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.

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.

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.

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.

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).

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.

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.

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.

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.

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.