Objective To study dIe ESBLs and plasmid-mediated AmpC enzymes in E.Coli and Klebsiella spp. with CLSI ESBL-screening test positive,confirmation test negative but cefepime susceptible.Methods Antimierobial susceptibility testing were performed by Kirby-Bauer(K-B)method.The genes encoding ESBLs and plasmid-mediated AmpC enzymes were detected by PCR Transfer of ESBLs or plagmid-mediated AmpC resistance was studied by conjugation experiments.The homology of donor (E.coli),recipient(E.coli J53)and their transconjugants were analyzed by ERIC-PCR DNA fingerprints of E.coli and Klebsiella pneumoniae were analyzed by PFGE as recommended bv PulseNet protocoL Results Of 18 isolates from Huashan Hospital,11 were E.coli.6 were Klebsiella pneumoniae and 1 was Klebsiella oxytoca.Antimicrobial susceptibility testing indicated all of 18 isolates were positive on the CLSI ESBL screening test but negative on the confirmation test.and all of isolates were susceptible to cefepime(a zoneof-inhibition diameter of≥18 mm wag considered to indicate susceptible).PCR results indicated that 9 of the 11 E.coli isolates predued CMY-2 AmpC enzyme.TEM,SHV,CTX-M,PER,VEB or SFO type β-lactamages were not identified.Of 6 Klebsiella pneumoniae isolates.5 were DHA-1 AmpC-producing strains.4 of the 5 DHA-1 AmpC-producing strains were coexistence of broad-speetrumβ-lactamaae or extended-spectrumβ-lactamase.including two producing SHV-11 and two producing CTX-M-14 and SHV-62 type ESBL respectively.One Klebsiella oxytoca wag also DHA-1 AmpC producing strain.Conjugation experiments indicated that both ESBLs and AmpC enzymes could be transfefred from donor to recipient.PFGE indicated that the DNA fingerprints of K.pneumoniae were difierent but seven CMY-2 AmpC-producing E.coli isolates from general surgieal ward were similar.Concluslons The main mechanism of antibiotic resistance in CLSI ESBLs-screening test-positive but eefepime.susceptible E.coli and KIebsiellaspp.is production of plagmid-mediated AmpC enzymes.Some strains produce both AmpC enzyme and ESBLs.Such strains should be reported as resistant to cefepime.The results suggest that laboratories should routinely conduct research on the ESBLs and plnsmid.mediated AmpC enzymes in Enterobacteriaceae in order to report antimicrobial susceptibility testing results more correcdy.

Objective To investigate the profile of antimicrobial susceptibility of the Mycoplasma pneumoniae (Mpn)strains isolated from pediatric patients with respiratory tract infection.Methods Antimicrobial susceptibility testing was conducted with a total of 112 Mpn clinical strains by broth microdilution method.Sequence analysis of full 23S rRNA genes was performed for all Mpn strains.Results One hundred and twelve Mpn strains were isolated from January 2009 to March 2011. Of these clinical isolates,98 (87.5%)were resistant to erythromycin and azithromycin.All macrolide-resistant Mpn strains harbored an A2063G or A2064G transition mutation in domain V of 23S rRNA genes.Mpn isolates were still very susceptible to the tetracyclines and fluoroquinolones tested.Conclusions The Mpn strains from pediatric patients are highly resistant to macrolides.The mechanism of macrolide resistance may be associated withthe transition mutation on 23S rRNA gene.

Objective To evaluate clinical and pathologic factors associated with pelvic lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervir.Methods From February 2004 to January 2007,135 patients with stage Ⅰ b-Ⅱ a cervical squamous cell carcinoma in Women's Hospital,School of Medicine,Zhejiang University,were retrospectively studied.The relationship between pelvic lymph node metastasis and age,clinical stage,tumor size,grade of differentiation,depth of muscular invasion,lymphatic vascular space invasion,pretreatment level of serum squamous cell carcinoma antigen,pretreatment plasma level of fibrinogen,pretreatment leveh of hemoglobin and platelet were evaluated by univariate and multivariate analyses.Results Totally 3996 lymph nodes were dissected in 135 patients,with an average of 29.6 lymph nodes in each patient.12.6%of the patients(17/135)had metastasized pelvic lymph nodes.Univariate analysis indicated that tumor size(P=0.003),depth of muscular invasion(P=0.004),vasular space invasion(P<0.01),pretreatment levels of platelet(P=0.006)and fibrinogen(P<0.01)were significantly related to pelvic lymph node metastasis.Multivariate logistic regression analysis showed that lymphatic vascular space invasion(OR:3.674,95%CI:1.825-7.393,P<0.01)and pretreatment plasma level of fibrinogen(OR:4.568,95%CI:1.779-11.725,P=0.002)were significantly related to pelvic lymph node metastasis in patients with early-stage squamous cell carcinoma of the uterine cervix.Conclusion In early-stage cervical squamous cell carcinoma,lymphatic vascular space invasion and higher pretreatment plasma levels of fibrinogen are risk factors of pelvic lymph node metastasis.

OBJECTIVE:To study on the effect of iodine (I2) on the acid content titration in the preparations,and establish a method of eliminating I2 for accurate determination of total acid content. METHODS:Taking an example of Onychomycosis paint,I2 was reduced by sodium sulfide(Na2S)before titration analysis and compared with the standard method of traditional titration. Ac-cording to the two methods of consumption of different sodium hydrate(NaOH)volume,the effect of I2 on the determination of to-tal acid content in preparation was explored. RESULTS:Reduction method of Na2S can eliminate the effect of I2,the color of indica-tor changed acutely. The determined total acid and theoretical values were the same;the linear range of the concentration of total ac-id in onychomycosis paint was 3.926-7.290 mol/ml(r=0.9999);RSDs of precision,stability and reproducibility tests were no more than 0.04%;average recovery was 99.91%-100.10%(RSD=0.036%,n=6). CONCLUSIONS:I2 has effect on the determina-tion of total acid content,the disproportion reaction of I2 can generate hydriodic acid that can interfere acid-base titration's results, and the generated hydriodic acid can oxidize indicator to make the end point is not sensitive or even invariant color. Using Na2S can eliminate the effect of iodine on the preparation of acid titration,which has improved precision and reproducibility with accurate and reliable determination results.

Objective To investigate the mechanism of the different levels of ciprofloxacin resistance in qnrA-containing transconjugants.Methods E. coli J53AzR as the recipient,4 qnrA-containing transconiugants were constructed by conjugation from 4 qnrA-carrying clinical isolates.MICs of the transconjugants were measured by E test.aac(6')-Ib-cr was detected by PCR,and qnrA mRNA expression level was determined by real-time RT-PCR.The promoter sequences of qnrA were amplified by PCR from qnrA-bearing plasmids and cloned into plasmid pKK232-8,then transformed into HB101.All promoter fragments were sequenced.Resuits The MICs of ciprofloxacin against 4 transconjugants demonstrated a 10-fold difference from 0.094 μg/ml to 1.000 μg/m1.Of 4 qnrA-bearing plasmids in E.coli J53,ciprofloxacin MICs of pHS4 and pHS5 were 0.094 μg/ml and 0.125 μg/ml,respectively;pHS3,which contained the aac(6')-Ib-cr gene as well,MIC was 0.25μg/ml;and pHS5,which had a high expression level of qnrA and the aac(6')-Ib-cr gene,MIC was 1.00μg/ml.The relative expression levels of qnrA mRNA in J53 pHS6 was 32.5,much higher than the other 3 transconjugants(from 1.0 to 2.5).The promoter in plasmid pHS6 was 12-fold stronger than that in the other 3 plasmids.Compared with pHS3,there was 7 bp(GTTAGCA)deletion between the transcription initiation site and the start of qnrA in pHS6.Conclusion Co-existence of qnrA and aac(6')-Ib-cr in a single plasmid and high level of qnrA expression can account for the different levels of ciprofloxacin resistance in transconjugants.

Objective To screen fosfomycin-resistant genes in the clinical isolates of Enterococcus faecium Efm-HS0661 and verify their functions. MethodsAntimicrobial susceptibility and conjugation experiments were carried out to determine if the antimicrobial resistance in clinical strain was transferable.By Solexa high-throughput sequencing,the genes conferring fosfomycin resistance were screened. The function of resistance gene was identified by cloning.ResultsThe clinical isolates of Enterococcus faecium Efm-HS0661 were resistant to glycopeptide antibiotics and fosfomycin, and the fosfomycin resistance was found to be transferred by conjugation. Within the 2414 bp nucleotide sequence obtained by high-throughput sequencing, fosB, a plasmid-mediated fosfomycin resistance gene was found. The fosB gene was 420 bp in length, which shared 99. 8％ amino acid identity with other fosB from Staphylococcus spp. The minimal inhibitory concentration (MIC) of DH5α transformant containing fosB gene against fosfomycin was higher than that of DHSa transformant without fosB gene. ConclusionsThe high-throughput sequencing can be used to screen unknown resistance genes in clinical isolates. The plasmidmediated resistance gene fosB can confer fosfomycin resistance in Enterococcus faecium.

Objective To learn the current in vitro antimicrobial susceptibility of Mycoplasma pneu-moniae in Shanghai and to understand the mechanisms of resistance to macrolides. Methods M. pneumoniae was isolated from pediatric patients with low respiratory tract infections(RTI) using broth and PPLO agar medi-um. PCR amplification and sequence analysis of P1 adhesion gene were performed to identify all M. pneumoniae strains. Susceptibility testing was carried out for macrolides, tetracyclines and fluoroquinolones using broth mi-crodilution method with SP4 broth. PCR amplification and sequence analysis of 23S rRNA genes were performed for all M. pneumoniae strains. P1 gene PCR-RFLP typing was performed to subtype the M. pneumoniae strains. Results One hundred and two M. pneumoniae strains were isolated in Shanghai from Oct 2005 to Dec 2008. All M. pneumoniae isolates were susceptible to the tetracyclines and fluoroquinolones tested. Of 102 clinical isolates, 83(81.4%) was resistant to erytbromycin and all 83 erythromycin-resistant strains had MIC>128 mg/L. An increasing trend of resistance rates were showed: 16.7% (1/6) in 2005, 76.5% (13/17) in 2006, 100.0% (24/24) in 2007 and 81.8% (45/55) in 2008. All macrolide-resistant M. pneumoniae strains harbored an A2063G transition mutation in domain V of 23S rRNA genes. The P1 gene RFLP type 1 is predominant (85.3%, 87/102) in M. pneumoniae clinical isolates. Conclusion The macrolide resistance rate of M. pneu-moniae is very high in Shanghai. The mechanism of macrolide resistance is associated with transition mutation on the 23S rRNA gene.

Objective To develop a method for rapid detection of Mycoplasma pneumoniae and its macrolide resistance mutation. Methods The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23s rRNA gene of Mycoplasma pneumoniae. By using recombinant plasmids containing 23s rRNA gene fragments, 102 Mycoplasma pneumoniae clinical isolates from 2005 to 2008, and 136 nasopharyngeal suction specimens from pediatric patients with low respiratory tract infections in Shanghai Children's Hospital from November to December in 2009 were investigated to determine the specificity and the sensitivity of the CycleavePCR method. PCR amplification and sequence analysis of 23S rRNA genes were performed for all Mycoplasma pneumoniae strains and Mycoplasma pneumoniae positive specimens to confirm the results of the CycleavePCR method. Results Of 102 clinical isolates, 83 was resistant to erythromycin and sequence results show that all macrolide-resistant Mycoplasma pneumoniae strains harbored an A2063G ( 82/83 ) or A2064G ( 1/83 ) transition mutation in 23S rRNA genes. Twelve was Mycoplasma pneumoniae detected positive by CycleavePCR in 136nasopharyngeal suction specimens. The CycleavePCR results were consistent with those of routine PCR and sequencing. There was no signal production from other bacterial species. Sensitivity and specificity were 100%. The detection limit of the CycleavePCR was 10 plasmid copies per reaction. Experiment can be done within 1.5 h. Conclusion A novel method is developed to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene using CycleavePCR.

Objective To understand the resistance mechanism and clinical feature of linezolid-resistant S.capitis isolated from blood samples.Methods Antimicrobial susceptibility testing was carried out to determine the susceptibility of clinical strains.PCR and sequencing analysis were used to analyze cfr gene and 23S rRNA mutation,which were associated with linezolid resistance.Patterns of pulsed-field gel electrophoresis (PFGE) were analyzed in combination with clinical data to understand the clinical feature of S.capitis strains.Results Five linezolid-resistant S.capitis strains were isolated from blood samples of 3 patients.These strains were resistant not only to linezolid,but also to most of the commonly used antimicrobial agents except glycopeptides,rifampin,and trimethoprim-sulfamethoxazole.Mutation was identified in 23S rRNA genes of all the five strains and cfr gene was found in four of the five strains.PFGE typing showed the same type,which supported the homology of the 5 strains.Three patients had deep vein indwelling catheter and two of them were treated with linezolid.Conclusions Linezolid-resistant S.capitis isolates showed the phenotype of resistance to multiple antimicrobial agents.Linezolid resistance may be mediated by cfr gene and 23S rRNA mutations in S.capitis.Long-term use of deep vein indwelling catheter and linezolid treatment may increase the risk of linezolid-resistant S.capitis infection.

Objective To investigate the resistance of clinical isolates from Shanghai Huashan Hospital in 2005.Methods Antimicrobial susceptibility test was carried out by Kirby-Bauer method.Results were analyzed according to CLSI 2005.Results Of the 3 896 clinical isolates,gram negative bacilli and gram positive cocci accounted for 68.1% and 31.9% respectively.About 93.2%(465/499)of S.aureus isolates were identified as methicillin-resistant Staphylococcus,94.9%(260/274)of coagulase negative Staphylococcus(CNS)isolates were methicillin-resistant.No vancomycin-resistant strain was found.The resistance rates of E.faecalis and E.faecium to high level gentamicin(120 ?g)were 67.4% and 82.8% respectively.Two strains of VRE were isolated.Both were VanA type.ESBLs-producing strains accounted for 47.6%(206/433)in E.coli and 69.6%(391/562)in Klebsiella spp(K.pneumoniae and K.oxytoca).Isolates of Enterobacteriaceae were still highly sensitive to imipenem and meropenem,resistance rates being 0-4% except Citrobacter isolates,9.1% of which were resistant.However,39.3% and 59.6% of P.aeruginosa strains were resistant to the above carbapenems,respectively.Conclusions The prevalence of MRSA and MRCNS is very high.ESBLs are prevalent in E.coli and Klebsiella spp.Two glycopeptide-resistant E.faecium isolates are identified firstly in Huashan Hospital.Our data will be useful for rational use of antimicrobial agents in the treatment of bacterial infections.