Listeria monocytogenes batch culture growth response to metabolic inhibitors.

In certain environments nutrient and energy sources available to microorganisms can be limited. Foodborne pathogens must efficiently adapt in order to be successfully transmitted through the food chain to their hosts. For the intracellular foodborne pathogen Listeria monocytogenes, little is known regarding its response to nutrient/energy-limiting conditions. The alternative stress responsive sigma factor σ(B) has been reported to contribute to survival under specific stresses. Therefore, the effects of several metabolic inhibitors on growth of L. monocytogenes wild-type and a ΔsigB mutant were examined. In the absence of inhibitors, both strains reached stationary phase after 18 h at 23°C and 10 h at 37°C. All of the metabolic inhibitors slowed growth of either strain, with few differences observed among the different inhibitors.

Antimicrobial effect and mode of action of terpeneless cold-pressed Valencia orange essential oil on methicillin-resistant Staphylococcus aureus.

AIMS: The objectives of this study were to evaluate the antistaphylococcal effect and elucidate the mechanism of action of orange essential oil against antibiotic-resistant Staphylococcus aureus strains. METHODS AND RESULTS: The inhibitory effect of commercial orange essential oil (EO) against six Staph. aureus strains was tested using disc diffusion and agar dilution methods. The mechanism of EO action on MRSA was analysed by transcriptional profiling. Morphological changes of EO-treated Staph. aureus were examined using transmission electron microscopy. Results showed that 0·1% of terpeneless cold-pressed Valencia orange oil (CPV) induced the cell wall stress stimulon consistent with the inhibition of cell wall synthesis. Transmission electron microscopic observation revealed cell lysis and suggested a cell wall lysis-related mechanism of CPV. CONCLUSIONS: CPV inhibits the growth of Staph. aureus, causes gene expression changes consistent with the inhibition of cell wall synthesis, and triggers cell lysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple antibiotics resistance is becoming a serious problem in the management of Staph. aureus infections. In this study, the altered expression of cell wall-associated genes and subsequent cell lysis in MRSA caused by CPV suggest that it may be a potential antimicrobial agent to control antibiotic-resistant Staph. aureus.

Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus.

Resistance to gastrointestinal conditions is a requirement for bacteria to be considered probiotics. In this work, we tested the resistance of six different Lactobacillus strains and the effect of carbon source to four different gastrointestinal conditions: presence of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides synthesized from lactose (GOS-La) and lactulose were used as carbon sources and compared with glucose. In general, all strains grew in all carbon sources, although after 24 h of fermentation the population of all Lactobacillus strains was higher for both types of GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a dependence on carbon source and Lactobacillus strain was observed for bile extract and low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when compared with other carbon sources. However, concentrations of lactic and acetic acids were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that the resistance to gastrointestinal conditions and hydrophobicity is directly related with the carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and lactulose could be an excellent alternative to monosaccharides to support growth of probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract.

Modulatory effects of Crataeva nurvala bark against testosterone and N-methyl-N-nitrosourea-induced oxidative damage in prostate of male albino rats.

BACKGROUND: Antioxidant properties of Crataeva nurvala bark contains a variety of the bioactive phytochemical constituents in medicinal plants which include flavonoids, phenolic compounds, tannins, anthracene derivatives, and essential oils. Components from Crataeva nurvala bark have been accounted to play an important role in scavenging free radicals generated by mutagens and carcinogens. Androgens are the key factors in either the initiation or progression of prostate cancer by inducing oxidative stress. In the present set of investigations, the antioxidative potential of Crataeva nurvala bark extract against androgen-mediated oxidative stress in male Wistar rats has been studied. MATERIALS AND METHODS: Oxidative damage in prostate was induced in rats by the injection of testosterone (100 mg/kg body weight [bw]) for 3 days followed by injection of chemical carcinogen N-Methyl N-Nitroso Urea (50 mg/kg bw) for 1 week. The oxidative damage in prostate-induced rats were treated with the ethanolic extract of Crataeva nurvala bark (150 mg/kg bw) and testosterone injection (2 mg/ kg bw) was also continued through the experimental period of 4 months. The prostate tissue was dissected out for biochemical analysis of lipid peroxidation and enzymic-antioxidants viz. catalase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase; the non-enzymic antioxidants viz. reduced glutathione, and Vitamin C. RESULTS: The results revealed that testosterone administration induced the oxidative stress in rat prostate; however, in drug (150 mg/kg bw) supplemented groups, a significant protective effect of Crataeva nurvala bark against testosterone-induced oxidative injury was recorded. CONCLUSION: Hence, the study reveals that constituents present in Crataeva nurvala bark impart protection against androgen-induced oxidative injury in prostate.

Immediate reduction of Salmonella enterica serotype typhimurium viability via membrane destabilization following exposure to multiple-hurdle treatments with heated, acidified organic acid salt solutions.

The antimicrobial activity of organic acids in combination with nonchemical treatments was evaluated for inactivation of Salmonella enterica serotype Typhimurium within 1 min. It was observed that the effectiveness of the multiple-hurdle treatments was temperature (P ≤ 0.05) and pH (P ≤ 0.05) dependent and corresponded to the degree of organic acid lipophilicity (sodium acetate being least effective and sodium propionate being the most effective). This led to the hypothesis that the loss in viability was due at least in part to cell membrane disruption. Evaluation of osmotic response, potassium ion leakage, and transmission electron micrographs confirmed treatment effects on the cell membrane. Interestingly, all treatments, even those with no effect on viability, such as with sodium acetate, resulted in measurable cellular stress. Microarray experiments explored the specific response of S. Typhimurium to sodium acetate and sodium propionate, the most similar of the tested treatments in terms of pK(a) and ionic strength, and found little difference in the changes in gene expression following exposure to either, despite their very different effects on viability. Taken together, the results reported support our hypothesis that treatment with heated, acidified, organic acid salt solutions for 1 min causes loss of S. Typhimurium viability at least in part by membrane damage and that the degree of effectiveness can be correlated with lipophilicity of the organic acid. Overall, the data presented here indicate that a combined thermal, acidified sodium propionate treatment can provide an effective antimicrobial treatment against Salmonella.

Effect of sublethal heat stress on Salmonella Typhimurium virulence.

AIMS: To determine the virulence gene expression of Salmonella Typhimurium in response to sublethal heat stress and determine the adhesion and invasion pattern of heat-stressed Salmonella in Caco-2 intestinal epithelial cells. METHODS AND RESULTS: Transcriptional profiling was employed to capture the virulence gene response of Salm. Typhimurium at 42°C sublethal heat stress. Data indicated an induction of SPI-2 and SPI-5 genes and a repression of SPI-1-encoded genes due to heat stress. Gene expression pattern also showed induced transcription of fimbriae genes and genes present within the stress-associated Rpo regulon. Changes in adhesion and invasion pattern of heat-stressed Salm. Typhimurium were tested in Caco-2 cells. Heat-stressed Salm. Typhimurium showed greater adhesion to Caco-2 cells compared with nonstressed control cells. CONCLUSIONS: Salmonella Typhimurium exposed to sublethal heat stress responds by altered virulence gene expression, which further enhances the adhesion of bacterial cells to intestinal Caco-2 cells. Results indicate a role of physiological stress in Salm. Typhimurium in promoting microbial virulence and host cell vulnerability to infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Studying the Salmonella virulence genes expression in response to sublethal heat stress is crucial for the understanding of the virulence status of Salmonella in temperature-abused foods. Results of this study provide information about the gene response and virulence status of Salmonella pathogenicity factors in response to sublethal heat stress towards host cells.

Genetic changes that correlate with the pine-oil disinfectant-reduced susceptibility mechanism of Staphylococcus aureus.

AIMS: To identify factors associated with the Staphylococcus aureus pine-oil disinfectant-reduced-susceptibility (PD(RS)) mechanism and to describe one possible PD(RS) model. METHODS AND RESULTS: Comparative genomic sequencing (CGS) and microarray analysis were utilized to detect mutations and transcriptome alterations that occur in a S. aureus PD(RS) mutant. Mutant analysis, antimicrobial gradient plates, growth studies and 3-hydroxy-3-methylglutaryl coenzyme A synthase assays were then performed to confirm the biological consequences of the 'omics' alterations detected in a PD(RS) mutant. CGS uncovered three mutations in a PD(RS) mutant in a(n): alcohol dehydrogenase (adh), catabolite control protein A (ccpA) and an NADPH-flavin oxidoreductase (frp). These mutations lead to increased growth rates; increased transcription of an NAD-dependent D-lactate dehydrogenase gene (ddh); and increased flux through the mevalonate pathway. PD(RS) mutants demonstrated reduced susceptibility to bacitracin and farnesol, and one PD(RS) mutant displayed upregulation of bacA, a bacitracin-resistance gene. Collectively, this evidence demonstrates altered undecaprenol metabolism in PD(RS) mutants. CONCLUSIONS: The PD(RS) mechanism proposed results from increased catabolic capabilities and increased flux through the mevalonate pathway as well as altered bactoprenol physiology. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel mechanism that bacteria utilize to overcome the killing effects of PD formulations is proposed that is unique from the PD(RS) mechanism of the enterobacteraciae.

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners.

AIMS: To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. METHODS AND RESULTS: Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. CONCLUSIONS: HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations.