Objective To investigate the effect of resistance nodulation division (RND) efflux pumps on reduced susceptibility to tigecycline in carbapenem-resistant Acinetobacter baumanii.Methods Totally 631 isolates of Acinetobacter baumanii were collected from 16 hospitals in 7 provinces in 2010.Genes oxa-51 and oxa-23 were detected by PCR method,and the ST profiles were determined by multilocus sequence typing (MLST).The disk susceptibility assay was used to determine the inhibition zone diameters of β-lactams,aminoglycosides,macrolides,tetracyclines,carbapenems,tigecycline and polymyxin.The minimum inhibitory concentration (MIC) of tigecycline was determined by E-test in strains with inhibition zone diameters ≤ 12 mm on tigecycline.The expression of operon genes adeB,adeG and adeJ was determined with efflux pump inhibitor NMP (N-methyl-2-pyrrolidone) for detection of efflux pump inhibitor phenotype.The isolates of Acinetobacter baumanii which were resistant both to tigecycline and carbapenems and with the inhibited phenotype of efflux pump inhibitor were collected as the experiment group,the isolates which were susceptible to tigecycline but resistant to carbapenems were collected as the control group,and ATCC 19606 was used as the reference strain.The expressions of adeABC,adeFGH and adeIJK were quantified by q-PCR at the transcriptional level.Genes adeR,adeS and adeL were amplified and sequenced using PCR method to find polymorphic locus and insertion sequences.Results There were 32 isolates of Acinetobacter baumanii with reduced susceptibility to tigecycline and carbapenem-resistant.Eight isolates were with the inhibited phenotype by efflux pump inhibitor.And 4 strains which were susceptible to tigecycline but resistant to carbapenems were selected as the control.The expressions of adeABC in A518,Z1219 and A527 of experiment group were 13-fold,5-fold and 7-fold higher than reference strain ATCC19606,respectively.The expressions of adeFGH and adeIJK were up-regulated slightly in some isolates.Transcript of adeABC was not found in control group strains A207 and A1731,and the expressions of adeABC,adeFGH and adeIJK were not up-regulated in other isolates.The single nucleotide polymorphism (SNP) was detected in adeR (E220K) and adeS (A130D),respectively.ISAab1 insertion sequence was identified in adeS of adeABC-over expressed isolates.No mutation was found in adeL.Conclusion High expression of adeABC pump may play an important role in tigecycline resistance in carbapenem-resistant Acinetobacter baumannii,mainly due to the insertion of ISAba1 sequence in its regulator gene adeS,but other mechanism of tigecycline resistance may not be excluded.

Objective To investigate the synergistic efficacy of different antibiotic combinations against KPC-2 carbapenemase producing Klebsiella pneumoniae strains in vitro and search for effective antibiotic combination.Methods During 2008 - 2009,a total of 24 strains of K.pneumoniae producing KPC-2 carbapenemase were collected from 8 hospitals in the First Affiliated Hospital of Medical School of Zhejiang University,Ningbo LiHuiLi Hospital,Zhejiang People's Hospital,Hangzhou Third Hospital,the Second Hospital of Shaoxing,Hangzhou First Hospital,Fudan University Huashan Hospital,General Hospital of Nanjing Military Region.MLST technique was used for epidemiological analysis.The MIC of antibiotics,such as amikacin,minocycline,imipenem,amoxicillin/clavulanic-acid,ceftazidime,meropenem,gentamicin,cefoxitin,cefepime,rifampicin,polymyxinB,ciprofloxacin were determined by an agar dilution method,the MIC of tigecycline and piperacillin/tazobactain were determined by Etest.The antibacterial activities of cefepime in combination with amoxicillin/clavulanic-acid,amikacin,or ciprofloxacin,amikacin with ciprofloxacin,imipenem with amikacin,ciprofloxacin,polymyxinB,or minocycline,polymyxin B with rifampicin,ceftazidime with amoxicillin/clavulanic-acid were assessed by chequerboard synergy agar dilution tests against all the isolates.Results MLST showed 5 STs among 24 strains of KPC-2 carbapenemase producing K.pneumoniae,and the most prevalent clone was ST11 (15 strains).All isolates were susceptible to polymyxin B and tigecycline,and the resistance rate of minocycline was 4.2％.The synergetic effects were observed in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin,ceftazidime-amoxicillin/clavulanic acid combinations as 19 isolates,13 isolates,and 13 isolates,respectively.Conclusions KPC-2 carbapenemase producing K.pneumoniae is sensitive to polymyxin B,tigecycline and minocycline.The synergetic effect is predominant in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin ceftazidime-amoxicillin/clavulanic acid combinations in vitro,their clinical efficacy are worthy of further observation.

Objective To characterize the prevalence of plasmid-mediated quinolone resistance determinants qnrA in extended-spectrum β-lactamase(ESBL)-producing Escherichia coli and Klebsiella pneumonia.Methods PCR was used to amplify qnrA gene in ESBL-rpoducing isolates(including 263isolates of Escherichia coli and 99 isolates of Klebsiella pneumonia).Conjugation experiments and southern blot hybridization were employed to definitude the location of the genes in ZJ96 isolate of Klebsiella pneumonia which had positive qnrA and CTX-M genes.Shot gun sequencing was performed for analyzing the complete nucleotide sequence of pKP96,a plasmid containing qnrA and CTX-M-24 genes in ZJ96 isolate.Results qnrA was detected in 5 out of 263(1.9%)Escherichia coli isolates and 8 out of 99(8.1%)Klebsiella pneumonia isolates.pKP96,a conjugative plasmid including qnrA gene and CTX-M-24 gene presented in ZJ96 isolate.The sequence of the plasmid pKP96 displayed the qnrA,CTX-M-24,aac(6')-Ib-cr,tetA and int Ⅰ 1 genes.Conclusion The plasmid-mediated genes,such as qnrA and CTX-M,may facilitate the prevalence of multi-drug resistant strains.

Objective To investigate the genotypes of β-lactamases produced by Klebsiella pneumoniae.Methods Plasmid conjugation,PCR amplification,gene cloning and DNA sequencing,isoelectric focusing electrophoresis and extended-spectrum β-lactamase(ESBLs)confirmatory test were carried out for analyzing the encoding gene of β-lactamases in clinical strains of Klebsiella pneumoniae collected from hospital wards.Results Totally 75 clinical strains of Klebsiella pneumoniae were collected,in which 48 strains were confirmed to produce genotype of β-laetamases(64.0%),including 39 ESBLs-producing Btraim(52.0%).Among 48 strains,17 isolates(35.4%)carried 2 types of ESBLs genes,7(14.6%)carried 3 types of ESBL8 genes,and 5(10.4%)carried 4 types of ESBLs genes.CTX-M was the most comon type(30/48,62.5%),followed by TEM(26/48,54.2%)and SHV(25/48,52.1%).Among 9 isolates with DHA-1 AmpC β-laetamase,8 produced AmpC β-lactamases and ESBLs.Class A carbapenemase KPC-2 was produced in 3 isolates.False negative rate of ESBLs confirmatory test was 23.1%(9/39).Condusion Genotypes of β-lactamases produced by Klebsiella pneumoniae are complicated,which results in multi-drug resistance in clinic.

Objective To investigate the resistance of Pseudomonas aeruginosa and genotyping of the main β-lactamases in China. Methods A total of 645 Pseudomonas aeruginosa isolates were collected from 28 hospitals in 16 cities in China from July 2006 to July 2007. The susceptibilities to 11 kinds of antimicrobial agents were detected by agar dilution or Kirby-Bauer disk diffusion method. The genotypes of β-lactamases including TEM, SHV, CTX-M and OXA of all the strains were detected by polymerase chain reaction (PCR) and sequence analysis. Results The resistance rates of 645 Pseudomonas aeruginosa isolates to antimicrobial agents were high, except those to amikacin and meropenem were lower than 30 %. Two hundred and seventy-five (42. 64 % ) strains were carbapenem and (or) meropenem-nonsusceptible Pseudomonas aeruginosa. Three hundred and sixty-eight (57.05 %) strains were multidrug-resistant Pseudomonas aeruginosa and 20 (3. 10%) strains were pandrug-resistant. The genotyping results of β-lactamases were as follows: 51 stains produced OXA-10 group β-lactamases, 37 were CARB type, 36 were TEM, 35 were PER, 11 were CTX-M, 9 were VEB, 5 were SHV, 24 were metallo-β-lactamases positive and 1 was GES. None of genotypes of plasmidmediated AmpC enzyme and other carbapenemases were detected. CTX-M-13, CTX-M-14,CTX-M-15, CTX-M-3 of extended spetrum β-lactamese were detected in Pseudomonas aeruginosa.Conclusions The situation of Pseudomonas aeruginosa resistances is severe in China. OXA-10 and PSE-1 are the most common genotypes of β-lactamases. The β-lactamases genotyping is different between carbapenem-nonsusceptible and carbapenem-susceptible strains.

Objective To investigate the value of cardiac troponin Ⅰ (cTn Ⅰ) in diagnosis of multi-trauma patients combined with myocardiac contusion.Methods A retrospective review was made on 98 cases of multi-trauma patients combined with blunt chest trauma.The groups were identified according to whether the patients were associated with myocardiac contusion or not,including myocardiac contusion group (n =48) and non-myocardiac contusion group (n =50).The detection and diagnosis of myocardiac contusion in the use of different cut-off points of cTn Ⅰ and creatine kinase MB isoenzyme/creatine kinase (CKMB/CK) or their combination were compared between groups.Results cTn Ⅰ ≥0.60 ng/ml had a specificity of 90.0％,a sensitivity of 64.6％ and a Youden index of 0.54 in diagnosis of myocardiac contusion,indicating a best diagnostic accuracy as a single parameter.As compared with the single use of cTn Ⅰ ≥ 0.60 ng/ml or CKMB/CK ≥ 6％ in diagnosis of myocardiac contusion,the combined use of two parameters presented a significantly higher diagnostic sensitivity (85.4％ vs 64.6％ ; 85.4％ vs 27.1％ respectively,both P ＜ 0.05),but no markedly lower specificity (84.0％ vs 90.0％ ; 84.0％ vs 88.0％ respectively,both P ＞0.05).cTn Ⅰ level was positively correlated with ISS score of the multi-trauma patients combined with myocardiac contusion (r =0.534,P ＜ 0.01).Mortality rate in patients with severely increased cTn Ⅰ was much higher than that in patients with mild-moderately increased cTn Ⅰ (P ＜ 0.01).Conclusions cTn Ⅰ ≥0.60 ng/ml presents a high sensitivity and preferable specificity for diagnosis of multiple trauma patients combined with myocardiac contusion.It can be served as a biomarker for diagnosis of MC and its combination with CKMB/CK≥6％ improves the diagnostic sensitivity.cTn Ⅰ can be used as an assessment indicator for the early risk stratification and outcome in multi-trauma patients combined with myocardiac contusion.

Objective This study was designed to evaluate the safety ,tolerability and efficacy of intravenous caspofungin for treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .Methods This was a non-controlled ,multicenter ,candidiasis .All the 63 patients were included in the safety set (SS) and the full analysis set (FAS) .In the SS ,19 SAEs occurred in 14 patients .All these SAEs were unrelated to caspofungin .There were 73 caspofungin-related non-serious AEs in 31 patients (49 .2% ) .Five patients (7 .9% ) had both clinical AEs and laboratory abnormalities .Eight patients (12 .7% ) had clinical AEs (mainly rashes) ,and 27 patients (42 .9% ) had laboratory abnormalities ,mainly increases in liver enzymes alanine transaminase and aspartate transaminase and reduction in blood potassium .About 91 .7% of the clinical AEs were mild to moderate .Treatment was discontinued in 1 patient (1 .6% ,1/63) due to AEs .The overall efficacy was 58 .1% (36/62) in the FAS and 70 .0% (35/70) in the per-protocol set (PPS) .In the FAS ,the therapeutic efficacy was 57 .6% (34/59) for invasive candidiasis and 66 .7% (2/3) for esophageal candidiasis .In the PPS , the therapeutic efficacy was 68 .8% (33/48 ) for invasive candidiasis and 100% (3/3 ) for esophageal candidiasis .Conclusions The AEs of caspofungin were mostly mild to moderate in the treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .Only one patient terminated therapy due to drug-related AE .Caspofungin is safe and effective for the treatment of invasive candidiasis and esophageal candidiasis in Chinese adults .

Objective To investigate the prevalence and dissemination mechanism of 16S rRNA methylase genes in extended-spectrum beta-lactamases(ESBLs)-producing Enterobacteriaceae in China.Methods PCR amplification and DNA sequencing were used for screening and identifing 16S rRNA methylase genes and ESBLs genes.Minimal inhibitory concentrations(MICs)of the antimicrobial agents were detected by Etest.Conjugation and plasmid extract were performed to study dissemination mechanism of 16S rRNA methylase genes and ESBLs genes.Results Only one strain.Klebsiella oxytoca strain ZJ157 was screened as positive for armA gene from 447 ESBLs-producing isolates,which also contained CTX-M-15 and TEM-1 genes.It was resistant to aminoglycesides,ciprofloxacin,and most β-lactams,except carbapenems,polymyxin E and tigecyeline.Resistance to amikacin and β-lactams was transferred to a recipient Escherichia coli 600 by conjugation experiment.arntA.CTX-M-15 and TEM-1 genes were detected in the transconjugant.A plasmid about 55 kb was extracted from Klebsiella oxytoca ZJl57 and the transconjugant.Conclusions A 16S rRNA methylase gene armA was detected in an isolate of Klebsiella oxytoca.armA,CTX-M-15 and TEM-1 genes can be co-transferred in the same plasmid leading to multi-drug resistance.

Objective To discuss the association of allele polymorphisms SNP-rs1799724(C>T)in the TNF-αand SNP-rs11559013(G>A)in the ALCAM with HCV chronic infection in Han population in Yunnan province. Methods 434 HCV chronic infectious patients and 444 healthy individuals of Han Chinese population in Yunnan province were recruited. Two single nucleotide polymorphisms(SNPs)in the SNP-rs1799724(C>T) of TNF-αgene and SNP-rs11559013(G>A)of ALCAM gene were determined by real-time TaqMan polymerase chain reaction. We evaluated the associations of the two SNPs with HCV chronic infection. Results The distributions of allele and genotype of SNP-rs1799724(C>T)in the TNF-αand SNP-rs11559013(G>A)in the ALCAM between hepatitis C virus(HCV)chronic infectious patients and the healthy controls were not statistically significant(P > 0.05). Conclusion SNP-rs1799724(C>T)in the TNF-αand SNP-rs11559013(G>A) in the ALCAM have no association with HCV chronic infection in the Han population in Yunnan province.

OBJECTIVE To study the mechanism of drug-resistance of multi-resistant Pseudomonas aeruginosa,and provide the guideline for treatment and control of P.aeruginosa infection in hospital.METHODS Fifty strains of multi-resistant P.aeruginosa were selected with K-B susceptibility method.The three-dimensional method was taken to differentiate the various beta-lactamases.The relative drug-resistance gene was detected by polymerase chain reaction(PCR).RESULTS Among 50 strains of multi-resistant P.aeruginosa,there were 2 strains(4%)producing ESBLs,20 strains(40%)producing AmpC beta-lactamases,and 11 strains(22%) producing ESBLs and AmpC beta-lactamases at the same time.There were 8 positive genes in the detected drug-resistance gene,the most common sources of gene were CTX(56%),OprD(60%) and aac(6′)-Ⅱ(60%),respectively.CONCLUSIONS The main beta-lactamases are AmpC beta-lactamases and the main genotype is CTX in the multi-resistant P.aeruginosa cultured in our area.The main course of imipenem-resistance was deletion of outer membrane proteins,and the aminoglycoside modifying enzyme gene and disinfectant-resistance gene in multi-resistant P.aeruginosa are acquired.In order to reduce the drug-resistance strains and control the infection of P.aeruginosa,antibiotics should be used reasonably according to drug susceptibility testing clinically.