Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Choline transport activity in Staphylococcus aureus induced by osmotic stress and low phosphate concentrations.

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from [14C]choline was accumulated as [14C]glycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na+ dependent and had a Km of 46 microM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaC1 concentrations.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

Very Low Ethanol Concentrations Affect the Viability and Growth Recovery in Post-Stationary-Phase Staphylococcus aureus Populations

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (≤0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC⁺ conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC⁻ conditions) for prolonged culture times (up to 8 days). VLEC⁺-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC⁺ conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC⁺ conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.     

Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and long-term survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimicrobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the catabolization of acetate during the stationary phase. clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary- and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.     

Peptidoglycan crosslinking relaxation plays an important role in Staphylococcus aureus WalKR-dependent cell viability

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.     

熟鸡肉中金黄色葡萄球菌生长预测模型的建立

【目的】研究不同浓度和不同温度条件下金黄色葡萄球菌接种在熟鸡肉中的生长情况,比较3种常见预测模型拟合的准确性,选择最适合的预测模型建立一级和二级模型,为进一步探讨建立三级模型提供数据基础。【方法】测定浓度为102、103和104 CFU/g的金黄色葡萄球菌接种在15-36°C熟鸡肉中的生长数据,使用Matlab软件分别建立修正的Gompertz、Logistic和Baranyi模型,通过比较残差和拟合度(RSS、AIC、RSE)选择最优模型,并且拟合出生长参数(迟滞期、最大比生长速率和最大细胞密度),在此基础上通过响应面方程建立二级模型。最后对模型的可靠性进行了内部和外部实验验证。【结果】36°C和29°C条件下,修正的Gompertz模型最适合;22°C和15°C条件下,最适合模型按接种浓度依次为修正的Gompertz、Logistic和Baranyi模型,综合考虑,最优模型选择修正的Gompertz模型。通过计算预测标准差(%SEP)、平方根误差(RMSE)、准确性因子(Af)和偏差因子(Bf)对建立的二级模型进行数学检验,检验结果均在可接受范围内。【结论】用修正的Gompertz方程和响应面方程建立的一、二级预测模型可以为建立三级模型提供有效、精确的基础。     

High phenotypic diversity in infecting but not in colonizing Staphylococcus aureus populations

In hostile environments diversity within a bacterial population may be beneficial for the fitness of the microbial community as a whole. Here we analysed the population diversity of Staphylococcus aureus in infecting and colonizing situations. In the study, performed independently in two German centres, the heterogeneity of the S. aureus population was determined by quantifying the occurrence of phenotypic variants (differences in haemolysis, pigmentation, colony morphology) in primary cultures from nose, oropharyngeal and sputum specimens from cystic fibrosis (CF) patients and in nose swabs from healthy S. aureus carriers. The proportion of heterogeneous samples, the number of clearly distinguishable isolates per sample and the qualitative differences between phenotypes was significantly higher in CF sputum specimens than in the other samples. The heterogeneity of the S. aureus population could be correlated with high bacterial densities in the sputum samples. In patients co-infected with Pseudomonas aeruginosa lower S. aureus bacterial loads and less heterogeneity in the S. aureus population were observed. Typing of all S. aureus isolates from heterogeneous samples by pulsed-field gel electrophoresis or spa typing revealed that the bacteria were polyclonal in 30%, monoclonal with minor genetic alterations in 25% or not distinguishable in 69% of the specimens. Some specimens harboured monoclonal and polyclonal variants simultaneously. Importantly, differences in antibiotic susceptibility were detected in phenotypic S. aureus variants within a single specimen. Diversification of a S. aureus population is highly favoured during chronic CF lung infection, supporting the general hypothesis that maintenance of intrahost diversity can be of adaptive value, increasing the fitness of the bacterial community.     

Effect of sub-inhibitory concentrations of antibiotics on the virulence of Staphylococcus aureus

The production of virulence factors by various bacteria can be influenced by sub-inhibitory concentrations of antibiotics. The effect of six antibiotics on the production of representative extracellular enzymes and toxins produced by Staphylococcus aureus was investigated. The production of the virulence determinants coagulase, protein A, alpha and delta haemolysin was monitored in the presence of ciprofloxacin, enoxacin, chloramphenicol, gentamicin, tetracycline and methicillin. The protein synthesis inhibitors reduced the production of coagulase and protein A, and almost completely inhibited the production of the haemolysins. Haemolysin production was also reduced by ciprofloxacin and enoxacin, but these antibiotics had little effect on the production of coagulase and protein A. Methicillin stimulated the production of alpha and delta haemolysins but had no effect on the production of coagulase and protein A.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.