Objective To investigate the clinical characteristics, risk factors and prevention measures of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection. Methods Thirty-nine cases whose CRKP isolation and culture were positive and who were confirmed with nosocomial infection in 2nd affiliated hospital medical school of Zhcjiang University between July 2006 and July 2008 were retrospectively investigated. Results There were 22 (56.4%) sputum specimens among CRKP samples. The average age of the patients was 64.0 years old and the average length of staying in hospital was 80.8 days. There wcre 36 patients who had been in intensive care unit (ICU) within 2 weeks and 26 were conscious disturbance,38 received urethral catheterization, 34 inserted with central venous catheter, 32 received tracheal intubation and 30 assisted with mechanical ventilation. There were 27 (69. 2 % ) patients who were co-infected with other bacteria and 8 were co-infccted with fungi. The mortality was 46. 2% in patients with CRKP infection. Before CRKP-positive culture, 27 (69.2%)experienced ＞4 kinds of antibiotics with the majority of β-lactams/β-lactamase inhibitors and carbapenems.Conclusions CRKP infection frequently occurrs in elder and patients who prolonged stay in hospital,especially patients in ICU. Invasive procedures could increase the infection opportunities.

Objective To investigate the prevalence of qnr and aac(6')-Ⅰ b-cr genes in water-borne environmental bacteria and clinical isolates of Citrobacter freundii, and the subtypes of qnr gene. Methods Environmental bacteria were isolated from surface water samples obtained from 10 distinct loca-tions in Hangzhou city, and clinical isolates of C. Freundii were isolated from several hospitals of 4 cities in China. Minimum inhibitory concentrations (MICa) of ciprofloxacin, levofloxacin and nalidixic acid were de-termined by agar dilution method, qnrA, qnrB, qnrS and aac(6')-Ⅰ b-cr genes were screened by PCR, and the genotypes were analyzed by DNA sequencing. Results Seventy-eight gram negative bacilli (including 33 Enterobacteriaceae, 21 Aeromonas spp., 10 Acinetobacter spp., 10 Pseudomonas app., 2 Alcaligenes app. , and 2 Plesiomonas app.) were isolated from water samples. Among these isolates, 8 of 10 C. Freundii were positive for qnrB gene. qnrS1 and aac (6')-Ⅰ b-cr were detected in two distinct Escherichia coil, and qnrS2 was detected in a Aeromonas punctata qnr and aac(6')-Ⅰ b-cr genes were present in 75 (72.8%) and 12 (11.6%) of 103 clinical isolates of C. Freundii, respectively. Three (2.9%) C. Freundii isolates were positive for qnrA1 gene, 65 (63.1%) qnrB, 1 (1.0%) qnrS2, 5 (4.8%) were positive for both qnrA1 and qnrB, and 1 was positive for both qnrS1 and qnrB. Within the subtypes of qnrB gene, qnrB9 predominated, followed by qnrB8 and qnrB6. Conclusion It was the first isolate of Aeromonas spp. Harboring qnrS2 gene outside Europe. The prevalence of qnrB in water-borne environmental and clinical isolates of C.freundii was particularly high and qnrB9, qnrB8 and qnrB6 were the most common subtypes, aac(6')-Ⅰ b-cr gene widely spread in clinical isolates of C. Freundii.

Objective To investigate the molecular epidemiology and mechanism of earbapenem resistance of Serratia marcescens,Klebsiella pneumoniae and Escherichia coli isolates from intensive care units(ICUs).Methods Twenty-one S.marcescens,ten K.pneumoniae and one E.coli isolates with carbapenem resistance or reduced carbapenem susceptibility were recovered from two ICUs in our hospital from April 2006 to Febmary 2007.Pulsed-field gel electrophoresis(PFGE)and enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR)were performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined bv agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained bv using an alkalinelysis technique and was digested by various endonucleases.Elimination of plasmid from S.marcesceus isolates were performed by repeated SDS treatment.The crude β-lactamase extracts of original isolates and E.coli transconjugants were subjected to isoelectric focusing(IEF);Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.Results ERIC-PCR indicated that all S.marcescens isolates belonged to a clonal strain.PFGE indicated that ten K. pneumoniae isolates were indistinguishable or closely related to each other.The MICs of imipenem and meropenero for all isolates were 2 to 8 μg/ml except K.pneumoniae K10(128 and 256 μg/ml).Conjugation studies with E.coli(EC600)resulted in the transfer of reduced carbapenem susceptibility from original isolates(MICs:from≤0.125 μg/ml to 1-2μg/ml).IEF,PCR and DNA sequence analysis confirmed that S.marcescens isolates produced KPC-2(pI of 6.7)and a β-lactamase(pI 6.5).k pneumoniae isolates produced TEM-1(pI 5.4),KPC-2,CTX-M-14(pI 7.9),and a β-lactamase(pI 7.3).E.coli El produced KPC-2,CTX-M-15(pI 9.0),and a β-laetamase(pI 7.3).Only a KPC-2 was detected in E.coli transeonjugants.Plasmid restricfion analysis using EcoR Ⅰ,Hind Ⅲ,and Bcu Ⅰ showed identical restrietion patterns among all E.coli transconjugants.SDS-PAGE and ompK 35/36 gene sequence analysis of OMPs revealed that K.pneumoniae K10 failed to express OmpK36 because of insertional inactivation by an insertion of ISEcp1.Conclusions Carbapenem-non-susceptible S.marcescens.K.pneumoniae and E.coli were epidemic in two ICUs in our hospital.Resistance or reduced susceptibility to carbapenems in these strains is mainly due to production of KPC-2.Presence of KPC-2 combined with porin deftciency result in high-level carbaoenem resistance in K.pneumoniae.The game blaKPC2-encoding plasmid spreads among the three different genera.

Objective To estimate the application for the rapid identification of common clinical bacteria by MALDI-TOF MS. MethodsFour hundred and twenty-six bacteria, including Salmonella spp strains collected from Zhejiang center for disease control and prevention were collected from blood, sputum,secretion and urine in 2nd Affiliated Hospital of Zhejiang University during December 2008 to August 2009. The isolates included 76 gram positive coccus and 350 gram negative bacilli. Species identification was performed with the Vitek system, and serotypes of Salmonella and Shigella were determined by serum agglutination test. 16s rDNA gene of 91 bacteria were amplified by PCR. The RCP products were sequenced. Then the results were compared with the reported sequences from GenBank. All strains were identified by MALDI-TOF MS. Results of three identification methods were compared with each other. Results Among 426 tested isolates, identification results from Vitek system and MALDI-TOF MS for gram positive coccus and 323 out of 350 gram negative bacilli (exception for Salmonella and Shigella spp.),were identical. For 23 Salmonella and Shigella spp. , only 2 Salmonella enterica subsp, enterica serovar Typhimurium were identified the same results by the three methods. Besides, results from Vitek system and serum agglutination test for 1 Salmonella enterica subsp, enterica serovar Typhi, 3 Salmonella enterica subsp.enterica serovar Paratyphi A, 1 Salmonella enterica subsp, enterica serovar Paratyphi B, 1 Salmonella enterica subsp, enterica serovar Enteritidis, and 1 Salmonella enterica subsp, enterica serovar Bovis-morbificans were consistent with that from 16S rDNA gene sequence. Four isolates which were confirmed as S. flexneri by Vitek system and serum agglutination test were identified as Escherichia coli by both 16S rDNA gene sequence and MALDI-TOF MS. ConclusionMALDI-TOF MS could be used for rapid and accurate identification of common clinical bacteria with good repeatabihty, excepting for the Salmonella and Shigella spp.

Objective To investigate mechanisms of carbapenem resistance in Klebsiella pneumoniae. Methods Two carbapenem-non-susceptible Klebsiella pneumoniae Z4 and Z5 isolated from Beijing Hospital in 2008 were investigated. MICs of antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. Elimination of plasmids was performed by repeated SDS treatment. The crude β-lactamase extracts were subjected to IEF. The genotype of β-lactamases were confirmed by PCRs and DNA sequence analysis. Outer membrane proteins (Omps) were isolated and examined by SDS-PAGE.The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. Results MICs of imipenem, meropenem and ertapenem for Z4 and Z5 were 32, 32 and 256 μg/mi, and 1, 1 and 2 μg/ml.Conjugation study with Escherichia coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from Z4 and Z5 ( MICs increased at least 8-fold). Klebsiella pneumoniae Z4 produced IMP-4 metallo-β-lactamase, TEM-1 and SHV-1 spectrum β-lactamase and Z5 produced IMP-4, TEM-1 and SHV-12 extended-spectrum β-lactamase. E. coli transconjugants of both Z4 and Z5 produced a single IMP-4.Elimination of IMP-4-encoding plasmid from Z5 resulted in carbapenem susceptibility in the isolate,however, Z5 whose IMP-4-encoding plasmid was eliminated exhibited reduced susceptibility to carbapenems ( MICs of imipenem, meropenem and ertapenem were 0. 25 μg/ml,0. 5 μg/ml and 4 μg/ml). Amplification of integron revealed that blaIMP-4 gene of both Z4 and Z5 located within two different class I integrons which were carried on two plasmids with a similar size of approximately 55 000 bp. SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that Z4 failed to express OmpK36, because of a nonsense mutation (CAG into TAG) in the ompK36 gene. Conclusion Production of plasmid-mediated metallo-β-lactamase IMP-4 or production of β-lactamase combined with porin OmpK36 deficiency can lead to reduced susceptibility to carbapenems. High-level carbapenem resistance in Z4 is mainly due to production of IMP-4 and the loss of OmpK36.

Objective To investigate the linezolid resistance mechanisms and molecular epidemiology of clinical isolates of methicillin-resistant coagulase-negative staphylococci (MRCoNS).Methods Seventeen MRCoNS,including 10 S.capitis,4 S.cohnii,2 S.haemolyticus,and 1 S.sciuri with various levels of linezolid resistance were isolated from intensive care units in our hospital from March to August 2011. Minimal inhibitory concentration (MIC) was determined by E-test method. Pulsed-field gel electrophoresis was performed to analyze the molecular epidemiology.PCRs and DNA sequencing were preformed to investigate the mechanisms of linezolid resistance in MRCoNS.Results Nine S.capitis with linezolid MIC of ＞256 μg/ml were indistinguishable,and another S.capitis with linezolid MIC of 4 μg/ml was closely related.Four S.cohnii with linezolid MIC of ＞256 μg/ml were belonged to the same clonal strain.MIC of linezolid for S.sciuri was 64 μg/ml,and were 4 μg/ml and 6 μg/ml for 2 S.haemolyticus,respectively.A commom G2576T mutation and a novel C2104T mutation were identified in 9 S.capitis with linezolid MIC of ＞256 μg/ml by DNA sequence analysis of domain V of the 23S rRNA gene.cfr gene was deteeted in all staphylococci except a S.sciuri whose 23S rRNA gene contained the G2576T mutation.Conclusion It is the first report of linezolid-resistant clinical isolates of staphylococci in China.Linezolid resistance in MRCoNS is related to the presence of DNA mutation in domain V of the 23S rRNA gene and cfr gene.It's a clonally dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital.

Objective To evaluate the capability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF MS) for rapid identification of methicillin-resistant Staphylo-coccus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) strains.Methods Twenty-five MRSA and thirty MSSA isolates were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine as the experiment group .Twelve MRSA and twenty-two MSSA clinical strains were isola-ted as the control .All strains were identified by MALDI-TOF MS and the results were further analyzed by ClinProTools 3.0 software.Results Four algorithms including support vector machine (SVM), genetic al-gorithm ( GA) , supervised neural network ( SNN) and quick classifier ( QC) showed similar results in dis-tinguishing MRSA from MSSA isolates .The sensitivity of GA was 100 .0%and the sensitivities of other algo-rithms were all greater than 95.0%.The specificities of GA, SVM and QC were all greater than 90.0%. The areas under the receiver operating characteristic ( ROC) curves of four characteristic peaks at mass-to-charge ratios (m/z) of 3279, 6485, 6555 and 3299 m/z were all greater than 0.9.The virtual gel view showed that the bands generated by MSSA isolates at 3279 , 6485 and 6555 m/z were obviously deeper in color than those generated by MRSA isolates .However , the bands of MSSA isolates at 3299 m/z were appar-ently lighter in color than those of MRSA isolates .83.3%of MRSA and 90.0%of MSSA isolates from the control group were correctly identified by the GA model .Conclusion MALDI-TOF MS could rapidly and accurately identify MRSA from MSSA isolates under the strictly controlled experimental conditions with the advantages of less time-consuming, high sensitivity and high specificity .The accurate identification of MRSA from MSSA isolates could be applied for the prevention and treatment of MRSA infection .

ObjectiveTo investigate the molecular epidemiology and mechanisms of carbapenem resistance of Morganella morganii.MethodsSeven carbapenem-non-susceptible M.morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital from October 2010 to February 2011.Pulsed-field gel electrophoresis (PFGE) was performed to analysis the molecular epidemiology of isolates.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNA was obtained by an alkalinelysis technique and examined by electrophoresis.Specific PCRs and DNA sequencing were preformed to confirm the genotype of β-lactamases.ResultsPFGE indicated that 6 M.morganii isolates from emergency care unit were indistinguishable or closely related and 1 isolate from intensive care unit was distinguishable.Seven M.morganii showed similar antibiotic susceptibility patterns.M.morganii isolates were resistant to imipenem,were susceptible to meropenem,and were susceptible or intermediate resistant to ertapenem,with MICs of 8 μg/ml,1 μg/ml,and 0.25-0.50 μg/ml,respectively.M.morganii isolates were resistant to penicillins,aztreonam,and ciprofloxacin,were resistant or susceptible to cephalosporins,and were susceptible to amikacin.E.coli (EC600) acquired an approximately 60 kb plasmid from M.morganii by conjugation studies and resistant or intermediate resistant to carbapenems and other β-lactams.PCRs and DNA sequence analysis confirmed that all M.morganii isolates and their E. coli transconjugants produced the KPC-2 carbapenemase and carried the qnrS1 gene.ConclusionIt is the first detection of KPC-2 in M.morganii isolates.Production of KPC-2 mainly contributed to the carbapenem resistance in M.morganii.

Objective To investigate the molecular epidemiology and resistance mechanism of reduced carbapenem susceptibility of Proteus mirabilis from intensive care unit (ICU).Methods Nineteen carbapenem resistance or reduced carbapenem susceptibility of Proteus mirabilis were isolated from two ICUs in Second Afiliated Hospital of Zhejiang University from August 2010 to October 2010.Pulsed-field gel electrophoresis (PFGE) was performed to analyze the molecular epidemiology of isolates.Antibiotic susceptibilities were determined by agar dilution method.Conjugation experiments were carried out in mixed broth cultures.Plasmid DNAs were obtained by using an alkalinelysis technique.Specific polymerase chain reaction (PCR) and DNA sequencing were preformed to confirm the genotype of β-lactamases. Results Nineteen Proteus mirabilis showed resistance or reduced carbapenem susceptibility,and were resistant or susceptible to cephalosporins.PFGE analysis indicated that nineteen carbapenem-non-susceptible Proteus mirabilis belonged to 3 clones,named as clone A (14 isolates),clone B (2 isolates of subclone B1 and 2 isolates of subclone B2 ) and clone C (1 isolate).Fourteen clone A isolates were indistinguishable,and subclone B1 and subclone B2 were closely related with only one fragment disparity.Escherichia coli (EC600) acquired an approximately 45 000 bp,54 000 bp and 45 000 bp plasmids from clone A,subclone B1 and subclone B2 isolates separately by conjugation studies.PCRs and DNA sequence analysis confirmed that all Proteus mirabilis isolates and their transconjugants produced the KPC-2 carbapenemase. Conclusion Carbapenem-non-susceptible Proteus mirabilis were epidemic in two ICUs in our hospital,and were clonally disseminated.

Objective To compare the capability of ertapenem-hydrolyzing in various concentrations in KPC-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry( MALDI-TOF MS).Methods Nineteen KPC-producing Enterobacteriaceae including ten Proteus mirabilis isolates,three Enterobacter aerogenes isolates,two Serratia marcescens isolates,two Citrobacter freundii isolates,one Klebsiella pneumoniae isolate and one Enterobacter cloacae isolate were isolated from 2nd Affiliated Hospital of Zhejiang University; Seven KPC-producing Morganella morganii were isolated from Hangzhou Traditional Chinese Medicine Hospital; Eleven Escherichia coli transconjugants produced the KPC-2 carbapenemase gene were conjugated from seven M.morganii and four P.mirabilis.MALDI-TOF MS was used to detect the ertapenem-hydrolyzing capability in different KPC-producing Enterobacteriaceae.Results When the concentration of ertapenem was 0.1 g/L,ertapenem can be hydrolyzed by KPC carbapenem within 1.5 h and the sensitivity was 100％,all the three peaks produced by ertapenem disappeared; when the concentration of ertapenem raised to 0.3 g/L,the sensitivity fell to 70.3％ (26/37) within 1.5 h,followed with 89.2％ ( 33/37 ) within 2.5 h and 94.6％ (35/37) within 3.5 h; when the concentration of ertapenem raised to 0.5 g/L,the sensitivity was 48.6％ (18/37) within 1.5 h,83.8％ (31/37) within 2.5 h,94.6％ (35/37 ) within 3.5 h.Two independent samples tests indicated that 0.1 g/L of ertapenem group has significant difference with 0.3 g/L and 0.5 g/L group,while there was no significant difference between 0.3 g/L group and 0.5 g/L group; As for the bacteria groups,there are no significant differences between the bacteria groups except the group of P.mirabilis and E.coli.Conclusion MALDI-TOF MS was easy to operate; it can rapidly detect KPC-producing Enterobacteriaceae with high sensitivity and a low false positive rate.0.1 g/L of ertapenem was recommended to detect KPC carbapenem rather than 0.3 g/L and 0.5 g/L; The detection time is short and can be widely applied clinically.