Objective To investigate the effects of Acinetobacter baumannii culture supernatants on planktonic cell growth and biofilm formation of Pseudomonas aeruginosa.Methods The standard isolates (ATCC 19606,ATCC 1195) and clinical isolates (AB23,AB39,AB53) of Acinetobacter baumannii were collected and the 6,12,16,24 and 48 hour-cultured supernatants were extracted.The effects of the culture supernatants on the biofilm formation of Pseudomonas aeruginosa PAO1 were detected on the 96-well plate combined with crystal violet staining.Two-fold concentration of LB medium was prepared to eliminate the effects of nutrition consumption of Acinetobacter baumannii during culture on Pseudomonas aeruginosa growth.The active ingredients in the supernatant of Acinetobacter baumannii culture medium were investigated by using the concentrated tube containing protein with relative molecular mass 3 000.Results The most suitable period for Acinetobacter baumannii culture supernatant extraction was between 12 to 24 hours,so the 16 hourcultured supernatant was chosen for next experiments.The 50％ culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC 19606 significantly inhibited the planktonic cell growth of Pseudomonas aeruginosa PAO1,in which the absorbance at 630 nm reduced from(0.688 ± 0.014) and(0.692 ± 0.014) to (0.431 ± 0.023) and (0.428 ± 0.020) respectively (t =16.780,P ＜ 0.05;t =18.500,P ＜ 0.05).The 50％ culture supernatant of Acinetobacter baumannii ATCC 1195 and ATCC 19606 also significantly inhibited the biofilm formation of Pseudomonas aeruginosa PAO1 with decreased absorbance at 570 nm from (2.071 ± 0.068) and (1.986 ±0.023) to (1.639 ± 0.042) and (1.525 ± 0.202) respectively (t =9.358,P ＜ 0.05;t =3.924,P ＜ 0.05).The biofilm inhibitory effect of the protein with relative molecular mass less than 3 000 was obviously observed by reducing amount of biofilm formation from (1.177 ± 0.040) to(1.056 ± 0.030) (t =4.192,P ＜ 0.05),while there was no inhibitory effect of the proteins with relative molecular mass more than 3 000 in the composition.Conclusion Acinetobacter baumannii culture supernatant could effectively inhibit the planktonic cell growth and biofilm formation of Pseudomonas aeruginosa and the relative molecular mass of active ingredients in the culture supernatant may be less than 3 000.

Objective To study the correlation between patellar stability and keen clinical manifestation in adults with Kaschin-Beck disease (KBD).Methods Through a cross-sectional study in September 2016,one hundred and forty-three adult patients with KBD were asked to accept a digital radiographic X-ray (DR) which included positive and lateral slices of knee joint and axis slice of patellar.Meanwhile,every patient must undergo a Lysholm function evaluation for knee joint and morphological measurement on the DR film which included Insall index,lateral patellofemoral angle,sulcus angle,congruence angle,lateral migrating ratio of patella,and patellofemoral index.Then,the regression equation was built and the correlation analysis was made with multiple linear regression test.Results One hundred and forty-three patients' average scores of Insall index,lateral patellofemoral angle,sulcus angle,congruence angle,lateral migrating ratio of patella,patellofemoral index and Lysholm score were 1.10 ± 0.17,(14.49 ± 1.47)°,(138.08 ± 3.86)°,(11.55 ± 2.17)°,(1.34 ± 0.13)％,1.18 ±0.10,and (62.96 ± 6.11) scores.By multiple linear regression test,Insall index (X1),congruence angle (X4),lateral migrating ratio of patella (X5),and patellofemoral index (X6) were selected to enter into the equation;while,lateral patellofemoral angle and sulcus angle were rejected.The multiple linear regression equation was as follows:Y =17.529 + 15.232X5 + 0.950X4 + 15.957X6-4.224X1.The adjusted determination coefficient (R2) of the equation was 0.559.Those indexes which were selected to enter into the equation were ranked from big to small based on the impact on Lysholm as follow:congruence angle,lateral migrating ratio of patella,patellofemoral index,and Insall index.Among them,Lysholm score had a negative correlation with Insall index.Through variance analysis,F valve was 46.642,and P ＜ 0.05,which meant the fitted equation had statistical significance.Conclusion There is significant correlation between the patellar stability and knee Lysholm scoring in adult with KBD,which is reflected with the following parameters from big to small in turn:patellofemoral index,lateral migrating ratio of patella,congruence angle and Insall index.

Objective To investigate the effects of 3 kinds of broth media,including tryptose soya broth(TSB),LB and M-H,on the biofilm formation of Staphylococcus epidermidis (S.epidermidis).Methods The effects of TSB,LB and M-H broth media on the biofilm formation of S.epidermidis were investigated by the construction of bacterial biofilm in 96-well and 6-well microplates and the crystal violet straining for the semi-quantitative analysis and microscopic observation of the bacterial biofilm.The effects of TSB,LB and M-H broth media on the expression of adhesion-related genes in S.epidermidis were determined by the extraction of bacterial total RNA,reverse transcription and real-time PCR.Results Compared with LB (0.149 ± 0.047) and M-H (0.323 ± 0.003) media,TSB medium (2.954 ± 0.287) could significantly promote the biofilm formation of S.epidermidis (TSB vs LB,t =16.706,P ＜ 0.01;TSB vs M-H,t =15.877,P ＜ 0.01).Compared with LB medium,TSB medium could significantly enhance the expression of icaA gene (t =9.667,P＜0.01) but inhibit icaR gene (t =13.283,P＜0.01).Conclusion Compared with LB and M-H broth media,TSB medium may significantly improve the biofilm formation and the expression of adhesion-related gene icaA of S.epidermidis.

Objective To investigate the effect of Lactobacillus delbrueckii fermented supernatant on Candida albicans biofilm and explore its possible mechanism and effective components.Methods Lactobacillus delbrueckii was isolated from vaginal Lactobacillus capsules (Ding Junsheng) and identified by mass spectrometer and sequencing.Lactobacillus delbrueckii fermented supernatant was prepared.The effect of Lactobacillus delbrueckii fermented supernatant on Candida albicans biofilm was tested by microplate crystal violet staining method and catheterization tube modeling method.Biofilm morphological changes were analyzed by crystal violet staining and Live/ Dead fluorescent dyes.Exopolysaccharides of Candida albicans biofilm was measured by the sulphruic acid-phenol method.The effect of Lactobacillus delbrueckii fermented supernatant on the adherence of Candida albicans biofilm was tested by the method of colony counting on plate.The fermented supernatant was treated with NaOH,catalase,typsin and proteinase K respectively.Then the treated fermented supernatant's effect on Candida albicans biofilm formation was detected.One-way ANOVA or Wilcoxon Rank Sum Test was used as statistical analysis for quantitative data.Results Lactobacillus delbrueckii was successful ly isolated and identified from Ding Junsheng.Lactobacillus delbrueckii fermented supernatant could significantly and dose-dependantly inhibited the formation of Candida albicans biofilm(F=10.804,P=0.000).The morphological changes showed that the fermented supernatant could decrease the formation of biofilm.When treated with Lactobacillus delbrueckii fermented supernatant,Candida albicans biofilm expolysaccharides was significantly inhibited (F=17.140,P=0.000) and adherence ability was reduced.Compared to untreated fermented supernatant (A490=0.486±0.112),the biofilm inhibition effect of fermented supernatant treated with NaOH (A490=0.675 ± 0.095),catalase(A490=0.577 ± 0.118),typsin(A490=0.600 ± 0.044) and proteinase K (A490-0.495±0.084)only decreased slightly,but when compared to the control group (A490=1.079 ± 0.158),the treated fermented supernatant still showed significant biofilm inhibition effect (x2=26.052,P=0.000),which suggested that the effective components of fermented supernatant were organic acid and bacteriocin-like substance.Conclusions Lactobacillus delbrueckii fermented supernatant can effectively inhibit Candida albicans biofilm formation.The action mechanisms are related with expolysaccharides decreasement and adherence reduction.The function components of fermented supernatant are possibly organic acids and bacteriocin-like substance.

Objective To investigate the identification of staphylococcus aureus lineage ST59 using the combined detection of delta hemolysin allelic variant G10S(HldG10S) and beta hemolysin(β-toxin). Methods Perspective study.A total of 82 non-duplicate clinical staphylococcus aureus were collected from November 2017 to April 2018 in the department of Clinical laboratory, the Third Xiangya Hospital of Central South University, China.The strains were routinely identified by MALDI-TOF MS and the mass spectra were obtained. According to the m/z expression intensity of delta hemolysin(Hld), all strains were divided into three groups:HldG10S (3036±2.0)m/z, Hld (3006±2.0)m/z and ND [no (3036±2.0)m/z and no (3006±2.0)m/z]. The distribution of ST59 in the three groups was detected by MLST. Reverse synergic hemolysis test was used to determine theβ-toxin phenotype. And the sensitivity, specificity and accuracy of HldG10S,β-toxin and the combined detection of HldG10S and Hld to identify ST59 were compared. Results Among the 82 strains, 21 strains expressed HldG10S toxin, accounting for 25.6％. 39 strains expressed Hld toxin, accounting for 47.6％.22 strains did not express HldG10S and Hld toxin, accounting for 26.8％. In HldG10S group,16 strains were ST59, accounting for 76.19％(16/21).ST59 was not found in both Hld and ND groups. All 16 strains of ST59 in HldG10S group producedβ-toxin, while none of the 5 strains of non-ST59 producedβ-toxin. The specificity(100％) and accuracy(100％) of the combined detection was significantly higher than that of HldG10S andβ-toxin single detection of specificity(92.4％, 77.3％) and accuracy(80.5％, 81.7％) (χ2=19.472, P<0.001;χ2=17.792, P<0.001). Conclusion The combined detection of HldG10S andβ-toxin can preliminarily and rapidly identify ST59, which can assist the routine monitoring of the change trend of staphylococcus aureus epidemic.

Objective:To investigate the effect of small molecule antibacterial agent Halicin against Staphylococcus aureus. Methods:The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of Halicin against S. aureus were detected by the microbroth dilution method. The time-kill assay of Halicin against S. aureus was detected by agar plate dilution method. Micro checkerboard dilution method was used to determine the synergistic antibacterial activity between Halicin and conventional antibiotics. Crystal violet staining method was used to assess the biofilm inhibitory and eradicating activity of Halicin. Hemolysis rate was used to detect the mammal cell toxicity of Halicin. Through the mouse skin abscess model, take the skin tissue around the abscess to grind and dilute the colony to detect the antibacterial effect of Halicin in vivo. Results:Halicin showed significant bacteriostasis effects against S. aureus with the minimum inhibitory concentration of 2-4 mg/L. Halicin could significantly reduce the average CFU counts of S. aureus about 5.5×10 6 CFU/ml in a concentration-dependent manner after 8 h treatment at the concentration of 16 mg/L. The fractional inhibitory concentration value between Halicin and ampicillin was 0.5, showing a synergistic antibacterial efficacy. Halicin effectively inhibited the formation of biofilms at the concentration of 4 × MIC, reducing the total biofilm biomass ( A570) from (2.89±0.09) to (1.35±0.17) ( t=11.12, P<0.05). However, there was no eradication effect against preformed biofilms. In addition, Halicin had almost no hemolytic activity on red blood cells even at the concentration up to 128 mg/L. It showed that 20 mg/kg Halicin reduced bacterial burden about 3.0×10 7 CFU/ml in vivo. Conclusion:Halicin had a strong antimicrobial activity against S. aureus with no hemolytic activity.

BACKGROUND: Postoperative cholangitis is a common but severe complication after Kasai portoenterostomy for biliary atresia (BA). This study aimed to identify its prognostic factors. METHODS: Two sets of liver paraffin-embedded tissue samples were collected from BA patients who received Kasai portoenterostomy (n = 25 and n = 31, respectively). Patients were divided into non-cholangitis and cholangitis groups. The infiltration of CD4+, CD8+, CD45RO+, CD68+ cells and expression of Beclin1 were quantitatively evaluated in immunohistochemical analysis. RESULTS: Cholangitis group had a significantly lower CD8+ T cell infiltration but a higher CD45RO+ cell infiltration, and a lower Beclin1 level than non-cholangitis group (all P < 0.01). Multivariate logistic regression analysis indicated that infiltration of CD8+ cells (odds ratio [OR], 0.112; 95% confidence interval [CI], 0.022–0.577) and CD45RO+ cells (OR, 3.88; 95% CI, 1.37–11.03), and Beclin1 level (OR, 0.088; 95% CI, 0.018–0.452) were independent influence factors for early postoperative cholangitis. Receiver operating characteristic (ROC) analysis showed that area under ROC curve (AUROC) values for CD8+ cells, CD45RO+ cells and Beclin1 were 0.857, 0.738 and 0.900, respectively. CONCLUSION: Our findings demonstrated the CD8+ cells, CD45RO+ cells and Beclin1 level possessed the prognostic value for early postoperative cholangitis following Kasai operation, which may be helpful to develop new prevention and treatment strategies for postoperative cholangitis.
Biliary Atresia* ; Cholangitis* ; Humans ; Liver ; Logistic Models ; ROC Curve ; T-Lymphocytes*

Objective To study the correlation between knee biomechanics and clinical manifestation in adults with Kaschin-Beck disease(KBD). Methods Three hundred and eighty-nine digital radiographic X-ray (DR)anteroposterior keen films of adult KBD patients diagnosed at the Center for Disease Control and Prevention of Gansu Province from Sep 2012 to Sep 2016 were taken as subjects of observation and were retrospectively reviewed. Every patient must undergo a WOMAC function evaluation for knee joint and biomechanic angle measure for the lower limbs on the DR film which included femur angle, tibial angle, femur-tibial angle and joint space angle, Insall-Salvati index (LT/LP), and LT/HI index. Then, the regression equation was built and the correlation analysis was made with multiple linear regression test. Results Three hundred and eighty-nine patients' average scores of femur angle(X1),tibial angle(X2),joint space angle(X3),femur-tibial angle(X4),LT/LP index(X5),LT/HI index (X6) and WOMAC evaluation were (76.32 ± 2.05)°, (110.65 ± 4.68)°, (4.73 ± 0.91)°, (137.56 ± 2.29)°, 0.98 ± 0.12, 1.73 ± 0.11, and (61.35 ± 7.68) scores. By multiple linear regression test, femur angle, tibial angle, femur-tibial angle,LT/LP index, and LT/HI index were selected to enter into the equation; while, joint space angle was rejected. The multiple linear regression equation was as follows: Y = - 77.307 - 6.327X6+ 1.360X1+ 1.310X5+ 0.331X4-0.147X2.The adjusted determination coefficient (R2) of the equation was 0.958. Through variance analysis, the fitted equation had statistical significance (F = 1 790.391, P < 0.05). Through two- tailed significance test, the above-mentioned 5 indexes that were adopted to enter this question were in significant correlation with WOMAC scores(P<0.05).Conclusion There is a significant correlation between knee biomechanic angle and WOMAC scoring in adults with KBD,which is reflected with the following parameters from big to small in turn:femur angle,tibial angle, LT/LP index,femur-tibial angle,LT/HI index,.

Objective:High-throughput screening to obtain small molecular compounds against Gram-negative bacilli by targeting BamA outer membrane protein.Methods:The sybyl-X2.1 software was used to perform high-throughput virtual screening of small molecular compounds in Chemdiv compound library based on the molecular docking. The top 150 hits by high-throughput screening were re-screened through in vitro biological experiments. The top 4 small molecules with obvious antibacterial activity were selected for in-depth molecular docking analysis, and the small molecule 8308-0401 with the highest docking score was selected for further experiments. The antibacterial effect of 8308-0401 combined with rifampicin was tested by checkerboard assay. Finally, the affinity between 8308-0401 and BamA was tested by plasma surface resonance assay. Results:The docking score of the top 150 hits calculated by high-throughput virtual screening had a mean value of 5.63. In vitro biological experiments showed that small molecules 8308-0401, 8365-1335, C066-2507 and L582-0346 exhibited strong antibacterial activity. Among those molecules, 8308-0401 showed the highest molecular docking score, and synergistic antibacterial activity against both types of strains and clinical isolates when combined with rifampicin. 8308-0401 has a strong affinity to BamA with binding a constant of 182 μmol/L. Conclusion:The small molecule 8308-0401 exerts antibacterial activity against Gram negative bacilli by targeting the outer membrane protein BamA.

Anti-Bacterial Agents/pharmacology* ; Biofilms ; Microbial Sensitivity Tests ; Peptides ; Plankton ; Staphylococcus epidermidis