OBJECTIVE To investigate the produce of extended-spectrum ?-lactamases(ESBLs) and the presence of genotype of the ?-lactamases-encoding genes in Klebsiella pneumoniae isolated from the 98th Hospital of PLA,Huzhou,Zhejiang Province,China.METHODS Twenty-five strains of K.pneumoniae were isolated from the inpatients between Sep 2005 and Apr 2006.ESBLs were tested by phenotypic confirmatory tests recommended by CLSI.Twenty-one kinds of ?-lactamases genes of blaTEM,blaSHV,blaLEN,blaOKP,blaCTX-M-1 group,blaCTX-M-2 group,blaCTX-M-9 group,blaOXA-1 group,blaOXA-2 group,blaOXA-10 group,blCARB,blaPER,blaVEB,blaGES,blaLAP,blaDHA,blaACT/MIR,blaCMY/MOX,blaFOX,blaCMY/LAT,and blaACC were analyzed by PCR and verified by DNA sequencing.RESULTS In 25 strains of K.pneumoniae,the positive,negative,and "uncertainty" rates of ESBLs were 56.0%,20.0%,and 24.0%,respectively.The positive rate of genes of blaTEM,blaSHV,blaCTX-M-1 group,blaOXA-10 group,blaLAP,and blaDHA were 80.0%,4.0%,4.0%,80.0%,4.0% and 32.0%,respectively.The 15 kinds of rest genes were all tested negative.The total positive rate of 21 kinds of ?-lactamases gene was 92.0%.Among them,the blaLAP-2 gene sequence of the HZ12593 strain has been registered in GenBank(GenBank Accession Number: EU529981).CONCLUSIONS There are higher rate of ESBLs-producing strains in K.pneumoniae isolated from the inpatients,and at least 6 kinds of ?-lactamases gene existed.Both genes of blaTEM and blaOXA-10 group are the most common genotypes.Carring blaDHA Gene may influence the result of phenotypic confirmatory test for ESBLs in K.pneumoniae.

Objective To explore the correlations of intracranial pressure (ICP) with changes of neuron specific enolase (NSE),D-Dimer (D-D) and C-reactive protein (CRP) levels in patients with severe traumatic brain injury.Methods A serial of 35 patients with severe traumatic brain injury,admitted to our hospital from January 2012 to January 2014,were chosen as experimental group,and 20 healthy subjects performed physical examination in our Physical Examination Center at the same period were as controls.ICP monitoring was performed in these 35 patients.The patents were divided into two groups according to ICP:severely elevated ICP group (＞40 mmHg) and moderately elevated ICP group (20-40 mmHg).The NSE,D-D and CRP levels were measured,and these data were compared with those from the control group.The correlations of ICP with changes of NSE,D-D and CRP levels were analyzed.Results The levels of NSE,D-D and CRP in the severely elevated ICP group and moderately elevated ICP group were obviously higher than those in the control group ([12.11 ±2.35] lg/L,[0.39±0.61] mg/L,[3.72±0.69] mg/L) (P＜0.05).The levels ofNSE,D-D and CRP in the severely elevated ICP group ([104.08±7.90] μg/L,[1.55±0.26] mg/L,[47.66±8.60] mg/L) were also obviously higher than those in the moderately elevated ICP group ([61.89±30.35] μg/L,[0.93±0.32] mg/L,[30.87±9.84] mg/L)(P＜0.05).Significant positive correlations were noted between ICP and changes ofNSE,D-D and CRP levels in the patient group (regression equation:ICP=18.598+0.256 NSE [t=7.200,P=0.000],ICP=10.779+23.955D-D [t=10.29,P=0.000],ICP=9.932+0.771 CRP [t=8.423,P=0.000]).Multivariant stepwise regression analysis indicated the closest correlation between ICP and D-D (multiple correlation coefficient=0.873,coefficient of determination=0.762,F=105.917,P=0.000).Conclusions Significant positive correlations can be noted between ICP and changes of NSE,D-D and CRP levels,and the closest correlation is between ICP and D-D in patients with severe traumatic brain injury.The combined application of ICP and NSE,D-D and CRP levels can promote the diagnosis and treatment of severe traumatic brain injury patients.

Objective To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33. Methods The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into ΔopaR and ΔaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-ΔaphA and C-ΔopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, ΔopaR, ΔaphA, C-ΔaphA, and C-ΔopaR. Results opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains. Conclusion A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.

Objective To establish a method for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33. Methods The entire coding region of opaR or aphA was amplified by PCR and cloned into pBAD33. The recombinant plasmid was transformed into ΔopaR and ΔaphA (the opaR or aphA null mutant strain, respectively) separately to construct the complemented mutant strain C-ΔaphA and C-ΔopaR, respectively. RT-PCR was used to verify the transcription of opaR and aphA in the corresponding complemented mutant strains. Primer extension experiments were performed to determine the relative mRNA levels of mfpA (a gene previously characterized to be negatively regulated by AphA and positively by OpaR) in the wild-type strain, ΔopaR, ΔaphA, C-ΔaphA, and C-ΔopaR. Results opaR and aphA were transcribed in the corresponding complemented mutant strains, and their mRNA levels were comparable to those detected in the wild-type strains. Conclusion A method has been established for gene complementation in Vibrio parahaemolyticus using the plasmid pBAD33.