OBJECTIVE To investigate the distribution and resistantce of three commonly encountered Gram-negative bacilli in our hospital,and provide doctors with the laboratory evidence of guiding antibiotic therapy.METHODS Bacterial identification and antibiotic susceptibility tests of isolates between Jan 2005 and Dec 2006 were performed by MicroScan WalkAway-40.RESULTS Totally 2024 common Gram-negative bacilli strains were isolated,807 of which were Escherichia coli,737 were Klebsiella pneumoniae,480 were Pseudomonas aeruginosa.The antimicrobial resistant rate of E.coli and K.pneumoniae to imipenem(2.2%) was lower than to the other antibiotics(2.7%).While 14.0% of P.aeruginosa isolates were resistant to imipenem.The antimicrobial resistant rate of all the isolated bacteria to piperacillin/tazobactam and amikacin was low,accounted less than 15%.CONCLUSIONS The resistant rate of three commonly encounted Gram-negative bacilli is increasing year by year.Therefore,monitoring bacterial drug resistance is very important for the rational use of antibiotics and the containment of multi-drug bacteria.

OBJECTIVE To investigate the in vitro antimicrobial susceptibility of Streptococcus pneumoniae and guide the rational use of drug clinically. METHODS We performed statistical analysis of the susceptibility of 62 strains of S.pneumoniae isolated in our hospital from May 2007 to Aug 2008. RESULTS S.pneumonia e could be isolated from various specimens,most of them were isolated from sputum(84.0%).S.pneumoniae could be detected from many hospital wards,but more strains isolated from pediatric(67.8%) and respiratory(11.4%) departments.A total of 67.7% of S.pneumoniae isolates were penicillin non-susceptible,the resistance prevalence to tetracycline was 87.1%,to erythromycin 79.0% and to trimethoprim/sulfamethoxazole 49%,while they were highly susceptible to ofloxacin(90.3%),vancomycin(91.9%),levofloxacin(95.2%),moxifloxacin(96.8%) and rifampicin(98.4%),and more strains showed multi-resistance from penicillin non-susceptible isolates. CONCLUSIONS The resistance to penicillin of S.pneumoniae is serious in our hospital.The tetracycline and erythromycin are not the best ckoice in treating S.pneumoniae infection,and the new fluroquinolones show strong activity against S.pneumoniae.

Objective To investigate and analyze the double-disk inhibiting synergy test for detecting AmpC β-lactamase pro-duced by Klebsiella pneumoniae and to evaluate its application value in clinical laboratory.Methods The cefoxitin disk agar diffu-sion method,cefoxitin three-dimensional method,double-disk inhibiting synergy test and drug resistance gene multiplex PCR assay were adopted to detect the clinically isolated bacterial strains.Results Among 137 clinically isolated strains of Klebsiella pneumoni-ae,22 strains were insensitive to cefoxitin and 11 strains were positive by the three-dimensional method;in the double-disk inhibiting synergy test,18 strains were positive for the FOX/FOX+PBA group and 11 strains were positive for the CTT/CTT+PBA group respectively;in the multiplex PCR assay,19 strains were positive.The coincidence rate of the cefoxitin three-dimensional method and multiplex PCR methods was 47.4%(9/19),in the double-disk inhibiting synergy test,the coincidence rate of the positive re-sults in the CTT/CTT+PBA group and the multiplex PCR methods was 57.9%(11/19);the coincidence rate of the FOX/FOX+PBA group and multiplex PCR methods was 94.7%(18/19).Conclusion The double-disk inhibiting synergy test is simple with highly accurate results,in which the FOX/FOX+PBA double-disk synergy test could be applied to detect AmpC β-lactamase pro-duced by clinical isolates of Klebsiella pneumoniae.

Objective To investigate the prevalence of qepA, quinolone efflux protein, among 41 unique clinical strains of K. pneumoniae producing ESBLs and to study the qepA-bearing isolates using the Diversilab system. Methods Identification and antimicrobial susceptibility were performed by Vitek-2 Compact System. Screening of qepA was carried out by PCR amplification. The NCBI BLAST program was utilized for sequence comparisons. qnr-bearing strains was evaluated by the Repetitive-sequence-based PCR(Rep-PCR) employing the Diversilab system. And the existence of rmtB was detected among these qepA contained isolates. Results qepA were detected in 5 isolates( 12.2% ). The Rep-PCR profiles produced by the Diversilab system showed that 2/5 of isolates were indistinguishable. And 60% of qepA-positive isolates were detected to harbor rmtB gene. Conclusion The data suggest the emergence of qepA-borne K. pneumoniae.

Objective To analyze the distribution and drug resistance of bile-isolated pathogens in Xiamen area ,providing evi-dence for clinical use of antibiotics .Methods Bile cultures and antibiotic susceptibility tests were performed on strains isolated from the First Affiliated Hospital of Xiamen University .WHONET 5 .6 was used for data analysis .Results In 35 out of 217 samples ,2 kinds of pathogens were isolated .Among these ,Enterococcus and Enterobacteriaceae coinfection was most common .There were 252 strains isolated totally ,with 83 gram-positive strains(32 .9% ) ,165 gram-negative strains (65 .5% ) and 4 fungi strains (1 .6% ) . Escherichia coli ,Enterococcus faecalis and Klebsiella pneumoniae were three of the most common pathogens isolated .Pseudomonas aeruginosa and Acinetobacter baumannii were two of the most common nonfermenters isolated .The resistance rates of Enterobacte-riaceae to aminoglycosides ,fourth generation cephalosporins ,carbapenems or piperacillin/tazobactam were lower than 40 .0% .The resistance rate of Escherichia coli to quinolones was higher than 50 .0% .Enterococcus faecalis or enterococcus faecium were less re-sistant to vancomycin ,linezolid and tigecycline .The resistance rates of enterococcus to high concentration of streptomycin or genta-micin were lower than 30 .0% .Conclusion The top three common pathogens isolated from bile are Escherichia coli ,Enterococcus faecalis and Klebsiella pneumonia in Xiamen area .Infections by Enterococcus together with Enterobacteriaceae account for large numbers of coinfection cases .The resistance rates to cephalosporin or quinolones of pathogens causing biliary tract infections have increased dramatically .

Objective To investigate the clinical distribution and the drug resistance characteristics of Pseudomonas aeruginosa . Methods The results of the bacterial culture and the antimicrobial susceptibility test in the hospital from July 2007 to October 2008 were performed the retrospective analysis.Results Totally 335 strains of Pseudomonas aeruginosa were isolated,accounting for 9.2% of isolated pathogenic bacteria.The main specimen source was sputum,accounting for 77.6%.The ICU ward was the high incidence area.The resistance rates of Pseudomonas aeruginosa to amikacin,piperacillin/tazobactam,tobramycin,levofloxacin, cefepime,gentamicin,ticarcillin and ciprofloxacin were less than 10%.The resistance rates of imipenem-insensitive Pseudomonas aeruginosa to aztreonam,ceftazidime,ciprofloxacin,levofloxacin,piperacillin/tazobzctam were significantly higher than those in imi-penem-sensitive Pseudomonas aeruginosa (P <0.05).Conclusion The multiple drug resistance phenomena of Pseudomonas aerugi-nosa generally exist,amikacin,piperacillin/tazobactam and tobramycin are recommended for the treatment of infections caused by Pseudomonas aeruginosa .

Objective To analyze the distribution and antimicrobial resistance situation of pathogens isolated from blood culture to provide the scientific basis for the accuratel use of antibacterial drugs and preventing and controlling nosocomial acquired blood‐stream infection .Methods The US BACTEK‐FK automatic blood culture instrument and the French VITEK 2 COMPACT auto‐matic bacteria identification/susceptibility system were used to conduct the culture ,identification of blood culture isolated bacterial strains and drug susceptibility test .The results of drug susceptibility test were judged by adopting the 2011 criteria of the Clinical Laboratory and Standards Institute(CLSI) .Results The main isolated bacteria from blood culture for the recent three years were E coli ,staphylococcus aureus and klebsiella pneumonia bacteria ,coagulase negative staphylococcus(CNS) ,etc .The proportions of hos‐pital‐acquired bloodstream infections during these period were 42 .2% ,46 .9% and 54 .1% respectively .The detection rate of methi‐cillin‐resistant staphylococcus aureus (MRSA) was 18 .8% ,which of multiple drug resistant acinetobacter baumannii (MDRAB) was 42 .9% ,which of producing extended spectrum beta lactamase (ESBLs) in E .coli and klebsiella pneumoniae were 71 .8% and 69 .8% respectively .Conclusion The bloodstream infections pathogens in this hospital are mainly Enterobacteriaceae bacteria ,the proportion of hospital‐acquired bloodstream infections increases year by year ;the detection rate of multi‐drug resistant Acinetobact‐er baumanni (MDRAB) is higher ,clinic should pay more attention to the change of blood culture pathogens and their drug resist‐ance trend ,meanwhile nosocomial bloodstream infection should be prevented and controlled .

Objective To investigate the genotype and epidemiology of plasmid‐mediated AmpC β‐lactamases produced by the clinical strains of Escherichia coli and Klebsiella pneumoniae .Methods A total of 176 clinical nonrepetitive cefoxitin non‐sensitivity isolates of Escherichia coli and Klebsiella pneumoniae was collected from July 2011 to August 2012 .Polymerase chain reaction (PCR) for AmpC enzyme gene amplification and DNA sequencing were carried out for genotype of AmpC beta‐lactamases .Results The results of PCR showed that the positive rate of ampC of the 176 strains of Escherichia coli and Klebsiella pneumoniae AmpC was 18 .2% ,mainly DHA type ,counting for 59 .4% ,CIT counting for 37 .5% ,EBC counting for 3 .1% .The positive rate of ampC of Escherichia coli was 11 .4% ,mainly CIT type ,counting for 77 .8% ,the positive rates of DHA type and EBC type both were 11 .1% .The positive rate of ampC of Klebsiella pneumoniae were 23 .7% ,mainly DHA type ,counting for 78 .3% ,CIT type count‐ing for 21 .7% .The results of DNA sequencing showed that there were 18 strains DHA‐1 type and 1 strain ampC gene type of Morganella morganii in DHA type strains ,the concordance rate was 97 .0% ,10 CIT type strains was CMY‐2 type ,1 strain was CMY‐42 ,one strain was CMY‐4 type ,EBC type was ampC gene type of Enterobacter cloacae ,the concordance rate was 99 .0% .A total of 32 strains of gene sequencing were registered as KJ127248 - KJ127279 in GenBank .Conclusion The main genotypes of plasmid‐mediated ampC enzyme produced by Escherichia coli and Klebsiella pneumoniae were CMY‐2 and DHA‐1 respectively .

Objective To construct a mutant strain of Streptococcus mutans ( S.mutans ) with clpC-deletion and to investigate the role of clpC gene in genetic competence.Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR.The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1.The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination.The pCKX2 vector was linearized with SalⅠ and transformed into S.mutans UA159 strain.The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA.The KMR cassette was excised after incubating at 30℃ for 48 hours.Then the pCrePA plasmid was removed after overnight incubating at 37℃for the prepara-tion of clpC-deletion mutant.Total RNA were extracted from the S.mutans UA159 strain and the clpC-dele-tion mutant strain respectively, and then reverse transcribed into first strand cDNA.The target gene frag-ments were amplified by RT-PCR and analyzed by the agarose gel electrophoresis and sequencing.After be-ing verified by PCR and sequencing, the S.mutans UA159 strain and the clpC-deletion mutant strain were re-spectively transformed with E.coli-S.mutans shuttle vector pDL276 to observe the competence development induced by the competence-stimulating peptide (CSP).Results The PCR and sequencing results showed that the pCKX2 vector and the mutant strain with clpC-deletion were constructed successfully via homologous recombination.No clpC gene was detected in the clpC-deletion mutant as indicated by RT-PCR analysis.The formation of competent clpC-deletion mutant was delayed and the competence state was prolonged as com-pared with its parent strains.Conclusion The clpC gene negatively regulated the formation of competent S.mutans.