Objective To establish and evaluate the method for detection of methicillin-resistant Staphylococcus aureus (MRSA) by the cefoxitin disk diffusion test.Methods The disks with 30 microgramme of cefoxitin and oxacillin recommended by NCCLS were used for the detection of MRSA with PCR by using mecA as the reference.The MIC of cefoxitin to Staphylococcus aureus was also determined.Results Of the 145 test isolates of Staphylococcus aureus,83 isolates showed the mecA-positive by PCR,62 isolates were negative.In cefoxitin disk diffusion tests,82 isolates showed MRSA-positive and 63 isolates were negative.The sensitivity was 98.8% and the specificity was 100%.In oxacillin diffusion tests,the sensitivity was 95.2% and the specificity was 96.8%.Conclusions For the detection of MRSA,the cefoxitin disk diffusion test was consistent with the results by PCR for mecA.The cefoxitin disk diffusion test is easy to be performed and can be used in routine diagnostic works.

Objective To investigate the resistance of Acinetobacter baumannii to clinical common antibiotics and new drug tigecycline. Methods Six hundred and two Acinetobacter baumannii isolates were collected from 2008 to 2009 in four teaching hospitals in Zhejiang province. Agar dilution method was used to detect the resistance of 13 clinical commom antibiotics, polymyxin B and tigecycline. Homology analysis of 24 multi-drug resistant Acinetobacter baumannii strains was used to investigate the relationship of each strain with the method of pulsed field gel electrophoresis. Results From 2008 to 2009, the Acinetobacter baumannii isolates of four teaching hospitals in Zhejiang province were mainly isolated from respiratory specimens with the number of 277 (86.0%) strains in 2009, the number of blood samples decreased from 15 (5.4%) strains in 2008 to 5 ( 1.5% ) strains in 2009, and there were no obvious change in other specimens. Acinetobacter baumannii strains were resistant to 13 clinical common antibiotics at different degree, fluctuated from 35.0% to 85.0%. Compared with the resistance in 2008, levofloxacin and tobramyxin decreased 0. 9% and 9.0% in 2009, respectively. However, the resistance of other antibiotics increased at different degree, the resistance of ceftriaxone and cefepime increased about 10.0%, and the resistance of imipenem and meropenem increased to 74.2% (239/602) and 70.8% (228/602) in 2009,respectively. Acinetobacter baumannii showed high resistance to tigecycline with the percent of 78.9% (475/602), while it was only 3.7% (22/602) for polymyxin B. There were six cloning types among the 24 Acinetobacter baumannii isolates, and the most common type was type A with the percent of 50%.Conclusions The resistance of tigecycline makes the situation of nosocomial infectious more serious. It is necessary to control the transmission of multi-drug resistant Acinetobacter baumannii immediately.

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 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 isolation and drug resistance of Chryseobacterium spp in our hospital,and to explore the mechanisms of drug resistance.METHODS Bacteria were identified in our hospital for the last five years(Jan 2001-Dec 2005) and the antimicrobial susceptibility was tested by Kirby-Bauer plate dilution method.Forty-three isolates of C.meningosepticum,16 isolates of C.indologenes and 10 isolates of C.gleum were isolated and selected for further studies.Minimum inhibitory concentrations(MICs) against 14 antibiotics were determined by the agar dilution method.Extended-spectrum ?-lactamase(ESBL) and carbapenemase were detected by three-dimensional test and 2-mercaptopropionic acid inhibitory test.RESULTS One thousand and one hundred twenty-eight Chryseobacterium and others strains in total were isolated during the described period.Among them C.meningosepticum,C.indologenes,C.gleum,and other Chryseobacterium species were 88.3%,8.0%,2.9%,0.6% and 0.2%,respectively.The resistant ratios against antibiotics containing enzyme inhibitors were lower than other antibiotics.The MIC50 and MIC90 against most antibiotics were high except for quinolones.As for carbapenemase,the positive rate was 60.5%,68.8% and 90.0% in C.meningosepticum,C.indologenes,and C.gleum,respectively.CONCLUSIONS Chryseobacterium are highly resistant against a variety numbers of antibiotics.Nevertheless,there exists a significant difference in the resistance against different antibiotics for different species of Chryseobacterium.The major drug resistant mechanism in Chryseobacterium is due to the production of ?-lactamases,especially metallo-?-lactamases.

Objective To compare the application of three automated microbiology identification and rapid susceptibility assessment systems,Vitek1,Viket2 and BD Phoenix,in detection of extended-spectrum beta-lactamases(ESBLs).Methods The genotypes of 67 clinical isolates of ESBLs-producing E.coli and K.phneumoniae,which were collected from the 1st and 2nd Affiliated Hospital of Zhejiang University,were determined by PCR amplification and sequencing.Meanwhile,these isolates were analyzed by the three automatic microbiology identification system,and the minimum inhibitory concentration(MIC)s for antibiotics were determined.Results Majority of the 67 isolates produced CTXM type of ESBL.Among them,CTX-M-14,CTX-M-22,CTX-M24 and CTX-M-3 were the most frequently detectable types.By using Vitek-AMS,60 isolates(89.6%)were detected to be ESBLs-producing.By using Vitek2,62 isolates(92.5%) were ESBLsproducing.No SHV-12,CTX-M-14 and SHV-28 were detected by the 3 automatic systems.The results of MICs analysis indicated these isolates showed either high resistance for multiple antibiotics or around the boundary areas of MICs test.Conclusions The three automatic analysis systems are able to provide a detectable rate of more than 98% for ESBLs.However,it is needed to further improve the detection for high resistant strain or the strains carrying multiple resistant genes.

Objective To investigate the resistance of clinical isolated gram negative bacilli to tigecycline.Methods One hun-dred and fifteen Escherichia coli isolates,110 Klebsiella pneumoniae isolates and 99 Acinetobacter calcoaceticus-Acinetobact-er baumannii complex isolates were collected from Affiliated Second Hospital of Zhejiang University College of Medicine from the year 2012.The other 393 A.calcoaceticus-A .baumannii complex isolates were collected from 15 hospitals of nine cities in Zhejiang province during September to October 2012.Species identification and susceptibility test were confirmed by VITEK-2 compact system,while the 393 A.calcoaceticus-A .baumannii complex strains isolated from Zhejiang province were identified by MALDI-TOF MS.Moreover,159 A.baumannii isolates with tigecycline MIC value ≥8 μg/ml or 4 μg/ml and part of the MIC value ≤0.5 μg/ml,1 μg/ml and 2 μg/ml which were firstly determined by VITEK 2 GN AST-16 Suscepti-bility card were then determined by Etest.Results The 393 A.calcoaceticus-A .baumannii complex strains were identified as 317 of A.baumannii,32 of A.pittii and 44 of A.nosocomialis.When using the FDA breakpoints,the resistance rate of tige-cycline against 115 E.coli isolates,110 K.pneumoniae isolates and 99 A.calcoaceticus-A .baumannii complex isolates were 0.0%,7.3% and 6.1%,respectively.Interestingly,85.7% of the tigecycline-resistant gram negative bacilli were resistant to carbapenems.However,only 10.0% of the carbapenem-resistant gram negative bacilli were resistant to tigecycline.Conclu-sion Tigecycline had a good activity against clinical isolated multi-drug resistant or even pan-drug resistant gram negative bacilli.No matter which criteria for tigecycline was referred to,the resistance rate oftigecycline was slightly lower by Etest than by GN AST-16 Susceptibility card.

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 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.