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 .

Objective To investigate the molecular types of carbapenem-non-susceptible Esche-richia coli ( E.coli) isolates and their mechanism of carbapenem resistance .Methods Twenty-two carbap-enem-non-susceptible E.coli strains were isolated from 3 hospitals in Hangzhou from 2007 to 2011.The mini-mum inhibitory concentrations ( MICs) of antimicrobials to those isolates were determined by agar dilution method and E-test.The molecular mechanisms of carbapenem resistance of E.coli isolates were analyzed by conjugation experiment,PCR and DNA sequencing.Pulsed-field gel electrophoresis (PFGE),multilocus se-quence typing ( MLST ) , and phylogenetic typing were performed to analyze the molecular epidemiology of those isolates.Results The MICs of imipenem and meropenem to 22 E.coli isolates were ranged from 1 μg/ml to 16 μg/ml,and the MICs of ertapenem were 2 μg/ml to 64 μg/ml.All E.coli isolates produced the KPC-2 carbapenemase and various β-lactamases , and some of them also produced plasmid-mediated AmpC enzymes.Carbapenem resistance was transferred by conjugation and transformation from 22 E.coli iso-lates to E.coli EC600 strains.The E.coli transconjugants or transformants acquired the blaKPC-2 gene and showed similar antibiotic susceptibility patterns in comparison with donor strains .Only a few isolates were in-distinguishable or closely related as indicated by PFGE .Four sequence types including ST131 (9 isolates), ST648 (5 isolates),ST38 (2 isolates) and ST405 (2 isolates) were identified by MLST.Phylogenetic analy-sis indicated that 9 ST131 isolates belonged to phylogenetic group B 2 and the other isolates belonged to group D (11 isolates),group B1 (1 isolate) and group A (1 isolate),respectively.Conclusion The sequence type of prevalent E.coli isolates producing KPC-2 from Hangzhou was ST131,which is an international epi-demic,multidrug-resistant clone,followed by ST648.

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.

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 detect carbapenemase production in enterobacteriaceae by using modified Hedge test (MHT) and investigate the distribution of carbapenemase genes.Methods A total of 3 718 isolates from enterobacteriaceae were collected from 4 hospitals,including Second Affiliated Hospital of Zhejiang University,People's Hospital of Dongyang,Beijing Hospital,and Huaxi Hospital of Sichuan University.Susceptibility of enterobacteriaceae to ertapenem was tested by K-B method.MHT was used to detect carbapenemase in enterobacteriaceae and the common carbapenemase genes were amplified by using PCR.Results The total resistance rate ( resistance and intermediate resistance) of enterobacteriaceae to ertapenem was 3.04％ (113/3718) and there were differences in resistance rate of enterobacteriaceae to ertapenem among 4 hospitals (5.09％,2.15％,2.59％,and 1.72％,respectively).Of the 3718 isolates,2.29％ (85) were positive to MHT,and there were differences in positive rate to MHT among 4 hospitals (4.73％,1.21％,1.06％,and 1.58％,respectivdy).Of 113 non-susceptible isolates to ertapenem,82 were positive to MHT and 31 were negative.Of 85 MHT-positive isolates,82 were resistant or intermediate resistant to ertapenem and only 3 were susceptible.Of 82 MHT-pesitive and non-susceptible isolates to ertapenem,65 were positive to KPC gene,15 were positive to IMP gene (two of them were positive to both KPC and IMP),and 4 were negative to all tested carbapenemase genes.Thirty-one MHT-negative and nonsusceptible to ertapenem and 3 MHT-positive and susceptible isolates to ertapenem were negative to all tested carbapenemase genes.Conclusions MHT is used to detect carbapenemase in enterobacteriaceae with high sensitivity and low false positive rate.KPC gene is the most occurred gene to be dominant in the production of carbapenemase in enterobacteriaceae, and the IMP gene is also responsible to the genesis ofcarbapenemase in enterobacteriaceae..

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 evaluate the effect of four carbapenems combined with EDTA for metallobeta-lactamases (MBLs) detection.Methods Thirty MBLs-producing gram-negative bacteria ( 16 strains of Enterobacteriaceae and 14 strains of Non-fermentative),9 KPC-producing Enterobacteriaceae and 10 OXA-producing Acinetobacter baumannii strains were collected from the Second Affiliated Hospital of Zhejiang University.A double disk screening test with EDTA was performed for MBLs detection,comparing the changes of inhibition zone diameter with or without EDTA.Results When the inhibition zone diameter difference of ≥5 nn as standard,the sensitivity of panipenem (PAN) group was relatively high (66.7％,20/30),followed by meropenem group (MEM) (63.3％,19/30) and imipenem (IPM) group (60.0％,18/30),etrapenem (ETP) group was the wont (43.3％,13/30).When the inhibition zone diameter difference of ≥4 mm as standard,the sensitivity of meropenem and panipenem group was 80.0％ (24/30)respectively.When the inhibition zone diameter difference of ≥ 3 mm as standard,the sensitivity of imipenem and meropenem group was 90.0％ (27/30) respectively,while 83.3％ ( 25/30 ) for etrapenem and panipenem group.Specificity of four groups under these three interpretation was 100％ respectively.Results of screening test for 16 MBLs positive strains showed that the sensitivity of panipenem group was the highest (75.0％,12/16).While using ≥ 5 mm as the MBLs positive interpretation,the sensitivity of panipenem group was the highest (75.0％,12/16).While using ≥4 mm as the MBLs positive interpretation,the sensitivity of panipenem group (93.8％,15/16) was much higher than that of imipenem (75.0％,12/16),meropenem (68.8％,11/16) and ertapenem (68.8％,11/16).Conclusions Detection of MBLs with EDTA is easy to operate,and it shows a low false positive rate against gram-negative bacteria.Ertapenem is not recommended to screen for MBLs,the best method for screening for MBL production in gram-negative bacteria is the MEM-EDTA or PAN-EDTA with a breakpoint of ≥4 mm.As for Enterobacteriaceae,PAN-EDTA with a breakpoint of ≥4 mm is better than the other three carbapenems in screening for MBL production,but panipenem is not recommended for MBLs screening tests for nonfermentative gram-negative bacteria.

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 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.