Objective:To evaluate the role of Nod-like receptor family pyrin domain-containing 2 (NLRP2) in the dorsal root ganglion (DRG) in neuropathic pain (NP) in rats.Methods:Thirty-two male Sprague-Dawley rats in which intrathecal catheters were successfully implanted, aged 2-3 months, weighing 200-250 g, were divided into 4 groups ( n=8 each) using a random number table method: sham operation group (S group), NP group, NP plus NLRP2-siRNA group (NP+ siRNA group) and NP plus NLRP2-scrRNA group (NP+ scrRNA group). The right sciatic nerve was only exposed but not ligated in group S. NP was induced by chronic constriction injury (CCI) to the sciatic nerve in anesthetized rats in group NP, group NP+ siRNA and group NP+ scrRNA.NLRP2-siRNA and NLRP2-scrRNA were intrathecally injected at 3 days before CCI in group NP+ siRNA and group NP+ scrRNA, respectively.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before CCI (T 0) and 1, 3, 7 and 10 days after CCI (T 1-4). The rats were sacrificed after the last measurement of pain threshold, and the L 4, 5 segments of the DRG on the operated side were removed for determination of the expression of NLRP2 and caspase-1 (by Western blot), the expression of NLRP2 mRNA (by real-time polymerase chain reaction) and interleukin-1beta (IL-1β) content (by enzyme-linked immunosorbent assay). Results:The MWT was significantly lower at T 2-4 than at T 0 in group NP, group NP+ siRNA and group NP+ scrRNA ( P<0.05). Compared with group S, the MWT was significantly decreased at T 2-4, the expression of NLRP2 protein and mRNA and caspase-1 was up-regulated, and the content of IL-1β was increased in group NP ( P<0.05). Compared with group NP, the MWT was significantly increased at T 2-4, the expression of NLRP2 protein and mRNA and caspase-1 was down-regulated, and the content IL-1β was decreased in group NP+ siRNA ( P<0.05), and no significant change was found in the parameters mentioned above in group NP+ scrRNA ( P>0.05). Conclusion:NLRP2 in DRG is involved in the development of NP, and the mechanism is related to NLPR2 inflammasomes-induced peripheral neuroinflammation in rats.

Objective To investigate the molecular mechanism of the inhibitory effects of rhizoma coptidis on multi-drug resistant uropathogenic Escherichia coli.Methods High-throughput RNA sequencing ( RNA-seq ) was performed to investigate the transcriptome in a multi-drug resistant uropathogenic Escherichia coli strain (NB8) treated with water decoction of rhizoma coptidis .Agar dilution test was used to determine the minimal inhibitory concentration ( MIC) of water decoction of rhizoma coptidis against the NB 8 strain.A growth curve was drawn to evaluate the effects of water decoction of rhizoma coptidis on the growth of NB8 strain.Total RNAs were extracted from the NB 8 strain after treated with the water decoction of rhizo-ma coptidis for 30 minutes and then synthetized to cDNA by reverse transcription after screening out the rRNAs.The HiSeq 2000 sequencing system was used for transcriptome sequencing .The TopHat software was used to map and analyze the RNA-Seq reads, and then Cufflinks was run to assemble transcripts and es-timate their abundances .The differential expression , GO enrichment and KEGG metabolic pathway were fur-ther analyzed .The NB8 strain dealt with normal saline was used as negative control .Results The MIC of water decoction of rhizoma coptidis to NB 8 strain was 12.5 mg/ml.There were 3665 genes expressed in NB8 strain treated with water decoction of rhizoma coptidis and 3430 genes expressed in NB8 strain treated with normal saline .The number of differentially expressed genes was 1428 including 921 up-regulated genes and 507 down-regulated genes .Those differentially expressed genes mainly enriched in the modules of binding and catalysis.The genes concerning cell adhesion , apoptosis and multicellular process were up-regulated, while those concerning the regulation of enzyme activities were down-regulated.Results of the KEGG meta-bolic pathway enrichment analysis showed that the genes concerning synthetic pathway of LPS were signifi -cantly up-regulated as well as those encoding the repair polymerase Ⅲthat was involved in DNA replication . However , the genes concerning fatty acid metabolism , histidine metabolism , thiamine metabolism , folate metabolism and iron carrier in ribosome synthesis showed overall down-regulation.Conclusion The tran-scriptome in uropathogenic Escherichia coli strain treated with rhizoma coptidis was profiled .The main mo-lecular mechanism of the inhibitory effects of rhizoma coptidis on uropathogenic Escherichia coli was to de-stroy the cell wall of Escherichia coli, affect the replication of DNA and regulate the transcription and transla-tion of proteins .This study illustrated that the inhibitory effects of rhizoma coptidis on uropathogenic Esche-richia coli were achieved in multiple levels .

Objective To investigate the distribution of β-lactamase genes in a pan-drug resistant Klebsiella pneumoniae isolate JM45.Methods Klebsiella pneumoniae JM45 was isolated from the blood sample of a patient admitted in the intensive care unit,the Second Affiliated Hospital,Zhejiang University School of Medicine on April 7,2010.The susceptibilities to 26 antibiotics were tested using E-test method.Cica-β-Test was performed to detect β-lactams,and modified Hodge test was performed to detect carbapenemase.Resistant genotypes were detected using PCR,DNA sequencing and BLAST algorithm.Whole genome sequencing (complete graph) was performed by high throughput Roche 454 sequencing approach to analyze the distribution of β-lactamase genes.Results Except polymyxin B and tigecycline,JM45 was resistant to other 24 kinds of antibiotics including cephalosporins and carbapenems.Several β-lactamases were positive in Cica-β-Test,and modified Hodge test was positive.Based on PCR typing,TEM-1,SHV-11,CTX-M-24 and VEB-3 were positive,but carbapenemase genes and metallo-β-lactamase genes were negative.A complete genome (chromosome) sequence (GenBank accession number:CP006656) and 2 plasmids sequences (GenBank accession number:CP006657,CP006658) were obtained by wholegenome sequencing.CTX-M-24 (Locus tag:N559_5233),TEM-1 (Locus tag:N559_5242) and VEB-3 (Locus tag:N559_5248) were positive in plasmid 1.CTX-M-24 located in insertion sequence (IS903-CTXM-24-ISEcp1),while TEM-1 and VEB-3 located in transposons (tnpA-TEM-1-rmtB and VEB-3-tnpA).SHV-11 (Locus tag:N559_2715) was positive in genome (chromosome),and 4 putative β-lactamase genes or β-lactamase domains were obtained:(1) metallo-β-lactamase domain protein (Locus tag:N559_0119,780 bp) ; (2) putative β-lactamase (Locus tag:N559_1633,1308 bp) ; (3) β-lactamase domain protein (Locus tag:N559_2279,813 bp); (4) β-lactamase domain protein (Locus tag:N559_3769,1101 bp).No insertion sequence or transposase gene was observed near SHV-11.Conclusion The resistance to antibiotics including cephalosporins and carbapenems is correlated with TEM-1,SHV-11,CTX-M-24,VEB-3 and 4 kinds of putative β-lactamase genes or β-lactamase domains.

Objective To analyze molecular evolution of carbapenemase KPC-12 and its binding free energies with β-lactams.Methods Class A beta-lactamases were divided into 2 clusters:those with carbapenemase activities and those without.Minimum Evolution method in MEGA4.1 software was used to analyze molecular evolution of class A beta-lactamases with carbapenemase activity,including KPC-2 to KPC-13,SFC-1,SME-1,IMI-1,NMC-A,and class A beta-lactamases without carbapenemase activity,including TEM-1,SHV-1.Then,tertiary structure of KPC-12 was predicted by homology modeling as reported in SWISS-MODEL database depending on tertiary structure of KPC-2.Moreover,DOCK module in ArgusLab 4.1 software was used to perform molecular docking of KPC-12 to 10 kinds of beta-lactams substrates,and the binding free energies (△ G) were calculated.Results Molecular evolution between KPC-12 and KPC-2 was the closest.The top three decline in binding free energies of β-lactams were penicillin G sodium salt (△G =-8.45149 kcal/mol),ertapenem (△G =-8.36383 kcal/mol) and ampicillin (△G =-8.19326 kcal/mol),while the last two decline in binding free energies of β-lactams were aztreonam (△G =-6.50614 kca]/mol) and clavulanic acid (△G =-6.88533 kcal/mol).Conclusion Carbapenemase KPC-12 has high catalytic activities to penicillin G sodium salt,ertapenem and ampicillin,while has low catalytic activities to aztreonam and clavulanic acid.

Objective To evaluate the killing efficacy of chlorine-releasing agents (CRAs) with different concentrations on multidrug-resistant Acinetobacter baumannii.Methods Totally 30 clinical strains of multidrug-resistant Acinetobacter baumannii were collected from November 2008 to December 2009.Killing efficacy of CRAs on these strains was evaluated by quantitative suspension test.The one-way analysis of variance was performed.Results The log values of killing to 30 strains of multidrug-resistant Acinetobacter baumannii were all ≥5.00,when the bacteria were exposed to available chlorine concentrations 400 mg/L of CRAs for 10 min,600 mg/L for 10 min,800 mg/L for 3 min and 1000 mg/L for 1 min,respectively.And effective rates were all 100％. Furthermore,there were significant differences among different available chlorine concentrations exposed for the same time ( except 10 min) ( F =72.72,64.79 and 32.33,P =0.00),and among different exposure time in the same available chlorine concentration ( except 1000 mg/L) ( F =110.42,20.41 and 3.20,P=0.00,0.00 and 0.03).Conclusion Satisfactory killing efficacy of CRAs to multidrug-resistant Acinetobacter baumannii can be achieved with available chlorine concentration 500 mg/L to 1000 mg/L and exposure time 10 min to 30 min.

ObjectiveTo investigate correlation between drug-resistance related genes and mobile genetic elements of Klebsiella pneumoniae resistant to β-lactams. Methods Forty-seven strains of multidrug-resistant Klebsiella pneumoniae were collected from 6 hospitals in Hangzhou and Huzhou of Zhejiang province from August 2008 to May 2010.Modified Hodge test was performed to detect phenotypes of carbapenemases.Forty kinds of β-lactamases (class A-D),ompK35,ompK36,and 12 kinds of mobile genetic elements were detected by PCR,and the results were analyzed by index cluster.ResultsThirty-five strains were positive in modified Hodge test,and 5 kinds of β-lactamases gene ( including KPC-2-like,GenBank:HQ258934) and 9 kinds of mobile genetic elements were detected.Mutations were observed in ompK35 and ompK36 when compared with sensitive strains.Index cluster analysis showed that correlation existed between KPC-2,KPC-2-like and ISKpn6,between TEM-1 and ISEcpl,IS26,int Ⅰ 1,trbC,IS903,and between CMY-2,OXA-30,DHA-1 and tnpU,tnp513,trbC.ConclusionsFive kinds of β-1actamases genes,and mutations in ompK35 and ompK36 may be associated with the resistance to β-1actams in multidrug-resistant Klebsiella pneumoniae.

Objective To investigate ehloramphenicol,tetracycline,rifampicin and trimethoprimsulfamethoxazole resistance and the related genes in multi-drug resistant Acinetobacter baumannii(MDR-ABA).Methods Sixty-two strains of MDR-ABA were isolated from clinical samples,and their susceptibilities to 22 antimicrobial agents were detected by Kirby-Bauer disk diffusion tests.Genes related to chloramphenicol(catB and cmlA),rifampicin(arr-2/3),tetracycline(tetA and tetB)and trimethoprimsulfamethoxazole(sull,dfrA1,dfrA5,dfrA7/17,dfrA12,dfr85)resistance and drug emux genes(tehA,emrB,emrD,emrE,smr-2,mdfA)were analyzed by PCR and verified by DNA sequencing.Results Resistant rates of these MDR-ABAs to chloramphenicol,rifampicin,tetracycline and trimethoprimsulfamethoxazole were 100.0%(62/62),100.0%(62/62),90.3%(56/62)and 82.3%(51/62)respectively,while62 strains(100.0%),46 strains(74.2%),36 strains(58.1%)and 8 strains (12.9%)were detected to carry mafA,tetB,sull and tehA genes,respectively.The lest 13 genes were all negative.tetB,sull,tehA and mafa genes(2 for each)chosen optionally from positive ones were verified by DNA sequencing and BLASTn.and all were identified as the same sequences in GenBank.Conclusions MDR-ABAs show hish resistance to chloramphenicol,tetracycline, rifampiein and trimethoprimsulfamethoxazole.Multi-drug resistant phenotypes of MDR-ABAs may be closely related to mdfA genes harboring in strains.

Objective To investigate the distribution of 22 beta-lactamase genes and enzyme activities in multi-drug resistant Acinetobacter baumannii (MDRAB). Methods Sixty-two MDRAB strains were isolated from the First Affiliated Hospital, College of Medicine, Zhejiang University. Twenty-two beta-lactamase genes were analyzed by PCR and verified by DNA sequencing. Enzyme activities of extended spectrum beta-lactamases (ESBLs) , cephalosporinase ( AmpC) and metallo-beta-lactamases ( MBL) were detected by the modified three-dimensional method using enzyme extraction. Results In 62 MDRABs, 51 (82.3% ) , 50 (80.6% ) and 36 (58.1% ) isolates were found to carry blaTEM, blaOXA-23 cluster, and blaADC, respectively. The rest 19 genes were not detected in this study. DNA sequencing and genomic comparison showed that 5 isolates carrying blaTEM had the same genotype as blaTEM-l , 6 isolates carrying blaOXA-23 cluster had the same genotype as blaOXA-23 carbapenemases (accession; CAB69042. 1) , and 2 isolates carrying blaADC had the same genotype as blaADC-like (accession: EU081908). Thirty-two isolates (51.6% ) produced ESBLs and AmpC, 19 isolates (30.6% ) produced ESBLs only, and 1 isolate (1.6%) produced AmpC only; and no isolate produced MBL. Conclusion MDRAB carrying blaOXA-23 carbapenemase and blaADC AmpC in this study are of high drug resistance.

Objective To investigate ADC type cephalosporinase genes in muhidrug-resistant strains of Acinetobater baumannii (MDR-ABA). Methods Sixty-two ABA strains were collected during November 2007 to February 2008 from the First Affiliated Hospital, College of Medicine, Zhejiang University. Kirby Bauer disk diffusion method was used to detect the susceptibilities of the strains to antimicrebial agents. PCR and DNA sequencing was performed to analyze the ADC type cephalosporinase gene. Results Sixty-two MDR-ABA strains showed high drag-resistance rate to most antimicrobial agents expect cefoperazone/sulbactam (69.4%), and 36 strains were ACD positive (58.1%). Conclusion MDR-ABA strains in this study are of high drug resistance, which is closely related to ADC type cephalosporinase.

OBJECTIVE To investigate the qacE△1-sul1 genes in multidrug-resistant Gram-negative bacilli and their significance.METHODS A total of 225 strains of multidrug-resistant Gram-negative bacilli were clinically isolated.The genes of qacE△1-sul1 were analyzed using polymerase chain reaction(PCR).RESULTS From them 120 strains of multidrug-resistant Gram-negative bacilli were found the qacE△1-sul1 genes.The positive rate of qacE△1-sul1 was 53.3%.CONCLUSIONS There are high percentages of qacE△1-sul1 genes in multidrug-resistant Gram-negative bacilli.