Hand Washing: What is the solution?

Patients in perioperative healthcare settings are at risk of acquiring or developing infections because of the performance of invasive procedures. Serious life threatening infections can arise when micro-organisms are transmitted onto susceptible sites, such as: surgical wounds or intravascular cannulation sites. Infection control practices help to protect patients and healthcare providers by reducing and/or eliminating sources of infection.

Enhancement of whole cell dioxygenase biotransformations of haloarenes by toxic ionic liquids

Accessing chirally pure cis-diols from arenes using micro-organisms over-expressing toluene dioxygenase (TDO) is now well established, but the conversions remain low for the more toxic and volatile substrates. For such arenes, improved production has already been achieved in the presence of hydrophobic non-toxic ionic liquids (ILs) acting in the form of a reservoir for the arene substrate. Yet, the costs associated with such ILs require extensive process development to render them viable. Herein, we show that optimization of the hydrophobic IL's cationic moiety and of the IL's concentration are key to enhanced conversion yielding between a 2-5 fold yield increase in the conversion of four haloarenes (Ph-X; X = F, Cl, Br, I). Additionally, we report that hydrophilic imidazolium-based ILs offer opportunities to achieve similarly high yielding biotransformations, with further improved reaction rates (<6 h), and this at very low ILs' concentrations (0.0015 V_IL/V_aq). We also demonstrate that the increased biotransformations are due to these ILs being inhibitors of cellular respiration processes and thus favoring the shunting of NADH and O₂ towards the overexpressed biocatalytic process. © 2014 the Partner Organisations.

Biofilm Eradication Kinetics of the Ultrashort Lipopeptide C12-OOWW-NH2 Utilizing a Modified MBEC Assay™

In this study, we report the antimicrobial planktonic and biofilm kill kinetics of ultrashort cationic lipopeptides previously demonstrated by our group to have a minimum biofilm eradication concentration (MBEC) in the microgram per mL (μg/mL) range against clinically relevant biofilm-forming micro-organisms. We compare the rate of kill for the most potent of these lipopeptides, dodecanoic (lauric) acid-conjugated C₁₂-Orn-Orn-Trp-Trp-NH₂ against the tetrapeptide amide H-Orn-Orn-Trp-Trp-NH₂ motif and the amphibian peptide Maximin-4 via a modification of the MBEC Assay™ for Physiology & Genetics (P&G). Improved antimicrobial activity is achieved upon N-terminal lipidation of the tetrapeptide amide. Increased antimicrobial potency was demonstrated against both planktonic and biofilm forms of Gram-positive micro-organisms. We hypothesize rapid kill to be achieved by targeting of microbial membranes. Complete kill against established 24-h Gram-positive biofilms occurred within 4 h of exposure to C₁₂-OOWW-NH₂ at MBEC values [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 15.63 μg/mL] close to the values for the planktonic minimum inhibitory concentration (MIC) [methicillin-resistant Staphylococcus epidermidis (ATCC 35984): 1.95 μg/mL]. Such rapid kill, especially against sessile biofilm forms, is indicative of a reduction in the likelihood of resistant strains developing with the potential for quicker resolution of pathogenic infection. Ultrashort antimicrobial lipopeptides have high potential as antimicrobial therapy.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: Potent activity against oral pathogens

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50. μM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10. μM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Removal of microorganisms and their chemical metabolites from water using semiconductor photocatalysis

Semiconductor photocatalysis has been applied to the remediation of an extensive range of chemical pollutants in water over the past 30 years. The application of this versatile technology for removal of micro-organisms and cyanotoxins has recently become an area that has also been the subject of extensive research particularly over the past decade. This paper considers recent research in the application of semiconductor photocatalysis for the treatment of water contaminated with pathogenic micro-organisms and cyanotoxins. The basic processes involved in photocatalysis are described and examples of recent research into the use of photocatalysis for the removal of a range of microorganisms are detailed. The paper concludes with a review of the key research on the application of this process for the removal of chemical metabolites generated from cyanobacteria.

Host Defence Peptide LL-37; Effects of truncation on antimicrobial activity

Background: The oral cavity is an ideal environment for colonisation by micro-organisms. A first line of defence against microbial infection is the secretion of broad spectrum host defence peptides (HDPs). In the current climate of antibiotic resistance, exploiting naturally occurring HDPs or synthetic derivatives (mimetics) to combat infection is particularly appealing. The human cathelicidin, LL-37 is one such HDP expressed ubiquitously by epithelial cells and neutrophils. LL-37 exhibits the ability to bind lipopolysaccharide (LPS) and displays broad spectrum activity against a wide range of bacteria. The current study focuses on truncation of LL-37 and defining the antimicrobial and LPS binding activity of the resultant mimetics. Objectives: To assess the antimicrobial and LPS binding activity of LL-37 and three truncated mimetics (KE-18, EF-14 and KR-12). Methods: Peptides were synthesised in-house by Fmoc solid phase peptide synthesis or obtained commercially. Antimicrobial activity was determined using a radial diffusion assay and ability to bind LPS was determined by indirect ELISA. Results: LL-37 and mimetics displayed antimicrobial activity against Streptococcus mutans and Enterococcus Faecalis. KE-18 and KR-12 were shown to possess antimicrobial activity against both pathogens whereas EF-14 was the least antimicrobial. In terms of LPS binding, KE-18 and KR-12 were both effective whereas EF-14 showed the least activity of the three mimetics. Conclusion: Truncation of LL-37 can yield peptides which retain antimicrobial activities and have the ability to bind LPS. Interestingly in some cases the truncation of LL-37 produced mimetics with greater potency than the parent molecule in terms of antimicrobial activity and LPS binding. This work was funded by DEL and the Diabetes Wellness Foundation.

Engineered alpha-defensin peptides display antimicrobial activity against oral pathogens

Introduction: Human alpha defensins are a family of neutrophil-derived antimicrobial peptides also known as human neutrophil peptides (HNPs). The defensin family of peptides are characterised by six invariant cysteine residues forming three disulphide bridges. The formation of the correct disulphide pairs complicates the synthesis of full length human alpha defensin and limits its therapeutic potential as an antimicrobial peptide. Objectives: The aim of this study was to determine whether truncated alpha defensins displayed antimicrobial activity against a range of micro-organisms including oral pathogens. Methods: Engineered peptides were synthesised by solid-phase methods using standard Fmoc chemistry. Antibacterial assays were performed using a previously described ultra sensitive radial diffusion method. A total of five engineered defensin peptides and full length alpha defensin were tested for their sensitivity against eight micro-organisms, including Gram negative bacteria, Gram positive bacteria and fungal pathogens Results: Antimicrobial activity was identified as clear zones around peptide-containing wells. Zone diameters were used to calculate minimum inhibitory concentrations (MICs) for each peptide. There was considerable variability in the susceptibility of the micro-organisms to the truncated analogues. Bacillus subtilis and Enterococcus faecalis were sensitive to the majority of the engineered peptides whereas Staphylococcus aureus, Escherichia coli and Candida albicans displayed resistance (defined as an MIC of greater than 250 ug/ml) to the truncated defensins. Of the five engineered peptides synthesised, the 2-aminobenzoic acid (Abz)-containing analogues based on the C-terminal sequence of alpha defensin displayed MIC values closest to that of the full length defensin in 5 out of 8 micro-organisms studied. Conclusion: This study demonstrates that truncated alpha defensins display variable antimicrobial activity against a range of micro-organisms, including oral pathogens. The generation of truncated defensins without disulphide bridges simplifies their synthesis and increases their therapeutic potential.

The response of reworked aerosols to climate through estimation of inter-particle forces

This paper describes the first use of inter-particle force measurement in reworked aerosols to better understand the mechanics of dust deflation and its consequent ecological ramifications. Dust is likely to carry hydrocarbons and micro-organisms including human pathogens and cultured microbes and thereby is a threat to plants, animals and human. Present-day global aerosol emissions are substantially greater than in 1850; however, the projected influx rates are highly disputable. This uncertainty, in part, has roots in the lack of understanding of deflation mechanisms. A growing body of literature shows that whether carbon emission continues to increase, plant transpiration drops and soil water retention enhances, allowing more greenery to grow and less dust to flux. On the other hand, a small but important body of geochemistry literature shows that increasing emission and global temperature leads to extreme climates, decalcification of surface soils containing soluble carbonate polymorphs and hence a greater chance of deflation. The consistency of loosely packed reworked silt provides background data against which the resistance of dust’s bonding components (carbonates and water) can be compared. The use of macro-scale phenomenological approaches to measure dust consistency is trivial. Instead, consistency can be measured in terms of inter-particle stress state. This paper describes a semi-empirical parametrisation of the inter-particle cohesion forces in terms of the balance of contact-level forces at the instant of particle motion. We put forward the hypothesis that the loss of Ca2+-based pedogenic salts is responsible for much of the dust influx and surficial drying pays a less significant role.

Antimicrobial activity of essential oils isolated from Portuguese endemic species of Thymus

Aims: Thymus species are wild species mostly found in the arid lands of Portugal. Possible antimicrobial properties of Thymus essential oils have been investigated. The chemical composition of the essential oils and the antimicrobial activity of Thymus mastichina (L) L. subsp. mastichina , T. camphoratus and T. lotocephalus from different regions of Portugal were analysed. Methods and Results: Hydrodistillation was used to isolate the essential oils and the chemical analyses were performed by gas chromatography (GC) and GC coupled to mass spectrometry. The antimicrobial activity was tested by the disc agar diffusion technique against Candida albicans , Escherichia coli , Listeria monocytogenes , Proteus mirabilis , Salmonella spp. and Staphylococcus aureus . Pure linalool, 1,8-cineole and a mixture (1:1) of these compounds were included. Linalool, 1,8-cineole or linalool/1,8-cineole and linalool/1,8-cineole/linalyl acetate were the major components of the essential oils, depending on the species or sampling place. The essential oils isolated from the Thymus species studied demonstrated antimicrobial activity but the micro-organisms tested had significantly different sensitivities. Conclusions: The antimicrobial activity of essential oils may be related to more than one component. Significance and Impact of the Study: Portuguese endemic species of Thymus can be used for essential oil production for food spoilage control, cosmetics and pharmaceutical use. Further studies will be required to elucidate the cell targets of the essential oil components.

Análise comparativa da designação, definição e classificação de resíduos hospitalares em legislações da União Europeia

RESUMO - O quadro legislativo de um país, no que concerne aos resíduos hospitalares (RH), contém a sua designação, definição e classificação. É essa a matriz de referência para a separação efectuada na origem e todo o circuito que, a partir desse momento, um determinado resíduo toma até ao seu tratamento. Assim, faz-se o estudo comparativo das definições e tipos de classificação de RH em quatro países da União Europeia: Alemanha, Reino Unido, Espanha (Região Autónoma da Catalunha) e Portugal. Reconhecem-se as diferentes designações deste tipo de resíduos e discute-se o seu significado e as suas implicações na percepção de risco por parte dos profissionais e do público. Identificam-se duas estratégias subjacentes à elaboração das definições: a contaminação de materiais com microrganismos patogénicos bem definidos, as suas fontes e as actividades que os produzem. Apresentam-se as classificações de RH propostas pelos organismos internacionais de referência e analisa-se comparativamente a evolução do enquadramento legal português e da Região Autónoma da Catalunha, evidenciando-se a variabilidade temporal e justificando-se a necessidade de se efectuar o estudo da variabilidade geográfica. Utilizam-se três critérios para a análise das classificações consideradas: a concordância definição-classificação, o número e tipo de grupos das classificações e os tipos de resíduos por grupos. Identificam-se os denominadores comuns às classificações analisadas, assim como as suas principais diferenças. Conclui-se que a definição de RH adoptada por cada país condiciona o tipo de classificação de RH nesse mesmo país. Verifica-se ainda que a inexistência de critérios claros de avaliação da contaminação pode dificultar a tarefa da triagem dos RH por parte dos profissionais de saúde.

Toxicity assessment of refill liquids for electronic cigarettes.

We analyzed 42 models from 14 brands of refill liquids for e-cigarettes for the presence of micro-organisms, diethylene glycol, ethylene glycol, hydrocarbons, ethanol, aldehydes, tobacco-specific nitrosamines, and solvents. All the liquids under scrutiny complied with norms for the absence of yeast, mold, aerobic microbes, Staphylococcus aureus, and Pseudomonas aeruginosa. Diethylene glycol, ethylene glycol and ethanol were detected, but remained within limits authorized for food and pharmaceutical products. Terpenic compounds and aldehydes were found in the products, in particular formaldehyde and acrolein. No sample contained nitrosamines at levels above the limit of detection (1 μg/g). Residual solvents such as 1,3-butadiene, cyclohexane and acetone, to name a few, were found in some products. None of the products under scrutiny were totally exempt of potentially toxic compounds. However, for products other than nicotine, the oral acute toxicity of the e-liquids tested seems to be of minor concern. However, a minority of liquids, especially those with flavorings, showed particularly high ranges of chemicals, causing concerns about their potential toxicity in case of chronic oral exposure.

Caractérisation moléculaire de la modulation spatio-temporelle des fonctions du phagosome

La phagocytose est un processus par lequel des cellules spécialisées du système immunitaire comme les macrophages ingèrent des microorganismes envahisseurs afin de les détruire. Les microbes phagocytés se retrouvent dans un compartiment intracellulaire nommé le phagosome, qui acquiert graduellement de nombreuses molécules lui permettant de se transformer en phagolysosome possédant la capacité de tuer et dégrader son contenu. L’utilisation de la protéomique a permis de mettre en évidence la présence de microdomaines (aussi nommés radeaux lipidiques ou radeaux membranaires) sur les phagosomes des macrophages. Notre équipe a démontré que ces radeaux exercent des fonctions cruciales au niveau de la membrane du phagosome. D’abord nous avons observé que la survie du parasite intracellulaire L. donovani est possible dans un phagosome dépourvu de radeaux lipidiques. Parallèlement nous avons constaté qu’un mutant de L. donovani n’exprimant pas de LPG à sa surface(LPG-) est rapidement tué dans un phagosome arborant des radeaux membranaires. Pour comprendre le mécanisme de perturbation des microdomaines du phagosome par la molécule LPG, nous avons provoqué la phagocytose de mutants LPG- du parasite et comparé par microscopie les différences avec le parasite de type sauvage. Nous avons ainsi démontré que le LPG de L. donovani est nécessaire et suffisant au parasite pour empêcher la maturation normale du phagosome. Nous avons également découvert que la molécule LPG permet d’empêcher la formation des radeaux lipidiques sur le phagosome et peut aussi désorganiser les radeaux lipidiques préexistants. Enfin, nous avons montré que l’action de LPG est proportionnelle au nombre d’unités répétitives de sucres (Gal(β1,4)-Manα1-PO4) qui composent cette molécule. Nos travaux ont démontré pour la première fois le rôle important de ces sous-domaines membranaires dans la maturation du phagosome. De plus, nos conclusions seront des pistes à suivre au cours des études cliniques ayant pour but d’enrayer la leishmaniose. Le second objectif de ce travail consistait à effectuer la caractérisation des radeaux lipidiques par une analyse protéomique et lipidomique à l’aide de la spectrométrie de masse. Nous avons ainsi entrepris l’identification systématique des protéines présentes dans les radeaux membranaires des phagosomes et ce, à trois moments clés de leurmaturation. Le traitement des phagosomes purifiés avec un détergent nous a permis d’isoler les «Detergent Resistent Membranes» (DRMs) des phagosomes, qui sont l’équivalent biochimique des radeaux membranaires. Nous avons ainsi établi une liste de 921 protéines associées au phagosome, dont 352 sont présentes dans les DRMs. Les protéines du phagosome sont partagées presque également entre trois tendances cinétiques (augmentation, diminution et présence transitoire). Cependant, une analyse plus spécifique des protéines des DRMs démontre qu’une majorité d’entre elles augmentent en fonction de la maturation. Cette observation ainsi que certains de nos résultats montrent que les radeaux lipidiques des phagosomes précoces sont soit très peu nombreux, soit pauvres en protéines, et qu’ils sont recrutés au cours de la maturation du phagosome. Nous avons aussi analysé les phospholipides du phagosome et constaté que la proportion entre chaque classe varie lors de la maturation. De plus, en regardant spécifiquement les différentes espèces de phospholipides nous avons constaté que ce ne sont pas uniquement les espèces majoritaires de la cellule qui dominent la composition de la membrane du phagosome. L’ensemble de nos résultats a permis de mettre en évidence plusieurs fonctions potentielles des radeaux lipidiques, lesquelles sont essentielles à la biogenèse des phagolysosomes (signalisation, fusion membranaire, action microbicide, transport transmembranaire, remodelage de l’actine). De plus, la cinétique d’acquisition des protéines de radeaux lipidiques indique que ceux-ci exerceraient leurs fonctions principalement au niveau des phagosomes ayant atteint un certain niveau de maturation. L’augmentation du nombre de protéines des radeaux membranaires qui s’effectue durant la maturation du phagosome s’accompagne d’une modulation des phospholipides, ce qui laisse penser que les radeaux membranaires se forment graduellement sur le phagosome et que ce ne sont pas seulement les protéines qui sont importées.

Facteurs de risque d´introduction et diagnostic de Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) est l'agent causal de la paratuberculose, maladie entérique, chronique et incurable des ruminants, avec un impact économique important. Une meilleure compréhension des facteurs de risque associés à l'introduction de la maladie dans un troupeau est essentielle pour sa prévention. L’amélioration des tests diagnostiques est aussi importante pour son contrôle. L’introduction des nouveaux animaux dans le troupeau et la présence et contact des différentes espèces sauvages et domestiques avec les vaches, semblent être des facteurs de risques d’introduction de la maladie. Nous avons réalisé une revue systématique dont l`objective était de recueillir l’information pertinente pour répondre à la question sur l’importance de ces facteurs et leur impact sur l’introduction de la maladie dans un troupeau. D`un autre côté, la détection de MAP dans les fèces par culture bactérienne demeure la méthode diagnostique de choix malgré les facteurs qui l`affectent. Une série de 3 étapes est requise afin de confirmer la présence du MAP : (1) culture (2) coloration, et (3) confirmation du MAP par PCR (si détecté à l´étape 2). Certains échantillons fécaux présentent une particularité en raison de leur forte charge de micro-organismes. Ces contaminants peuvent interférer avec la croissance et la détection de MAP. Une étude visant à : a) estimer l'impact des certain covariables sur les résultats de la culture de MAP parmi l`analyse rétrospective d`un banque des données et b) évaluer la possibilité d'optimiser le processus de diagnostic du MAP en effectuant l'analyse PCR sur les cultures déclarées comme contaminées a été réalisée.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

In this thesis, the production and characterization of ligninolytic enzymes using the fungi isolated from mangrove area are studied. The objective of the present work are isolation and screening of dye decolorizing micro-organisms from mangrove area, screening of the selected microorganisms for the production of lignin degrading enzymes, identification of the potent micro-organisms, characterization of the crude enzyme, lignin peroxidase, of the selected fungi—Aspergillus sp. SIP 11 and Penicillium sp. SIP 10 etc. This included the determination of the optimum pH, temperature, veratryl alcohol and H2O2 concentration. Besides the stability of crude LiP at different pHs and temperatures were studied. The immense applications, particularly in bioremediation, to which the lignin degrading micro-organisms could be used make this study important, the ascomycetes and deuteromycetes fungi, especially form the marine environment were studied with respect to their ligninolytic enzyme system making this study an initial step in unraveling the vast hidden potential of these microbes in bioremediation, the marine microbes are halophilic in nature which make them better suited to cope with the high salinity of industrial effluents thereby giving them added advantage in the filed of bioremediation. The thesis deals with the isolation and screening of lignin degrading enzyme-producing microbes from mangrove area. The identification of the most potent fungal isolates and characterization of LiP from these are also done.

Production and Characterization of Lignin Peroxidases from Mangrove Ascomycetes

The continually growing worldwide hazardous waste problem is receiving much attention lately. The development of cost effective, yet efficient methods of decontamination are vital to our success in solving this problem.Bioremediation using white rot fungi, a group of basidiomycetes characterized by their ability to degrade lignin by producing extracellular LiP, MnP and laccase have come to be recognized globally which is described in detail in Chapter 1.These features provide them with tremendous advantages over other micro-organisms.Chapter 2 deals with the isolation and screening of lignin degrading enzyme producing micoro-organisms from mangrove area. Marine microbes of mangrove area has great capacity to tolerate wide fluctuations of salinitie.Primary and secondary screening for lignin degrading enzyme producing halophilic microbes from mangrove area resulted in the selection of two fungal strains from among 75 bacteria and 26 fungi. The two fungi, SIP 10 and SIP ll, were identified as penicillium sp and Aspergillus sp respectively belonging to the class Ascomycetes .Specific activity of the purified LiP was 7923 U/mg protein. The purification fold was 24.07 while the yield was 18.7%. SDS PAGE of LiP showed that it was a low molecular weight protein of 29 kDa.Zymogram analysis using crystal violet dye as substrate confirmed the peroxidase nature of the purified LiP.The studies on the ability of purified LiP to decolorize different synthetic dyes was done. Among the dyes studied, crystal violet, a triphenyl methane dye was decolorized to the greatest extent.