Objective To determine the ability of biofilm formation of Staphylococcus ephtermidis isolates and analyze the correlation between the icaA gene and its expression and biofilm formation. Methods Collecting 205 Staphylococcus epidermidis isolates identified with normal laboratory tests (coagulase-negative, biochemical identification, polymyxin-resistant and novobiocin-sensitive ), the suspected isolates were con-formed with API-Staph. Biofilm production was assessed by incubating the strains on Congo Red Agar (CRA) plates and quantitative biofilm production determined by a 96-well tissue culture plate and biofilm morphous were detected by scanning electron microscope ( SEM ) ; Amplifying partial fragments of icaA genes with PCR; Analyzing the expression levels of icaA gene with RT-PCR through Bio-Rad system and Quantity One software. Results 24 isolates showed positive in CRA tests, 22 isolates were positive in semiquantita- tive adhesion assays and 28 isolates existed icaA gene among 205 isolates of Staphylococcus epidermidis. The icaA-positive strains demonstrated biofilm formation (microcolonies on silica films ) while icaA-negative strains only adhered as individual cells under scanning electron microscope. All 22 strains which showed positive in semiquantitative adhesion assays harbored the icaA gene. The expression levels of icaA gene with RT-PCR in 6 Staphylococcus epidermidis isolates showed a higher tendency in 4 strains which demonstrated positive in semiquantitative adhesion assays than 2 negative strains in semiquantitative adhesion assays. Conclusion The isolations of Staphylococcus epidermidis have the abilities of forming biofilm, and the icaA gene and its normal expression is the important molecular biology foundation of biofilm formation. Other fac-tors maybe involve in the expression of icaA gene in Staphylococcus epidermidis isolates.

Objective To investigate the relationships between the expressions of cidA and lrgA and the biofilm formation of Staphylococcus epidermidis clinical isolates. Methods Thirty-nine S. epidermidis clinical isolates were divided into biofilm formation group and non-biofilm formation group according to adhesion assays and icaA amplification, 20 strains for biofilm formation group and 19 strains for another group respectively. Amplify cidA and lrgA fragment with PCR, detect mRNA levels of cidA and lrgA with RT-PCR and then calculate the ratios of cid/lrg mRNA. Results All of the 39 isolates of S. epidermidis existed cidA and lrgA gene.Select 2 strains of S. epidermidis, Y36 and Y26, at random for detecting the cidA and lrgA mRNA levels of different phases, the highest level of cidA mRNA shows on 4 h's culture while lrgA shows on6 h. The relative expression levels of cidA of 39 isolates of S. epidermidis were 0.340 + 0.250 in biofilm formation group and 0. 406 +0. 408( P =0. 541 ) in non-biofilm formation group for 4 h culture, while the relative expression levels of lrgA were 0.325 ± 0.218 and 0.253 ± 0.211 respectively ( P = 0. 299). There were no significant differences between these two groups. The ratios of cid/lrg mRNA were 1.067 ±0.529 and 1.958 ±1.877 respectively, the difference of population distribution was significant( P = 0.001 ). Conclusion The expressions of cidA and lrgA of S. epidermidis clinical isolates may be time-limited. There were no significant differeces of expressions of cidA and lrgA between biofilm formation group and non-biofilm formation group. The ratios of cid/lrg mRNA maybe have some biological significance.

Objective To investigate the diagnostic value of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in critically ill patients complicated with sepsis. Methods Fifty-six cases of systemic inflammatory response syndrome (SIRS) who admitted in intensive care unit (ICU) of the Second Hospital of Tianjin Medical University between May 2009 and June 2010 were recruited. The SIRS patients were divided into two groups: sepsis group (n= 32) and non-sepsis group (n= 24). The levels of procalcitonin (PCT), C-reactive protein (CRP) were measured within 24 hours of hospitalization.Levels of sTREM-1 were measured by enzyme linked immunoabsorbant assay (ELISA). Based on the receiver operating characteristic curve (ROC), cut-off value, sensitivity and specificity of sTREM-1 in diagnosis of sepsis were calculated. Comparison between groups was done by t test and Mann-Whitney U test. Count data were compared by chi square test. ResultsThe serum level of sTREM-1 were significantly higher in sepsis group than that in non-sepsis group (250.9 ng/L vs 103.6 ng/L, Z= 12. 864,P=0. 002). Areas under the curve (AUC) of sTREM-1, PCT and CRP were 0. 935, 0. 891 and 0. 602, respectively. With a cut-off value of 135.0 ng/L, the sensitivity and specificity of sTREM-1 in diagnosing sepsis were 93. 8％ and 84. 7％, respectively. With a cut-off value of 2. 0 μg/L, the sensitivity and specificity of PCT were 84. 4％and 87. 8％, respectively.ConclusionSerum level of sTREM-1 could be used as an early indicator for diagnosing sepsis.

Objective To investigate the relationship between the serum levels of vascular endothelial growth factor(VEGF) and severity and prognosis by evaluating its changes in severe sepsis patients. Method Us-ing control study design, a total of 29 severe sepsis patients who admired in ICU department of Second Hospital of Tianjin Medical University from July 2006 to November 2007 were enrolled. The patients were divided into survival group (n=16) and death group (n=13) according to the clinical outcomes at 28 days after onset.A total of 31 healthy persons were enrolled into the control group. Clinical and laboratory data including blood routine test,blood-gas analysis,blood chemistry,C-reactive protein,lactic acid were collected on the first,third and 7th day after on-set,respectively. APACHE Ⅱ score were calculated. VEGF levels were determined using ELISA method. Quantita-tive data were analyzed by Ftest. Results The VEGFlevels ofthe control groupwere (78.77±8.15) pg/mL, the VEGF levels of the survival group on the first,third and 7th day were (210.47±59.40) pg/mL, (161.79 ±32.58) pg/mL and (85.33±12.13) pg/mL, respectively. The peak value of VEGF levels appeared on the first day, Then,it decreased with the progression of the disease. The VEGF levels did not differ significantly between the control group and survival group on the 7th day (P>0.05). The VEGF levels in the death group on the first, third and 7th day were (324.12±44.35) pg/mL,(185.40±30.92) pg/mLand (273.32±55.23) pg/mL, respectively. The peak value of VEGF levels in the death group also appeared on the first day, but it did not de-crease significandy on the 7 th day as compared that on theist day. The value of VEGF levels on the 7 th day were significantly higher than that of the control group (P<0.01 ). The levels of VEGF were positive correlated with APACHEⅡ score(rs = 0.510,P<0.01), both VEGF levels and APACHEⅡ score were risk factors for the clinical outcomes of these patients. Conclusions The serum levels of VEGF are elevated at early stage in severe sepsis patients. The VEGF levels, which might be a potential prognositic factor for sepsis patients are significantly correlated with APACHE Ⅱ score.

ObjectiveTo determine the ability of biofilm formation of Staphylococcus epidermidis isolates and study the influence of different extracellular DNA(eDNA) levels in S.epidermidis isolates on the ability of biofilm formation.MethodsDetect the biofilm-formation ability of 227 S.epidermidis isolates with adhesion assays,amplify the icaA gene fragment with PCR.The S.epidermidis isolates were divided into biofilm formation(BF) group and non-biofilm formation (NBF) group according to adhesion assays and icaA amplification.Detect eDNA levels of S.epidermidis in planktonic culture and microtitre plate static culture.The eDNA in S.epidermidis biofilms stained by AO-PI was observed by CLSM.Results26 isolates were positive in adhesion assays and 32 isolates existed icaA gene among 227 S.epidermidis isolates.Select 20 isolates with positive adhesion assays and positive icaA amplification for BF group.Select 19 isolates with negative adhesion assays and negative icaA amplification for NBF group.The eDNA levels were (32.2±10.1)μg/ml,(33.6±11.9) μg/ml,(34.3±10.0) μg/ml in BF group when cultured in planktonic condition for 2,4,6 h,while the eDNA levels in NBF group were (28.7±8.9) μg/ml,(31.5±11.7) μg/ml,(31.8±12.7) μg/ml respectively.There were no significant differences between the two groups for these three phases(P＞0.05),though the eDNA levels of BF group were higher than that of NBF group.The eDNA levels were (740.0±264.4) ng/A600 in BF group when cultured in static microtitre plate,higher than that of NBF group,(80.1 ±31.1) ng/A600,and the difference between these two groups was significant.The eDNA in BF isolate Y36 biofilms could be visualized by staining with AO and PI when observed by CLSM,while neither biofilm structure nor eDNA appeard when NBF isolate Y26 was cultured for 24 h.ConclusionS.epidermidis isolates have the ability of biofilm formation.eDNA is one of the important matrix components in the S.epidermidis biofilm-forming process.The eDNA of static culture in microtitre plate was more efficient than planktonic culture in the case of estimating the ability of biofilm formation of S.epidermidis.

Objective To study the Genotype of local clinical isolates of Enterobacteriaceae producing extended spectrum ? lactamases. Methods K B test, conjugative transfer test, plasmid profile analysis, PCR, PCR RFLP, and DNA sequencing were used to detect the phenotype and genotype of 33 isolates of Enterobacteriaceae producing ESBLs. Results Eighty five percent of 33 isolates was resistant to cefotaxime which was obviously higher than that to ceftazidime. blaCTX M ESBLs was detected in 28 isolates by PCR, blaTEM DNA in 24 isolates, and blaSHV DNA in 9 isolates. Mutation of E104K was only identified in one TEM ? lactamase produced by EC98A7 by PCR RFLP. No substitution of G238S occurred in 9 SHV ? lactamases. DNA sequencing and DNA alignment showed the blaCTX M DNA fragments from 4 clinical isolates of EC98A7,EB56,CFR78 and, KP9941 belonged to CTX M type 1, with highest identity to blaCTX M 3 or blaCTX M 12 respectively. Conclusions CTX M ESBLs is carried in 85% isolates of Enterobacteriaceae producing ESBLs in this city. Most of the isolates carry 2 or more ? lactamases. E. coli EC98A7 produces two ESBLs, a TEM ESBL and a CTX M ESBL.

Objective To study the state, feature and risk factors of bacterial infection in patients with liver cirrhosis, find out the influence of infection on prognosis, and provide scientific basis for its prevention and treatment. Methods Three hundred and twenty-three patients with liver cirrhosis were analyzed. The number of the patients with infection, the location of infection, clinical feature as well as the kind of pathogenic bacteria were analyzed. Unconditional Logistic regression analysis was used to assess the risk factors of bacterial infection. Results The overall infection rate was 39.94% (129/323),of which community acquired infection rate and nosocomial infection rate were 22.60% (73/323) and 17.34%(56/323) respectively. The most common location of infection in turn were respiratory tract,gastrointestinal tract, urinary tract,biliary tract and abdominal cavity. The main pathogenic bacteria was Gram-negative bacillus, most of which had drug resistance for cefquinome and quinolones. The risk factors related with bacterial infection included liver cancer, Child-Pugh class B and C grade of liver function, gastrointestinal tract bleeding, diabetes mellitus,invasive operations and the length of staying in hospital. Conclusions The incidence rate of infection in patients with liver cirrhosis is higher. Multiple factors are likely to affect the incidence rate of infection in patients with liver cirrhosis.

Objective To study the distribution of CTX-M-ESBLs and class Ⅰ integron in enterobacter cloacae resistant to third generation cephalosporins,and discuss the relationship between CTX-M-ESBLs and class Ⅰ integron. Methods K-B test for resistance;DDST and PCR for CTX-M-(ESBLs);PCR,nested-PCR and sequence analysis for finding class Ⅰ integron carrying CTX-M-ESBLs gene cassette. Results Among 37 strains,there are 21 isolates producing CTX-M-ESBLs,20 isolates with class Ⅰ integron and 13 isolates carrying CTX-M-ESBLs as well as class Ⅰ integron.In 3 isolates: CH4,CH11 and Q1,each class Ⅰ integron haboured CTX-M-ESBLs gene cassette. Conclusions There is few report on class Ⅰ integron carrying CTX-M-ESBLs,which caused more dangers of horizontal transmission of ESBLs and was important factor to nosocomial outbreak of multiresistant clinical isolates.

Objective To study the antibiotic resistance mediated by the mar mechanism in clinical isolated Escherichia coli. Methods To analyse the antibiotic resistance pattern and the out membrane protein spectrum of clinical isolated E. coli . The plasmid pNJR3 2 carrying the wild type gyr A gene was transferred into clinical multiple resistant strain LC 1. The resistant pattern and plasmid pattern of LC 1 before and after plasmid transferring were compared. The mar OR of clinical strain LC 1 was cloned and sequenced through PCR. The nucleotides of mar gene were compared with the wild type issued by GenBank. Results Of all the 49 clinical strains , six strains express Mrp5, a mar specific out membrane protein. No changes were detected on susceptibility to fluoroquinolone of clinical strain LC 1 after pNJR3 2 transferred suggesting that gyr A was not the main reason contributing to fluoroquinolone resistance. Sequence analysis showed three mutant spots in mar OR of clinical strain LC 1. Conclusion There does exist mar mechanism in clinical isolated Escherichia coli resistant to fluoroquinolone. The mutation in mar OR may contribute to the mar phenotype of LC 1.

OBJECTIVE To investigate the repressor SmeT and other sequences of efflux pump SmeDEF in the clinic isolates of Stenotrophomonas maltophilia to involve in antibiotic resistance. METHODS The mRNA level of smeD examined the resistance profile of the clinical strains to antimicrobials was by agar dilution and their reaction to pump inhibitor was investigated by RT-PCR,and the smeD-smeT fragment was amplified by PCR,and then sequenced.Adding poly(A) to 3′ end of RNA,applying 3′RACE,nested PCR,then sequence-analysis. RESULTS The amino acid sequences of the forepart of SmeT were conservative in all the 7 strains.In antibiotic-resistant strains with positive reaction to pump inhibitor,the intergenic sequences of smeD-smeT were different from those in sensitive ones.The variations in(-165 to-82)nt of smeT and several nucleic acids before the start codon of smeT,possibly involved in antimicrobial resistance.All isolates didn′t have Leu166Gln mutation within the SmeT protein of D457R,which was reported possibly associated with antibiotic resistance.The resistant strains with efflux positively inhibited had Asp218Glu substitution,different from the negatively-inhibited ones. CONCLUSIONS The expression level of SmeDEF is related with the antibiotic resistance of S.maltophilia.The possible resistance mechanism includes the variations in the intergenic smeD-smeT and/or smeT codon sequence.