Construction of Δ0948 complementary strain of Streptococcus suis 2 and its effect on hemolysin secretion and virulence / 中华微生物学和免疫学杂志

Objective:To construct Streptococcus suis type 2 Δ0948 complementary strain and verify its effect on suilysin (SLY) secretion and virulence. Methods:The SSU05_0948 gene sequence with promoter was amplified by PCR and ligated to pAT18 vector to construct complementary strain and verify its expression through Western blot. Growth curve was drawn to compare the growth of complementary strain against the wild-type strain and mutant strain in different periods. CD1 mice challenge model was used to verify whether complementary strain could restore the virulence of mutant. SLY hemolytic activity and Western blot were compared the effect of complementary strain and wild-type strain and mutant strain on SLY protein secretion at different time points.Results:The complementary strain was successfully constructed, but the expression of SSU05_0948 was lower than the wild-type strain. The growth rate of the complementary strain was significantly slower than the wild-type strain and mutant strain in the logarithmic growth phase, but the same in the platform phase. The CD1 mice challenge model showed the complementary strain could basically restore the virulence of the mutant strain. The hemolytic activity of SLY and Western blot showed that SSU05_0948 could inhibit the secretion of SLY protein in the early and middle logarithmic phase, but did not affect the secretion of SLY in the late logarithmic and platform phase, while the complementary strain could restore the secretion of SLY protein.Conclusions:The complementary strain CΔ0948 of Streptococcus suis can restore the virulence of mutant strain Δ0948, and SSU05_0948 affects the virulence of Δ0948, which provides a new idea for the prevention and treatment of Streptococcus suis.

Construction of plasmids for differential expression of green fluorescent protein based on different transcription efficiency of IRES / 中华微生物学和免疫学杂志

Objective:To construct four retroviral plasmids for differential expression of green fluorescent protein (GFP) based on different terminal sequences of internal ribosome entry site (IRES) and provide reference for subsequent flow analysis or imaging.Methods:Based on the fact that the transcription efficiency of encephalomyocarditis virus IRES depends on its terminal sequence, IRES and enhanced GFP (EGFP) were fused into four fragments with different connection modes by overlapping PCR, and then cloned into retroviral plasmid pMSCV-NGFR. NGFR fragment was amplified by PCR and inserted in front of the retroviral plasmids pMSCV-IRES(1-4)-EGFP. These retrovirus plasmids pMSCV-NGFR-IRES(1-4)-EGFP were transfected into 293T cells. The expression ratio and mean fluorescence intensity (MFI) of EGFP were analyzed, and the expression of NGFR was also detected.Results:Four retroviral plasmids pMSCV-NGFR-IRES(1-4)-EGFP were successfully constructed. No significant difference in the expression efficiency of EGFP at 24 or 48 h was observed in 293T cells transfected with the four different retroviral plasmids, but there was significant difference in fluorescence intensity. Moreover, the expression of NGFR was not significantly different, indicating that the addition of different nucleotide sequences between IRES and EGFP would make a significant difference in the fluorescence intensity of EGFP.Conclusions:The expression intensity of EGFP was affected by the sequence between IRES and EGFP. Retroviral plasmids expressing EGFP of different intensity could meet different experimental requirements.

Detection of mecA gene and drug-resistance analysis / 中华老年医学杂志

Objective To determine whether coagulase-negative non-epidermal staphylococcus is methicillin-resistant coagulase-negative staphylococcus by mecA gene test,when the minimal inhibitory concentration(MIC)of oxacillin is between 0.5-2.0 mg/L.Methods The mecA gene was detected and analyzed by the cefoxitin disk diffusion,E-test,VITEK-2 Compact and polymerase chain reaction (PCR)purification.Results A total of 300 strains of coagulase-negative staphylococci were screened from 4032 patients(7.4％),of which 45 strains of Staphylococcus saprophyticus and 80 strains of Staphylococcus hemolyticus were identified by Compact VITEK-2.There was a statistically significant difference in the positive rate of mecA gene detection between Staphylococcus saprophyticus and Staphylococcus hemolyticus(P ＜0.05).The results of detection of cefoxitin disk diffusion(inhibitory zone diameter ≥ 25 mm),E-test(MIC of oxacillin between 0.5-2.0 mg/L)and Compact VITEK-2 (MIC of oxacillin between 0.5-2.0 mg/L)showed that there were 81 strains of coagulase-negative non-Staphylococci,of which 10 strains with positive mecA gene were confirmed by PCR.Conclusions When the minimal inhibitory concentration (MIC)of oxacillin against coagulase-negative non-Staphylococci stains is between 0.5-2.0 mg/L,the guidelines of the American clinical laboratory standardization institute(CLSI)should be strictly implemented in clinical microbiology laboratory and the mecA gene must be tested.Based on the wide dissemination of the mecA gene in Staphylococcus aureus population,if the mecA gene test is negative,it is reported as methicillin-susceptible coagulase-negative Staphylococcus(MSCNS),and the reverse result is reported as methicillin-resistant coagulase-negative staphylococcus(MRCNS).

Species distribution and drug-resistance analysis of urinary fungal infections in older adults / 中华老年医学杂志

Objective To analyze the detection rates,species distribution and drug-resistance of urinary fungal infection in elderly patients at Beijing Hospital from 2011 to 2013,in order to provide the basis for the reasonable clinical use of anti-epiphyte medicines.Methods Totally 263 patients with an average of 79.6 years old were collected from Beijing Hospital.The urine from freshly voided midstream or bladder puncture was collected under aseptic condition for fungal culture,then the strains of epiphytes were identified by using API 20C AUX.The drug sensitivity was tested with ATB fungus3.Results 263 strains of epiphytes were isolated from the 2 983 urine samples,of which 92 were C.tropicalis,85 were C.glabrata,77 were Candida albican,and 9 were other fungus candida.The rates of drug resistance to fluconazole were 14.1 ％ (13 strains),37.6 ％ (32 strains) and 15.6％ (12 strains),and to itraconazole were 16.3％(15 strains),35.3％(30 strains) and 9.1％(7 strains),respectively.All of the 263 strains were not found to have drug resistance to amphotericin.Conclusions The isolation rate of urinary fungal infections is 8.8％ in Beijing Hospital.The majority of the tested fungal are C.glabrata,C.tropicalis and Candida albican,the former has higher resistance rate to azoles,and the two latter have better sensitivity to azole,and all of them have the sensitivity to amphotericin.

The molecular characteristics and genetic background of VanB phenotype-van A genotype vancomycin resistant Enterococcus / 中华检验医学杂志

Objective To investigate the structure of Tn1546 like elements,the molecular features and genetic background of VanB phenotype-vanA genotype VREs and to explain the difference between phenotype and genotype. Methods Twenty-one VREs were collected in Beijing Hospital from March 2008 to January 2009. Etest was used to determin MICs of ten antibiotics. PCR product sequencing, PFGE and MLST were performed to study the molecular features and genetic background of the 21 VREs. Results All VREs were vanA genotype, but 3 of them( 14. 3% ) exhibited the VanB phenotype. Based on PFGE analysis, 21 VREs belonged to 9 different patterns. Six STs were identified by MLST analysis. The analysis of the structure of Tn1546 like elements showed the deletion of vanY, vanZ and the insertion of ISEfa4 in orf2-vanR intergenic region may be related to the formation of VanB phenotype-wanA genotype. Conclutions VanB phenotype-vanA genotype VREs were rarely found in China. The results of vanA cluster rearrangements could partly explain the causes of difference between phenotype and genotype.

Antibiotic resistance of clinical isolated bacteria in elderly inpatients in recent 3 years / 中华老年医学杂志

Objective To analyze the antibiotic resistance of clinical isolated bacteria in elderly inpatients in Beijing Hospital from 2006 to 2008.Methods A total of 5710 strains isolated from elderly inpatients received antibiotic sensitivity test (AST) by using K-B method, and the data were analyzed with WHONET 5.4 software.Results During the 3 years, in constituent ratio of bacteria, P.aeruginosa, E.coli and Stenotrophomonas maltophilia were at the top of gram-negative bacteria.And S.aureus, coagulase-negative Staphylococcus (CONS), S.pneumoniae and Enterococcus spp.were at the top of gram-positive bacteria.The results of AST in vitro showed that 19 of 121 strains of S.pneumoniae were penicillin-insensitive S.pneumoniae (PNSP).In 690 strains of S.aureus, the methicillin-resistant S.aureus (MRSA) ratio was 80.2%, and vancomycin-insensitive strains were not found.And 114 strains of Enterococeus faecalis and 95 strains of Enterococcus faecalis were isolated, while the antibiotic resistance of the latter was stronger than the former, and 19 strains were vancomycin-resistant strains.The detection ratios of E.colt producing ESBLs were 41.7%, 55.0% and 56.8%, and the detection ratios of Klebiella pneumonia producing ESBLs were 16.0%, 22.4% and 27.3 %.The antibiotic resistance of ESBL-producing bacteria was much stronger than non-ESBLs producing bacteria.Multi-antibiotic resistant strains of Pseudomonas aeruginosa and Acinetobacter spp.were found.Conclusions It is necessary to detect the drug-resistant strains periodically for understanding the changes in bacterial resistance and providing a theoretical basis for the medication by the clinical experience.

Influence of sulbactam content on susceptibility testing results of cefoperazone/sulbactam combination disks / 中华检验医学杂志

Objective To study the discrepancy influence of the sulbactam content on susceptibility testing results of cefoperazone/sulbactam combination disks.Methods Agar dilution method was used to determine MICs of cefoperazone,cefoperazone/sulbactam(2:1 and 1:1),and disk diffusion was used to detect the zone diameters of cefoperazone,cefoperazone/sulbactam(75/30 and 75/75μg/disk)disks against 534 clinical gram-negative isolates.The discrepancy within the results of MICs,zore diameters,the method of agar dilution and disk diffusion was analyzed.Results By standard agar dilution method,MIC_(50) of cefoperazone,cefoperazone/sulbactam(2:1 and 1:1)were 32,16,16μg/ml,and MIC_(90) of those were ≥256.128,64 μg/ml respectively.No statistic discrepancy was found for MICs between the ratios of 2:1 and 1:1 combination by Wilcoxon ranks test(Z=-0.248,P=0.804).Susceptibility rate,resistance rate,and intermediate rate with 75/30μg disk were 55.3%,24.5%and 20.2%respectively,which were similar to those determined by agar dilution method.Susceptibility rate,resistance rate,and intermediate rate(I%)with 75/75μg disk were 72.5%,12.4% and 15.1% respectively,compared with the susceptibility rate from 75/30μg disk was 17.2% higher.Statistic discrepancy were tested by paired t-test (t=21.613,P＜0.01)with two groups of whole strains' zone diameters from 75/30μg and 75/75μg disks,and resulting in the difference of susceptibility or resistance rates for ESBL-producing strains,Acinetobacter bauamnnii and Enterobacteriaceae without ESBL tested isolates.On the contrary,for Pseudomonas aeruginosa,Stenotrophomonas maltophilia and ESBL negative isolates,the zone diameters discrepancy was not statistically significant between the results from 75/30μg and 75/75μg disks.Conclusions There is no statistic discrepancy between the susceptibility results from cefoperazone/sulbactam(2:1 or 1:1 ratios)in dilution method and in diffusion method with 75/30μg disk.When the 75/75μg disk is used to be tested for ESBL-producing strains,Acinetobacter bauamnnii and other Enterobacteriaceae,the results should be shown with sulbactam content.

Mutant Prevention Concentration of Three Cephalosporins Against Staphylococcus aureus and Streptococcus pneumoniae / 中华医院感染学杂志

OBJECTIVE To determine the mutant prevention concentration(MPC) of 3 cephalosporins against clinical isolates of Staphylococcus aureus and Streptococcus pneumoniae.METHODS Agar dilution was used to determine the minimum inhibitory concentration(MICs) and MPCs of cefaclor,cefprozil and cefuroxime against S.aureus and the MICs to Str.pneumoniae.The MPCs against Str.pneumoniae were determined by mixing-bacterium.RESULTS The MPCs and the ratio of MPC90/MIC90 of cefaclor,cefprozil and cefuroxime against S.aureus were 32,16;16,16;2,8;and the MPCs and the ratio of MPC90/MIC90 against Str.pneumoniae were 8,16;4,32;2,8,respectively.CONCLUSIONS The ability of cefuroxime for restricting the selection of resistant mutant of Str.aureus and S.pneumoniae is stronger than cefaclor and cefprozil.

A rapid quantitative ELISA for measuring D-dimer to exclude pulmonary embolism / 中华检验医学杂志

494 ng/ml. The test showed a sensitivity of 100%, a negative predictive vale of 100%. Conclusion VIDAS D dimer is well sited as diagnostic tool for the exclusion pulmonary embolism.

Drug Resistance of 96 Fungal Strains from Urinary System / 中华医院感染学杂志

OBJECTIVE To study the distribution and the drug resistance of fungus species in hospital in one year,and to provide the evidence for clinical treatment.METHODS The fungus isolations were detected by colored separated-cuture,API 20C AUX,VITEK-60 identification system,and YBC identification card.The drug sensitivity was detected by ATB Fungus 3 INT strip.RESULTS Of the 96 islatens,37.5% were Candida glabrata,30.2% C.albicans and 27.1% were C.tropicalis.All the tested isolates were sensitive to 5-flucytosine(5Fc) and amphotericinB(AMB).CONCLUSIONS The hospital fungal infection of urinary system is caused by C.glabrata,C.albicans and C.tropicalis mostly.The clinical treatment should base on the drug sensitivity results.