Objective To investigate the genotypes and epidemic of metallo-β-lactamase-(MBL )-producing Pseudomonas aeruginosa (P .aeruginosa)in Changsha.Methods P .aeruginosa isolated from seven comprehensive hospitals in Changsha were collected and performed identification and antimicrobial susceptibility testing,pheno-types of MBL were detected with EDTA-disk synergy test and E-test,genotypes were determined by polymerase chain reaction (PCR),homology analysis were conducted by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR).Results Preliminary screening by EDTA-disk synergy test and E-test showed that only 10 of 81 iso-lates were strong positive;PCR result showed that 18 isolates were positive for MBL,11 of which were IMP-9-type MBL,1 was IMP-1-type,and 6 were VIM-2-type.SIM,SPM,GIM,and NDM-1-types were not found.ERIC-PCR showed that 12 strains of IMP-producing P .aeruginosa has multiple types,6 VIM-2-producing strains were of the same type.Conclusion IMP-9 and VIM-2 are main genotypes in P .aeruginosa in Changsha.

OBJECTIVE To acquire the information about the gene type and epidemic condition of the hospital to provide scientific proof for monitoring and controlling nosocomial infection.METHODS Meticillin-resistant Staphylococcus aureus(MRSA) was identified by its resistance to cefoxitin of disk diffusion and mecA PCR,randomly amplified polymorphic DNA(RAPD) was carried out with the optimization condition.RESULTS The rate of MRSA infection was 72.15% and the main gene type was A in the hospital.CONCLUSIONS The nosocomial infection may exist in the hospital and the hospital must take effective measure to decline nosocomial infection of the MRSA;RAPD is suitable for molecular epidemiology with high powerful discrimination,simplicity and rapidness.

OBJECTIVE To investigate the drug resistance and the prevalence of inducible clindamycin resistance in clinical isolates of Staphylococcus aureus and evaluate the clinical value of cefoxitin disk diffusion method and oxacillin disk diffusion for detection of meticillin-resistant S.aureus(MRSA).METHODS Bacteria identification and susceptibility test were performed by VITEK-2 system and K-B disk method.The PBP2a was detected by latex agglutination and MRSA was identified by cefoxitin disk diffusion method and oxacillin disk diffusion.The inducible resistance of erythromycin to clindamycin was checked by D-test according to the standards of CLSI(NCCLS).The statistical analysis was performed by WHONET 5.4 and SPSS 13.0 software.RESULTS Resistant rate to penicillin and ampicillin was 98.9% and 100.0%,respectively.Vancomycin-resistant(VRE) or intermediate strains were not found.Of the 93 S.aureus isolates,MRSA and meticillin-sensitive S.aureus(MSSA) were 58(62.4%) and 35(37.6%),respectively.The resistant rate of MRSA to 11 antibiotics was higher than MSSA.The sensitivity and specificity of cefoxitin disk diffusion method were 98.3% and 97.1%,respectively,those of oxacillin disk diffusion were 75.9% and 94.3%.Of the 9 isolates resitant to erythromycin but susceptible to clindamycin,5(55.6%) showed inducible resistance to clindamycin.CONCLUSIONS Resistance of S.aureus is quite serious.Cefoxitin disc diffusion method is a simple and reliable method for the detection of MRSA.The inducible resistance of erythromycin to clindamycin in S aureus should be checked by D-test in clinical microbiology laboratory routinely.

Objective To study the relationship between the expression of mtrF gene and high-level multiple resistant Neisseria gonorrhoea.Methods ① The susceptibility of 58 clinical Neisseria gonorrhoeae to 5 kinds of antibiotic agents was tested by disc diffusion method.② The minimum inhibitory concentration(MIC) of erythromycin was determined by tube dilution method.③ The expression of mtrD and mtrF gene in susceptive group,inter-mediated resistant group and high-level multiple resistant group was detected by semi-quantitative RT-PCR.Results ① There were 30 strains presenting resistance to two or more than two antimicrobial agents,which accounted for 51.7% of the 58 clinical strains.② The number of strains sensitive,intermediate and resistant to erythromycin was 7,21 and 30,respectively,and there were 17 strains with erythromycin MIC≥32.0 ?g /mL.③ Compared with that in susceptive group and inter-mediated resistant group,mtrF expression was up-regulated in high-level multiple resistant group(P

Objective To investigate the impact of functional enhancement of efflux pump system and reduced permeability of outer membrane on high-level multiple resistant Neisseria gonorrhoeae.Methods Several high-level multiple resistant isolates with erythromycin MIC=128.0 mg/L,accompanied by concurrent resistance to several antimicrobial agents,were selected.13 bp inverted sequence positioned within the mtrR promoter region were amplified by PCR and directly sequenced to detect the possible gene change.The outer membrane proteins of the strains were extracted to analyze the constitutive profiles by SDS-PAGE.Results There were no gene mutations in 5 sensitive strains.All the 3 high-level multiple resistant strains contained the same mutation and exhibited a single A/T base pair deletion in 13 bp inverted sequence positioned within the mtrR promoter region.Meanwhile porin protein 31 ku deficiency was found in all the 3 resistant strains.Conclusion The functional enhancement of efflux pump system induced by a single A/T base pair deletion in 13 bp inverted sequence positioned within the mtrR promoter region and the decreased cell envelope permeability induced by the absence of porin protein may have some effect on mediating high-level multiple resistance in Neisseria gonorrhoeae.

Objective:To investigate relationship between AdeABC efflux pump and resistance of Acinetobacter baumannii against carbapenem.Methods:Carbapenem-resistant strains were acquired from multistep selection resistance test by meropenem in vitro.The quantitation test for sensitivities of strains before and after induction was determined by the E-test,and carbonylcyanide-m-chlorophenylhydrazone (CCCP) inhibition test was used to screen efflux pump.PCR,sequencing analysis,or real-time PCR was used to analyze the changes of regulatory genes adeR and adeS of the AdeABC efflux pump system,or expressions of adeA,adeB,adeR,and adeS in the strains before and after induction,respectively.Results:The minimal inhibitory concentrations (MICs) of meropenem were at 0.38 μg/mL and 0.25 μg/mL in parental sensitive strain S25595 and S7257,respectively,and the MICs of meropenem for both S25595 and S7257 after induction were more than 32 μg/mL.Compared with parental sensitive strains,the expression level of adeA,adeB,adeR,and adeS mRNA were elevated from 2.45 to 9.44 times,but there were no gene mutations or insertion sequences in the regulatory gene adeS and adeR.Conclusion:High expression of the AdeABC efflux pump system in Acinetobacter baumannii is closely associated with meropenem resistance,The upregulation of adeA and adeB expression is not due to gene mutations in the regulatory gene adeS and adeR and other mechanisms might account for it.

Objective To study the relationship between biofilm-forming ability,distribution of quorum sensing related genes and antibiotic resistance in clinical isolates of Pseudomonas aeruginosa.Methods The biofilm-forming ability of 94 clinical isolates was analyzed semi-quantitatively by crystal violet staining.The antibiotic resistance of the isolates was determined by K-B method.Quorum sensing related genes,lasI,lasR,rhlR and rhlI,were detected by PCR.The diffe,rences of drug resistance of Pseudomonas aeruginosa with different biofilm-forming ability and the effects of quorum sensing related genes on biofilm-forming ability were analyzed.Results Of the 94 isolates,89(94.7％) showed biofilm-forming ability.The 89 isolates consisted of 22(23.4％) isolates with weakly positive biofilm-forming ability,44 (46.8 ％) with positive biofilm-forming ability and 23 (24.5 ％) with strongly positive biofilm-forming ability.The strains of Pseudomonas aeruginosa with different biofilm-forming ability showed different drug resistance rates to amikacin,tobramycin and gentamicin (P ＜ 0.05).The drug resistance rate of the strains with strong positive biofilm-forming ability to amikacin was higher than that of the strains with positive and weakly positive biofilm-forming ability(P ＜ 0.05),and the drug resistance rates to tobramycin and gentamicin were higher than those of the strains with weakly positive biofilm-forming ability(P ＜ 0.05).Of the 94 isolates,91 strains carried lasI,lasR,rhlI and rhlR gene and 2 strains only lost lasR gene,and 1 strain lost all the 4 genes.The strains with only lasR gene deficiency or all the lasI,lasR,rhlI and rhlR gene deficiencies showed negative biofilm-forming ability,and were sensitive to conventional antimicrobial agents.Conclusion Most of the clinical isolates of Pseudomonas aeruginosa in this study showed strong ability of biofilm-forming ability which may correlate positively to partial antibiotic resistance.The quorum sensing related genes may affect biofilm formation of Pseudomonas aeruginosa.

Objective To investigate the drug resistant mechanism and homology of three strains of carbapenem-resistant Klebsiella pneumoniae (K.pneumoniae) isolated from different sites of one patient.Methods Three strains of carbapenem-resistant K.pneumoniae were isolated from femoral vein catheter tip,wound secretions and sputum of a patient with severe burns,respectively.Their carbapenemase,metallo-β-lactamase (MBL) and drug resistance genes were detected by modified Hodge test,double-disk synergy test and combination disk diffusion and PCR,respectively,and homology and biological typing were analyzed by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) assay and multilocus sequence typing (MLST) technology,respectively.Results The carbapenemase and MBL of three strains of carbapenem-resistant K.pneumoniae were negative and positive,respectively.The blaNDM-1 gene was identified from the three strains,but other drug resistance genes such as blanC,blaGES,blaIMP,blaSPM,blaVIM,blaGIM and blaOXA-48 were not detected.ERIC-PCR showed that three isolates belonged to the same genotype,and MLST showed that they were type ST17.Conclusion Carring blaNDM-1 gene is the main cause leading to the drug resistance of three strains of carbapenem-resistant K.pneumoniae,and they belong to the same genotype.

Objective To understand clinical distribution and antimicrobial resistance of clinically isolated Serratia marcescens(S .marcescens ),and provide basis for rational use of antimicrobial agents,as well as prevention and control of infection.Methods 427 S .marcescens strains isolated between January 1 ,2012 and December 31 ,2015 were analyzed,antimicrobial susceptibility testing were performed by disk diffusion method.Results 427 S . marcescens strains were mainly from respiratory tract (70.26%),among which the majority were from sputum (64.87%).S .marcescens were primarily from intensive care unit(ICU,19.44%),department of integrated tradi-tional Chinese and Western medicine(15.46%)as well as rehabilitation department (13.58%).The resistance rates of S .marcescens to cefoperazone/sulbactam,ertapenem,cefepime,ceftazidime,amikacin,imipenem,levofloxacin, and piperacillin/tazobactam were all<10%;resistance rates to ciprofloxacin,gentamicin,tobramycin,ceftriaxone, sulfamethoxazole/trimethoprim (SMZ/TMP),and aztreonam were 10%-30%.Difference in the resistance rates of S .marcescens to cefoperazone/sulbactam,ciprofloxacin,ceftriaxone,amikacin,aztreonam,and SMZ/TMP dur-ing 4 years were statistically significant (P <0.05).In 2012-2013,resistance rates of S .marcescens to cefopera-zone/sulbactam,ciprofloxacin,ceftriaxone,aztreonam,and SMZ/TMP increased obviously,then resistance rates tend to be stable,while resistance rates to cefoperazone/sulbactam decreased.Conclusion Susceptibility of S.marcescens to most antimicrobial agents are high,but resistance had increasing tendency;susceptible rates of S .marcescens to ertapenem,ceftazidime,levofloxacin,and piperacillin/tazobactam are all high,and can be used as the empirical medication for the treatment of related infection.

Objective To investigate the genotype distribution of CTX-M-type extended-spectrum β-1actamase(ESBLs) by denaturing high-performance liquid chromatography(DHPLC) in Hunan Province and the accuracy of DHPLC assay. Methods The blaCTX-M genes of standard strains and clinical ESBLs-producing Enterobacteriaceae were amplified by multiplex PCR followed by DHPLC and genotype determination. 25 isolates randomly selected were sequenced to assess the accuracy of DHPLC method. Results Among 142 ESBLs-producing isolates, 109 isolates carried blaCTX-M gene (76. 8% ). Four different CTX-M genotypes were detected by DHPLC, including CTX-M-3 (33 isolates), CTX-M-15 (19 isolates), CTX-M-14 (52 isolates) and CTX-M-9 (5 isolates). The DHPLC typing of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing, but one CTX-M-15 isolate typed by DHPLC was shown to be CTX-M-82 by sequencing. Conclusion DHPLC is a powerful tool for genotyping of the resistance gene and is worth being applied in the clinical and scientific research with accurate, rapid and economic advantages.