A thermostable salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.This work was supported by the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013 from "Fundacao para a Ciencia e Tecnologia" (FCT), Portugal. The authors thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER. Hugo Oliveira acknowledges the FCT grant SFRH/BD/63734/2009. Maarten Walmagh held a PhD scholarship of the IWT Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide

<p>Abstract</p> <p>Background</p> <p>Hydrogen peroxide (H₂O₂) produced by vaginal lactobacilli is generally believed to protect against bacteria associated with bacterial vaginosis (BV), and strains of lactobacilli that can produce H₂O₂are being developed as vaginal probiotics. However, evidence that led to this belief was based in part on non-physiological conditions, antioxidant-free aerobic conditions selected to maximize both production and microbicidal activity of H₂O₂. Here we used conditions more like those <it>in vivo </it>to compare the effects of physiologically plausible concentrations of H₂O₂and lactic acid on a broad range of BV-associated bacteria and vaginal lactobacilli.</p> <p>Methods</p> <p>Anaerobic cultures of seventeen species of BV-associated bacteria and four species of vaginal lactobacilli were exposed to H₂O₂, lactic acid, or acetic acid at pH 7.0 and pH 4.5. After two hours, the remaining viable bacteria were enumerated by growth on agar media plates. The effect of vaginal fluid (VF) on the microbicidal activities of H₂O₂and lactic acid was also measured.</p> <p>Results</p> <p>Physiological concentrations of H₂O₂(< 100 μM) failed to inactivate any of the BV-associated bacteria tested, even in the presence of human myeloperoxidase (MPO) that increases the microbicidal activity of H₂O₂. At 10 mM, H₂O₂inactivated all four species of vaginal lactobacilli but only one of seventeen species of BV-associated bacteria. Moreover, the addition of just 1% vaginal fluid (VF) blocked the microbicidal activity of 1 M H₂O₂. In contrast, lactic acid at physiological concentrations (55-111 mM) and pH (4.5) inactivated all the BV-associated bacteria tested, and had no detectable effect on the vaginal lactobacilli. Also, the addition of 10% VF did not block the microbicidal activity of lactic acid.</p> <p>Conclusions</p> <p>Under optimal, anaerobic growth conditions, physiological concentrations of lactic acid inactivated BV-associated bacteria without affecting vaginal lactobacilli, whereas physiological concentrations of H₂O₂produced no detectable inactivation of either BV-associated bacteria or vaginal lactobacilli. Moreover, at very high concentrations, H₂O₂was more toxic to vaginal lactobacilli than to BV-associated bacteria. On the basis of these <it>in vitro </it>observations, we conclude that lactic acid, not H₂O₂, is likely to suppress BV-associated bacteria <it>in vivo</it>.</p

International Society of Sports Nutrition Position Stand: Probiotics.

Position statement: The International Society of Sports Nutrition (ISSN) provides an objective and critical review of the mechanisms and use of probiotic supplementation to optimize the health, performance, and recovery of athletes. Based on the current available literature, the conclusions of the ISSN are as follows: 1)Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (FAO/WHO).2)Probiotic administration has been linked to a multitude of health benefits, with gut and immune health being the most researched applications.3)Despite the existence of shared, core mechanisms for probiotic function, health benefits of probiotics are strain- and dose-dependent.4)Athletes have varying gut microbiota compositions that appear to reflect the activity level of the host in comparison to sedentary people, with the differences linked primarily to the volume of exercise and amount of protein consumption. Whether differences in gut microbiota composition affect probiotic efficacy is unknown.5)The main function of the gut is to digest food and absorb nutrients. In athletic populations, certain probiotics strains can increase absorption of key nutrients such as amino acids from protein, and affect the pharmacology and physiological properties of multiple food components.6)Immune depression in athletes worsens with excessive training load, psychological stress, disturbed sleep, and environmental extremes, all of which can contribute to an increased risk of respiratory tract infections. In certain situations, including exposure to crowds, foreign travel and poor hygiene at home, and training or competition venues, athletes' exposure to pathogens may be elevated leading to increased rates of infections. Approximately 70% of the immune system is located in the gut and probiotic supplementation has been shown to promote a healthy immune response. In an athletic population, specific probiotic strains can reduce the number of episodes, severity and duration of upper respiratory tract infections.7)Intense, prolonged exercise, especially in the heat, has been shown to increase gut permeability which potentially can result in systemic toxemia. Specific probiotic strains can improve the integrity of the gut-barrier function in athletes.8)Administration of selected anti-inflammatory probiotic strains have been linked to improved recovery from muscle-damaging exercise.9)The minimal effective dose and method of administration (potency per serving, single vs. split dose, delivery form) of a specific probiotic strain depends on validation studies for this particular strain. Products that contain probiotics must include the genus, species, and strain of each live microorganism on its label as well as the total estimated quantity of each probiotic strain at the end of the product's shelf life, as measured by colony forming units (CFU) or live cells.10)Preclinical and early human research has shown potential probiotic benefits relevant to an athletic population that include improved body composition and lean body mass, normalizing age-related declines in testosterone levels, reductions in cortisol levels indicating improved responses to a physical or mental stressor, reduction of exercise-induced lactate, and increased neurotransmitter synthesis, cognition and mood. However, these potential benefits require validation in more rigorous human studies and in an athletic population

Bacterial Anti-Adhesion of Coated and Uncoated Thin-Film-Composite (TFC) Polyamide (PA) Membranes

This study investigates the bacterial anti-adhesion performance of uncoated and coated reverse osmosis (RO) membranes. All the membranes were commercially available fully-aromatic thin-film-composite (TFC) polyamide (PA) membranes. Two of the TFC PA membranes (SW30 and BW30) were coated using polyvinyl alcohol (PVA) coating, while the other three membranes (LE, XLE and NF90) were uncoated. Among the characterised TFC PA membranes, the PVA coated were more hydrophilic and their surface energy was higher in comparison to uncoated. In addition, the PVA coated membranes had lower surface roughness. AFM interaction force measurement demonstrated higher repellence performance for the more polar surface. Bacteria attachment test showed differences between the coated and the uncoated membranes. Indeed, the increase in hydrophilicity and surface polarity showed decrease in the attachment of Pseudomonas aeruginosa cells. Moreover, the results demonstrated that the surface polarity showed better correlation with the attachment of the bacteria. In addition, the type of the surface roughness may somehow contribute to the bacteria repellence

Surface modification of thin film composite RO membrane for enhanced anti-biofouling performance

Anti-adhesion and antimicrobial coatings were prepared and applied on commercial thin-film-composite (TFC) polyamide (PA) membrane to enhance anti-biofouling performance. Polyvinyl alcohol (PVA) coating was modified with cationic polyhexamethylene guanidine hydrochloride (PHMG) polymer to obtain antimicrobial performance. ATR-FTIR, SEM and AFM investigated the surface chemistry and morphology of the coated membranes. The contact angle measurement was used to determine hydrophilicity and surface energy. All coated membranes revealed more hydrophilic and lower surface roughness compared to uncoated membrane. Lower number of adhered Pseudomonas aeruginosa ( P. aeruginosa) bacteria was detected on coated membranes, indicating anti-adhesion performance. The colony forming unit (CFU) and diffusion inhibition zone (DIZ) tests determined antimicrobial activity of the coated membranes against Escherichia coli ( E. coli) and Bacillus subtilis ( B. subtilis), showing the antimicrobial performance of PHMG. The results suggested that an optimal anti-fouling surface could be obtained applying a coating, which combines anti-adhesion and antimicrobial performance. © 2013 Elsevier B.V.link_to_subscribed_fulltex

Morphological changes in Salmonella Typhimurium caused by the lantibiotic bovicin HC5 in association with EDTA

Bacteriocins, particularly those produced by Gram-positive bacteria, have in recent years been considered promising antimicrobial agents to inhibit bacterial growth in food, and thus are potential food preservatives. These peptides generally exhibit a spectrum of action limited to Gram-positive bacteria. However, their action can be extended to Gram-negative bacteria through association with chelating agents. In the present study, we evaluated the occurrence of morphological changes on the cell envelope of Salmonella Typhimurium cells treated with bovicin HC5—a lantibiotic from Streptococcus bovis HC5—in association with EDTA. The morphological changes of the cells were visualized by atomic force microscopy (AFM), and the increase in cell membrane permeability was confirmed by the leakage of potassium ions (K+). The images displayed changes in the cell envelope, with increased surface roughness and a decreased cell volume. These changes indicate that EDTA plays a role in the destabilization of the outer membrane, allowing bovicin HC5 to act on the cytoplasmic membrane through the formation of pores, which was confirmed by the detection of potassium in the cell supernatant. These results suggest that bovicin HC5 combined with EDTA has potential for use on Salmonella cells