Preliminary study on high throughput screening small molecules targeting Gram-negative bacilli outer membrane protein BamA / 中华检验医学杂志

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.

Inhibitory effects of simeprevir on Staphylococcusepidermidis and itsbiofilm in vitro / 中南大学学报(医学版)

OBJECTIVES@#Staphylococcus epidermidis (S. epidermidis) is a Gram-positive opportunistic pathogen that often causes hospital infections. With the abuse of antibiotics, the resistance of S. epidermidis gradually increases, and drug repurposing has become a research hotspot in the treating of refractory drug-resistant bacterial infections. This study aims to study the antimicrobial and antibiofilm effects of simeprevir, an antiviral hepatitis drug, on S. epidermidis in vitro.@*METHODS@#The micro-dilution assay was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of simeprevir against S. epidermidis. Crystal violet staining assay was used to detect the biofilm inhibitory effect of simeprevir. The antimicrobial activity of simeprevir against S. epidermidis and its biofilm were explored by SYTO9/PI fluorescent staining. The combined effect between simeprevir and gentamycin was assessed by checkerboard assay and was confirmed by time-inhibition assay.@*RESULTS@#Simeprevir showed significant antimicrobial effects against S. epidermidis type strains and clinical isolates with the MIC and MBC at 2-16 μg/mL and 4-32 μg/mL, respectively. The antimicrobial effects of simeprevir were confirmed by SYTO9/PI staining. Simeprevir at MIC could significantly inhibit and break the biofilm on cover slides. Similarly, simeprevir also significantly inhibit the biofilm formation on the surface of urine catheters either in TSB [from (0.700±0.020) to (0.050±0.004)] (t=54.03, P<0.001), or horse serum [from (1.00±0.02) to (0.13±0.01)] (t=82.78, P<0.001). Synergistic antimicrobial effect was found between simeprevir and gentamycin against S. epidermidis with the fractional inhibitory concentration index of 0.5.@*CONCLUSIONS@#Simeprevir shows antimicrobial effect and anti-biofilm activities against S. epidermidis.

Antibacterial effects of small molecule antidiabetic agent Halicin against Staphylococcus aureus / 中华检验医学杂志

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.

Identification of staphylococcus aureus lineage ST59 by the combined detection of delta hemolysin allelic variant G10S and beta hemolysin / 中华检验医学杂志

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.

Effect of Lactobacillus delbrueckii fermented supernatant on Candida albicans biofilm formation / 中华检验医学杂志

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.

Effects of three kinds of liquid media on the biofilm formation of Staphylococcus epidermidis / 临床检验杂志

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.

Effect of Acinetobacter baumannii secretions on Pseudomonas aeruginosa cell growth and biofilm formation / 临床检验杂志

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.