamase with pI of 7.7. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin. CONCLUSION: The plasmid-mediated ampC gene cloned was identified as bla(DHA-1). IS26 observed on the flanks of the bla(DHA-1) maybe relate to the translocation of bla(DHA-1) gene region from the chromosome to plasmid.

Nosocomial spread of multi-resistant Klebsiella pneumoniae containing a plasmid encoding multiple beta-lactamases.

Six Klebsiella pneumoniae isolates that exhibited resistance to a wide spectrum of antibiotics were recovered from the intensive care units in the First Affiliated Hospital, Zhejiang University, Hangzhou, China. All isolates contained two plasmids of approximately 95 kb and 200 kb. The 95 kb plasmid was shown to be transferable by conjugation experiments. Isoelectric focusing patterns of the beta-lactamases extracted from the six transconjugants were identical, displaying five pI bands: 5.4, 7.75, 8.0, 8.2 and 8.4. The band corresponding to a pI of 7.75 could be inhibited by cloxacillin but not clavulanic acid, while the other bands could be inhibited by clavulanic acid but not cloxacillin. The 95 kb plasmid was digested with HindIII and a recombinant plasmid pT948 was obtained. The insert was found to contain blaDHA-1, regulatory gene ampR and an insertion element (IS26), which was downstream of blaDHA-1. PCR and DNA sequencing results confirmed that the 95 kb plasmid encoded at least four beta-lactamase genes: blaTEM-1, blaSHV-12), blaCTX-M-3 and blaDHA-1. Epidemiological typing by PFGE of the six clinical isolates of K. pneumoniae demonstrated identical genotypic patterns. In conclusion, all results indicated that the six multi-drug resistant clinical isolates of K. pneumoniae most probably originated from one clone and caused a localized epidemic in the intensive care units.