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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.
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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.
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Objective:To study the molecular mechanism of β-1, 4-galactosyltransferase 6 (β4galt6) in regulating lymphocyte migration under inflammatory conditions.Methods:CRISPR/Cas9 system was used to knock out the β4galt6 gene of mouse islet vascular endothelial cells (MS1). Adhesion assay was performed to compare the adhesion ability of lymphocytes to wild-type cells and gene knockout cells. Expression of adhesion molecules on the surface of wild-type cells and gene knockout cells were compared using RT-PCR and flow cytometry. Transwell model was used to compare the transmigration ability of lymphocytes across wild-type cells and gene knockout cells.Results:The β4galt6 gene knockout cell line, β4galt6 KO, was successfully constructed. The percentage of lymphocytes adhereing to wild-type MS1 cells was significantly higher than that to β4galt6 KO cells under inflammatory conditions. The expression of adhesion molecules including intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1) and P-selectin on the surface of wild-type MS1 cells was significantly higher than that on β4galt6 KO cells. Moreover, the percentage of lymphocytes passing through wild-type MS1 cells was significantly higher than that through β4galt6 KO cells.Conclusion:Under inflammatory conditions, β4galt6 could promote the migration of lymphocytes.
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Objective:To study the function of gene 0267 of Streptococcus suis type 2 98HAH33. Methods:Growth rates of the wild type strain Streptococcus suis type 2 98HAH33, the mutant stain Δ0267 and the complemented strain CΔ0267 at different stages were compared. Bacterial adhesion, whole blood killing and macrophage phagocytosis assays were used to compare the adhesion and anti-phagocytosis of these strains to host cells. A piglet model was used to evaluate their differences in virulence. Results:The growth rates of the wild type strain, the mutant strain and the complemented strain were the same. The adhesion rates of the wild type strain and the complemented strain to A549 cells, Hep2 cells and human brain microvascular endothelial cells (HBMEC) were 2.59, 4.87 and 3.08 times, and 2.65, 4.65 and 2.86 times higher than those of the mutant strain, respectively. The survival rates at 1 h, 2 h and 3 h of the wild type strain, the mutant strain and the complemented strain in whole blood were 36.91%, 28.12% and 41.61%, 58.44%, 44.06% and 58.26%, and 93.02%, 70.08% and 95.85%, respectively. Results of the phagocytosis assay showed that the bacterial loads of the wild type strain, the mutant strain and the complemented strain in murine RAW264.7 macrophages were 2 767, 5 322 and 2 567, respectively. The competitive infection experiment using piglets showed that gene 0267 was associated with the virulence of Streptococcus suis type 2 98HAH33. Conclusions:Streptococcus suis type 2 98HAH33 gene 0267 is associated with bacterial adhesion and anti-phagocytosis, suggesting that it is a new virulence factor.
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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).
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Objective@#To construct the mutant strain ATP binding cassette transporter SSU05_0948 of Streptococcus suis type 2 and comprehensively study its pathogenicity, and to provide useful insights for understanding the mechanism that Streptococcus suis avoid host innate immunity. @*Methods@#The mutant strain 05ZYH33Δ0948 was constructed through homologous recombination technology. The differences between the mutant strain and the wild type strain were evaluated through bacterial adhesion, whole blood killing, mice meningitis assay, mice and piglets virulence assay. Chi-square test and t test were used. @*Results@#Successfully constructed the mutant strain 05ZYH33Δ0948. The adhesion results showed that the adhesion rate (0.663±0.047)% of the wild strain to A549 cell was significantly higher than that of the mutant (0.246±0.074)%, the difference was statistically significant (χ2=5.267, P=0.014); the adhesion rate (16.540±2.320)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.970±0.320)%, the difference was statistically significant (χ2=0.014, P<0.01); the adhesion rate (5.497±0.174)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.950±0.335)%, the difference was statistically significant (χ2=0.016, P<0.01). The killing rate (32.970±3.589)% of the wild strain in whole blood is no difference with the mutant (29.560±3.737)% (χ2=1.200, P=0.133). Piglets competitive infection showed that, the competitive index at 12 h, 24 h and 36 h were 0.046±0.003, 0.107±0.003, 0.064±0.001, respectively. 12 h and 24 h was significant differences(t=15.490, P=0.041), 24 h and 36 h was significant differences(t=5.660, P=0.047), 12 h and 36 h was no differences(t=1.445, P=0.285). @*Conclusions@#Streptococcus suis type 2 ABC transporter SSU05_0948 is a new adhesion factor and virulence factor of Streptococcus suistype 2, and also a new meningitis factor, which plays important roles in Streptococcus suis against host innate immunity.
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Objective To study the function of gene 0955 in Streptococcus suis type 2 98HAH33. Methods Growth condition of wild-type, mutant and complemented strains of Streptococcus suis type 2 98HAH33 was compared at different stages. Differences in adhesion ability to host cells and anti-phagocyto-sis among these strains were compared by using bacterial adhesion test and analyzing their survival rates in blood. Mouse and piglet models were used to evaluate their virulence. Results The growth of the mutant and the complemented strains was slightly slower than that of the wild type strains in logarithmic growth phase, but no significant difference was found in plateau phase. Bacterial adhesion test showed that gene 0955 might encode a new adhesion factor of Streptococcus suis type 2 98HAH33. Blood bactericidal test sug-gested that gene 0955 was not associated with anti-phygocytosis. Animal experiments showed that gene 0955 might be a novel virulence gene of Streptococcus suis type 2 98HAH33. Conclusion Gene 0955 might en-code a novel adhesion factor and virulence factor of Streptococcus suis type 2 98HAH33.
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Objective To study the mechanism of platelet aggregation induced by Streptococcus suis serotype 2 muramidase-released protein (MRP) and to provide scientific proof and theoretical basis for clinical treatment of patients with Streptococcus suis infection. Methods Nickel column affinity chromatogra-phy was used to purify recombinant proteins of MRP-N and MRP-C. Platelet aggregometer, thromboelastog-raphy (TEG) and scanning electron microscope were used to observe the platelet aggregation induced by MRP. Results Streptococcus suis 2 wild type strain,but not the mutant strain ΔMRP,could induce platelet aggregation. It was MRP-N but not MRP-C that induced platelet aggregation. GPRP,an inhibitor of β2inte-grin receptor,could significantly inhibit the platelet aggregation induced by MRP. Conclusion Streptococ-cus suis 2 MRP induces platelet aggregation through β2integrin receptor pathway.
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Objective To study the main types and their distribution of molecular typing of carbapenem-resistant Acinetobacter baumannii (CRAB)in Beijing.Methods Seventy-eight non-repeated CRAB strains were isolated and collected from patients aged over 60 years in hospitals in Beijing from 2010 to 2014.The drug susceptibilities of 11 antimicrobial agents were tested by microdilution method.A modified Hodge assay was used to preliminarily screen the carbapenemases.A multiplex PCR assay was used for detection of the carbapenemases genes:OXA-23-like,OXA-24-like,OXA-51-like,OXA-58-like,IMP-1,and VIM-2 of Acinetobacter baumannii.We also detected the insertion sequence IsAbal of OXA-23-like and OXA-51-like,and the preliminary classification of homology of carbapenem-resistant Acinetobacter baumannii (CRAB)was conducted by 3LST technique.According to the drug susceptibility,carbapenemases gene typing,3LST primary screen,and hospital distribution,22 strains were selected to conduct MLST classification.Results The test of OXA enzyme gene in 78 strains of CRAB showed that all the strains(100％)carried oxa-51-like,and 74/78 strains(94.8％)carried oxa-23-like.Additionally,all products of ISAbal-oxa-23 were positive in OXA-23-like positive strains.In the examined five β-lactamase genes,74 strains(94.8％)showed positive AmpC gene;50 strains (64.1％) showed positive TEM-1 gene;5 strains (6.4％) showed positive PER-1 gene;48 strains(61.5％)showed positive genes of TEM-1 and AmpC and 3 strains showed positive genes of TEM-1,Amp C,and PER-1.By using 3LST technique for preliminary classification,we found 74 strains were in type Ⅰ of group types belonging to the European Ⅱ cloning spectrum.Of the six ST types found in MLST classification,the ST195,ST208,ST218 and ST368 belonged to the clone complex 92(CC92);the ST103 and ST500 were two newly discovered types in Chinese population.Conclusions CC92 clone cluster is the major epidemic strain of CRAB in Beijing,which belongs to the classic European Ⅱ cloning spectrum,and its insertion sequence,Abalinduced OXA-23-like carbapenemases is the major molecular of carbapenem resistant Acinetobacter baumannii(CRAB) in Beijing.Both AmpC and TEM-type 1 β-lactamase genes are detected to be positive in most of strains.The newly discovered two types are mainly CC103 clone complex.
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Objective To study the mechanism of carboxypeptidase E ( CPE ) in promoting the migration of lymphocytes and their subsets through vascular endothelial cells. Methods CRISPR/Cas9 technology was used to prepare cpe gene-knockout MS1 (Cpe-/-MS1) cells. Adhesion ability of lymphocytes to MS1 and Cpe-/-MS1 cells was analyzed with adhesion assay. Expression of adhesion molecules on these cells were detected by RT-PCR and flow cytometry. Transwell model was used to compare the difference in the transmigration of lymphocytes and their subsets through MS1 and Cpe-/-MS1 cells. Results Cpe-/-MS1 cells were successfully obtained. Under the stimulation of TNF-α, the adhesion ability of lymphocytes to MS1 cells was much better than that of Cpe-/-MS1 cells. Moreover, adhesion molecules expressed on MS1 cells were significantly more than those on Cpe-/-MS1 cells. The percentages of lymphocytes and their sub-sets that transmigrated through MS1 cells were significantly higher than those through Cpe-/-MS1 cells. Con-clusion CPE involved in the adhesion of lymphocytes to vascular endothelial cells and the transmigration of them through vascular endothelial cells, which was of great significance for understanding the migration of lymphocytes across vascular endothelial cells to peripheral lymph nodes.
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Objective To construct a mutant strain of Streptococcus suis type 2 05ZYH33 express-ing ABC transporter SSU05 0946 and to study the pathogenicity of ABC transporter SSU05 0946 for better understanding the immune evasion strategies by Streptococcus suis. Methods Genome of the Streptococcus suis type 2 05ZYH33 strain was extracted and used as a template to amplify SSU05 0946 upstream and down-stream homeodomains. Chloramphenicol-resistance gene was amplified by using pSET1 plasmid as the tem-plate. These three amplified fragments were fused and integrated with the thermo-sensitive plasmid pSET4s by using overlap extension PCR. Homologous recombination method was used to construct the mutant strain 05ZYH33Δ0946. Differences between the mutant and wild type strains were evaluated through bacterial ad-hesion assay,whole blood killing assay and challenge test in mice and piglets. Results The mutant strain 05ZYH33Δ0946 was successfully constructed. Results of bacterial adhesion assay demonstrated that SSU05 0946 was not involved in the adherence of Streptococcus suis to human epithelial cells. SSU05 0946 was an ovel anti-phagocytic factor and virulence factor of Streptococcus suis. Conclusion Streptococcus suis type 2 ABC transporter SSU05 0946 is a newly discovered virulence factor of Streptococcus suis, playing an impor-tant role in the evasion of host innate immunity by Streptococcus suis.
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Objective To explore the effects of willed movement on neurological performance and the extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) pathway in rats following focal cerebral ischemia.Methods Reversible middle cerebral artery occlusion was induced in 144 male SpragueDawley rats using intraluminal sutures,and they were randomly divided into a control group,a swimming exercise group,an environment modification group,and a willed movement group.The observation time points were at 7,15 and 30 days after reperfusion.A behavioral test was performed to evaluate any neurological deficiency.Reverse transcription PCR (RT-PCR) and immunofluorescence were used to detect the ERK and CREB responses in terms of mRNA and phosphorylated ERK (pERK) and phosphorylated CREB (pCREB) protein in the peri-ischemic brain tissue.Results The climbing frequency of the willed movement group was significantly higher than that of the environment modification group.Three days after reperfusion the neurological deficit scores of all groups began to decrease,and that of the willed movement group had decreased significantly more than in the other three groups at all time points.ERK/CREB mRNA and pERK and pCREB protein expression were dramatically up-regulated in the willed movement group at 7,15 and 30 days after reperfusion,significantly more than in the other three groups.Conclusions Willed movement may promote motor recovery by up-regulating and activating the ERK/CREB pathway following focal cerebral ischemia.
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Objective To compare and evaluate two type-specific primers PCR genotyping methods of hepatitis B virus ( HBV) which were established by Naito et al ( Naito method) and our lab (new method). Methods The two genotyping methods were applied for detecting the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2 and the plasmids mixed with different proportion of subgenotypes B1 and C2. In addition, the genotypes of 113 serum samples of patients with chronic HBV infection from Shenzhen, Handan and Urumqi cities of China were identified by the two methods, respectively. The results were verified by PCR product based sequencing. Results The sensitivity of the two methods showed no difference when they were applied to detect the plasmids containing the HBV genomes of genotype A or D or subgenotype B1 or C2. While detecting the plasmids mixed with different proportion of subgenotypes B1 and C2, the sensitivity of the new method was superior than that of Naito method. Meanwhile, the specificity of the new method was obviously superior than that of Naito method. Both of the two methods were highly sensitive in identification of HBV genotypes of serum samples with a single genotype. However, the new method showed more sensitive in identification of the B/C mix strains than that of Naito method. The total coincidence rate between the two methods was 83. 2% (94/113), with the discrepancy of 16. 8% (19/113). Fifteen of the 19 inconsistent genotypes by the two methods were selected and their PCR products were sequenced directly. The sequencing results were identical with that of the new methods, but not with that of the Naito method. Conclusion The new method is more sensitive, and its specificity is superior to the Naito method. It could be used for clinical and epidemiological studies on HBV genotype and subgenotype in China.
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OBJECTIVE To investigate the prevalence,antibiotic resistance and genotype of the extended-spectrum ?-lactamases(ESBLs)-producing clinical isolates of Klebsiella pneumoniae.METHODS A total of 104 isolates of K.pneumoniae were examined for the ESBLs production and the susceptibilities of the bacteria to 15 antimicrobial agents.PCR was performed to detect the genes encoding the ESBLs belonging to SHV and TEM families as well as CTX-M-1 and CTX-M-9 groups.RESULTS The ESBLs-producers of K.pneumoniae were 54.0% in the total of 104 isolates.Almost all of the ESBLs-producing isolates were resistant to the antibiotics commonly used,and only remained susceptible to carbapenems and the combination of cefoperazone with sulbactam.The genes of SHV,CTX-M-1 and TEM groups were detected in the ESBLs-producing isolates by 64.3%,46.4%,and 32.1%,respectively,and 35.7% and 8.9% of ESBLs-producing K.pneumoniae strains carried two and three genes.CONCLUSIONS The clinical isolates of K.pneumoniae in Tianjin Nankai Hospital are shown a high rate of ESBLs-producing and antibiotic resistance.SHV and CTX-M-1 groups of ESBLs are the dominant genotypes in the isolates of ESBLs-producing K.pneumoniae.