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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.
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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.
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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.