Conditions for nisin adsorption by Streptococcus lactis cells

Some conditions for absorption of nisin, a polypeptide antibiotic by the cells of Str. lactis were studied. The amounts of nisin adsorbed by the cells depended on the culture age: at the late stationary phase the adsorption level was 2 times higher than that at the logarithmic phase. The cells grown on a "poor" medium adsorbed 85-90 per cent of nisin added to the solution, while the cells grown on the "rich" medium adsorbed 50 per cent of the antibiotic. The adsorption level of nisin by the cells subjected to a thermal shock was higher than that by the live cells. Desorption of nisin from the cells with acid ethanol and bivalent cation solutions was insignificant. Nisin is adsorbed by the cells of other microorganisms, the adsorption levels by the cells of Bac. brevis being the same as those by the streptococcal cells, while the levels adsorbed by Bac. polymyxa being 4 times lower.     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。     

香芹酚抑制金黄色葡萄球菌生物被膜的形成

【背景】生物被膜是细菌的一种自我保护形式,可以增强细菌对药物及宿主免疫应答的抵抗力,引起细菌耐药性和持续性感染。【目的】探究香芹酚对金黄色葡萄球菌生物被膜的作用机制,为开发新型抗生物被膜药物提供可靠的理论依据。【方法】通过结晶紫染色法检测香芹酚对供试菌株生物被膜形成的抑制和对成熟生物被膜的清除作用;使用刚果红平板法探究香芹酚对供试菌株生物被膜形成过程中细胞间多糖黏附素(polysaccharide intercellular adhesion,PIA)合成的作用;通过分光光度法检测香芹酚对胞外DNA (extracellular DNA,eDNA)分泌的抑制作用;利用RT-PCR技术检测香芹酚对供试菌株的生物被膜相关基因icaA、cidA和sarA转录水平的影响。【结果】香芹酚对生物被膜形成的抑制和生物被膜的清除均有较强作用效果。256μg/mL香芹酚抑制PIA合成和e DNA释放的效果显著。香芹酚可通过抑制相关基因转录从而抑制生物被膜的形成,当64μg/mL的香芹酚作用后,sarA的转录水平降低了60.44%±2.91%,cidA的转录水平降低了76.48%±1.67%,icaA的转...     

Proton motive force in growing Streptococcus lactis and Staphylococcus aureus cells under aerobic and anaerobic conditions.

Measurements of the electrochemical gradient of hydrogen ions, which gives rise to the proton motive force (PMF), were carried out with growing Streptococcus lactis and Staphylococcus aureus cells. The facultative anaerobe was chosen in order to compare the PMF of cells growing aerobically and anaerobically. It was expected that during aerobic growth the cells would have a higher PMF than during anaerobic growth, because the H+-translocating ATPase (BF0F1) operates in the direction of H+ influx and ATP synthesis during respiration, whereas under anaerobic conditions the BF0F1 hydrolyzes glycolytically generated ATP and establishes the proton gradient by extruding H+. The electrical component of the PMF, delta psi, and the chemical gradient of H+, delta pH, were measured with radiolabeled tetraphenylphosphonium and benzoate ions. In both S. lactis and S. aureus cells, the PMF was constant during the exponential phase of batch growth and decreased in the stationary phase. In both species of bacteria, the exponential-phase PMF was not affected by varying the growth rate by adding different sugars to the medium. The relative contributions of delta psi and delta pH to the PMF, however, depended on the pH of the medium. The internal pH of S. aureus was constant at pH 7.4 to 7.6 under all conditions of growth tested. Under aerobic conditions, the delta psi of exponential phase S. aureus remained fairly constant at 160 to 170 mV. Thus, the PMF was 250 to 270 mV in cells growing aerobically in media at pH 6 and progressively lower in media of higher pH, reaching 195 to 205 mV at pH 7. Under anaerobic conditions, the delta psi ranged from 100 to 120 mV in cells at pH 6.3 to 7, resulting in a PMF of 150 to 140 mV. Thus, the mode of energy metabolism (i.e., respiration versus fermentation) and the pH of the medium are the two important factors influencing the PMF of these gram-positive cells during growth.     

Effect of folinate on thymidine uptake by Pediococcus cerevisiae

Uptake of (3)H-thymidine by resting cells of Pediococcus cerevisiae was found to be energy- and temperature-dependent. The pH optimum was between 6.5 and 8.0, and after 2 min of incubation most of the radioactivity was found in the deoxyribonucleic acid (DNA) fraction. Iodoacetate at a concentration of 10(-2)m caused a 50% inhibition of uptake. Preincubation of resting cells for 10 min with folinate (10(-3)mu mole/ml) diminished the (3)H-thymidine uptake by 75%. In growing cells, the folinate-induced inhibition was still more striking. Deoxyuridine augmented the folinate effect, whereas fluorodeoxyuridine and aminopterin or amethopterin abolished it. Preincubation with folinate did not interfere with the uptake of (3)H-amethopterin, and thus the inhibitor did not compete for uptake sites within the cell. The role of these inhibitors in reversing the folinate effect is discussed. Cells preincubated with folinate showed an increased incorporation of (14)C-uracil into DNA, presumably after prior conversion to thymidylate. We concluded that the folinate effect was due to stimulation of de novo thymidylate synthesis with concomitant inhibition of the uptake of external thymidine.     

冬凌草甲素对金黄色葡萄球菌生物膜的抑制机制

【背景】金黄色葡萄球菌是一种常见的食源性致病菌,易在食品及加工器具表面形成生物膜,引起食品腐败和疾病的传播,威胁食品安全。【目的】研究冬凌草甲素抑制金黄色葡萄球菌生物膜形成的作用机制。【方法】使用结晶紫染色法和扫描电镜观察冬凌草甲素对金黄色葡萄球菌生物膜形成的抑制作用,刚果红平板法定性检测冬凌草甲素对细胞间多糖黏附素(polysaccharideintercellular adhesion,PIA)合成的影响,分光光度法测定冬凌草甲素对供试菌株胞外DNA (eDNA)释放量的影响,RT-PCR技术检测冬凌草甲素对供试菌株ica A、cid A、agr A和sar A基因表达量的影响。【结果】冬凌草甲素对金黄色葡萄球菌生物膜形成有较强的抑制作用;冬凌草甲素能显著抑制PIA的合成,且呈浓度剂量依赖;冬凌草甲素能抑制供试菌株e DNA的释放量,其中1/4最小抑菌浓度(minimum inhibitory concentration,MIC)的冬凌草甲素作用金黄色葡萄球菌16 h后,与对照组相比,e DNA的释放量降低了48.62%;冬凌草甲素可显著抑制金黄色葡萄球菌生物膜形成相关基因的表达,其中1/2MIC的冬凌草甲素作用金黄色葡萄球菌16 h后,ica A、cid A、agr A和sar A基因的表达量分别比对照降低了91.6%、94.7%、77.6%和70.4%。【结论】冬凌草甲素通过抑制ica A和cid A基因的表达,影响PIA的合成和eDNA的释放,进而干预生物膜的形成。     

和厚朴酚抑制耐甲氧西林金黄色葡萄球菌生物被膜形成

【目的】研究和厚朴酚(HNK)抑制MRSA生物被膜(BF)形成的作用机制。【方法】使用TTC法测定了HNK对供试菌株BF的形成和成熟BF的抑制作用;刚果红平板法定性检测了HNK对PIA合成的影响;分光光度法测定了HNK对供试菌株eDNA释放量的影响;RT-PCR技术检测了HNK对供试菌株icaA、cidA以及agrA基因表达量的影响。【结果】HNK对MRSA 41573 BF的形成和成熟BF均有较强的抑制作用,其中,HNK抑制MRSA 41573 BF形成的MIC和MBC分别为10μg/mL和20μg/mL;抑制成熟BF的MIC和MBC分别为50μg/mL和100μg/mL。当用亚抑菌浓度的HNK与万古霉素联合作用后,可显著提高成熟BF对万古霉素的敏感性。HNK能显著抑制PIA的合成,且呈浓度剂量依赖。HNK能抑制供试菌株eDNA的释放量,其中1/8 MIC的HNK作用供试菌株16 h后,与对照组相比,e DNA的释放量降低了28.3%。HNK可抑制供试菌株BF形成的相关基因,其中1/2 MIC的HNK作用供试菌株16 h后,与对照相比,icaA的表达量降低了59.1%,cidA的表达量降低了56%,agrA的表达量降低了72.3%。【结论】HNK能显著抑制MRSA 41573 BF的形成,其作用机制主要是通过抑制icaA和cidA基因表达量,影响PIA和eDNA的合成,进而抑制BF的形成。此外HNK也可通过调控细菌的QS系统影响BF的形成。     

Plasmid DNA in Strains of Pediococcus cerevisiae and Pediococcus pentosaceus

Five parental strains of Pediococcus were examined for plasmid content. Each strain contained three to six resident plasmids, ranging in size from 4.5 to 39.5 megadaltons. A bacteriocin-like substance produced by Pediococcus cerevisiae FBB63 was tentatively linked to a 10.5-megadalton plasmid after being cured with novobiocin.     

Inhibition of Carotenoid Hydroxylation in Staphylococcus aureus by Mixed-Function Oxidase Inhibitors

Compounds known to be inhibitors of mixed-function oxidase systems inhibited the aerobic synthesis of hydroxylated carotenoids in Staphylococcus aureus U-71. Growth of the cells in the presence of 2-diethylaminoethyl-2,2-diphenyl valerate, 2,4-dichloro-6-phenylphenoxyethylamine, 2,4-dichloro-6-phenylphenoxyethyldiethylamine, and piperonyl butoxide reduced the levels of the rubixanthins found in stationary-phase cells by 75 to 97%. In cells grown with mevalonate-2-(14)C, the turnover rate of phytoene was reduced and the turnover rate of phytofluenol was increased in the presence of these inhibitors. The zeta- and delta-carotenes, which turn over in the absence of the inhibitors, accumulated (14)C in the presence of the inhibitors. This suggested that a mixed-function oxidase was responsible for the aerobic hydroxylation of delta-carotene in S. aureus U-71.     

Inhibition of Fibrinogen Reaction by Polysaccharide of Encapsulated Staphylococcus aureus

Encapsulated and nonencapsulated strains of Staphylococcus aureus which lack coagulase or clumping factor (bound coagulase), or both, were examined for the antigen associated with the fibrinogen-cell clumping reaction. Extracts of the cells were tested for the ability to react with fibrinogen or to inhibit fibrinogen precipitation. Antisera prepared against encapsulated (coagulase-positive, clumping factor-negative) variants, as well as against nonencapsulated wild-type (coagulase-positive, clumping factor-positive) S. aureus strains, contained high titers of clumping-inhibiting antibody. When coagulase-negative, clumping factor-negative mutants were the immunizing agents, antisera contained no demonstrable clumping-inhibiting antibody. Phenol extracts of all coagulase-positive strains tested precipitated fibrinogen, regardless of the ability of cells to clump in the presence of fibrinogen. Polysaccharide extracts of encapsulated, clumping factor-negative strains inhibited this fibrinogen-precipitating activity, whereas similar extracts of nonencapsulated staphylococci did not inhibit the fibrinogen reaction. From these results, it appeared that the coagulase-positive, encapsulated staphylococci which do not clump in fibrinogen solution possess clumping factor, but that their capsular polysaccharide inhibits clumping activity. These findings suggested a closer association of clumping factor and coagulase than is now recognized.     

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.     

Interference between Streptococcus pneumoniae and Staphylococcus aureus: In vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae

The bactericidal activity of Streptococcus pneumoniae toward Staphylococcus aureus is mediated by hydrogen peroxide. Catalase eliminated this activity. Pneumococci grown anaerobically or genetically lacking pyruvate oxidase (SpxB) were not bactericidal, nor were nonpneumococcal streptococci. These results provide a possible mechanistic explanation for the interspecies interference observed in epidemiologic studies.     

Inhibition of Vitamin K2 and Carotenoid Synthesis in Staphylococcus aureus by Diphenylamine

Diphenylamine at concentrations which did not effect the growth rate inhibited the synthesis of vitamin K(2) in both anaerobic and aerobic cultures by about 50%. At this concentration, diphenylamine inhibited the synthesis of the cyclic carotenoids delta-carotene and the rubixanthins 25 to 35% anaerobically and 60 to 90% aerobically. The inhibition of synthesis of cyclic carotenoids and vitamin K(2) by diphenylamine had no detectable effect on the formation of the membrane-bound electron transport system.     

Induction of stable L-forms of Staphylococcus aureus and Streptococcus faecalis

Stable L-forms were induced from Staphylococcus aureus and Streptococcus faecalis. These formed typical foamy L-colonies and showed large and small round bodies. They grew continuously on routine antibiotic-free nutrient broth and blood agar media for 12 passages without reversion to their parental forms. At different concentrations of penicillin various morphological forms were observed. Effect of sucrose, normal horse serum and penicillin on their adaptation and stabilization is discussed.