Confocal spectral microscopy, a non-destructive approach to follow contamination and biofilm formation of mCherry Staphylococcus aureus on solid surfaces.

Methods to test the safety of wood material for hygienically sensitive places are indirect, destructive and limited to incomplete microbial recovery via swabbing, brushing and elution-based techniques. Therefore, we chose mCherry Staphylococcus aureus as a model bacterium for solid and porous surface contamination. Confocal spectral laser microscope (CSLM) was employed to characterize and use the autofluorescence of Sessile oak (Quercus petraea), Douglas fir (Pseudotsuga menziesii) and poplar (Populus euramericana alba L.) wood discs cut into transversal (RT) and tangential (LT) planes. The red fluorescent area occupied by bacteria was differentiated from that of wood, which represented the bacterial quantification, survival and bio-distribution on surfaces from one hour to one week after inoculation. More bacteria were present near the surface on LT face wood as compared to RT and they persisted throughout the study period. Furthermore, this innovative methodology identified that S. aureus formed a dense biofilm on melamine but not on oak wood in similar inoculation and growth conditions. Conclusively, the endogenous fluorescence of materials and the model bacterium permitted direct quantification of surface contamination by using CSLM and it is a promising tool for hygienic safety evaluation.

Survival of Bacterial Strains on Wood (Quercus petraea) Compared to Polycarbonate, Aluminum and Stainless Steel.

Healthcare-associated infections (HAI) remain a burden in healthcare facilities, environmental surfaces being a potential reservoir for healthcare-associated pathogens. In this context, exploration of materials with potential antimicrobial activities represents a way forward for the future. Here, we explored the survival of four bacterial species commonly involved in HAI (Acinetobacter baumannii, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus), on oak versus three other materials (aluminum, polycarbonate, stainless steel). Twenty microliters of each bacterial suspension (approximatively 107 bacteria) were deposited on each material. Bacterial counts were measured by grinding and culturing on day 0, 1, 2, 6, 7 and 15. Analyses were performed in triplicate for each material and each time evaluated. It appeared that the bacteria viable count decreased rapidly on transversal and tangential oak compared with the other materials for all bacterial species. Furthermore, no difference was noticed between transversal and tangential oak. These results underline the potential for use of oak materials in healthcare facilities, a consideration that should be supported by further investigations.

Wood materials for limiting the bacterial reservoir on surfaces in hospitals: would it be worthwhile to go further?

Aim: To assess the activity of Quercus petraea (oak) on five bacterial species/genus frequently involved in hospital-acquired infections for evaluating the interest of going further in exploring the possibilities of using untreated wood as a material in the hospital setting. Materials & methods: We studied the activity of Q. petraea by the disk diffusion method. Results:Q. petraea was active on Staphylococcus aureus and Acinetobacter coalcoaceticus-baumannii complex, two bacterial species particularly resistant in the hospital environment, independently from their resistance to antibiotics, and was slightly active on Pseudomonas aeruginosa. Concurrently, Q. petraea was not active on Enterococci and Escherichia coli. Conclusion: Overall, untreated wood material presented antimicrobial properties that could have an impact on the cross-transmission of certain bacterial species in healthcare settings.

Experimental Parameters Influence the Observed Antimicrobial Response of Oak Wood (Quercus petraea).

The present investigation aimed to utilize a direct wood disc diffusion method to study the influence of plane of cutting, cutting method, sterilization method, and origin of tree on the antimicrobial activity of wood material. Six oak wood trees (Quercus petraea) were collected from 3 different locations in France. They were cut into 4 mm thick slices with either transverse (RT), tangential (LT) or radial (LR) faces. Round discs (diameter 9.95 ± 0.1 mm) were cut from the slices via a laser machine or a manual punch machine, and were sterilized with gamma irradiation (25 kGy) or autoclaving (121 °C). The antimicrobial activity of wood was tested using a direct diffusion method against Staphylococcus aureus and Acinetobacter baumannii isolates. The zone of inhibition around the wooden disc was recorded following the recommendations used for antibiotics tests. The results showed that S. aureus was more susceptible than A. baumannii, to the chemicals that diffused from the wood. The transverse face discs exhibited higher antimicrobial activity. Samples that had been sterilized by autoclaving showed significantly (p < 0.05) lower antimicrobial activity, whereas the cutting method and origin of tree did not influence the antimicrobial activity of wood material. Therefore, the choice of sterilization method and cutting planes must be taken into account while studying and interpreting the antibacterial properties of wood material.

Comparative study of microbiological transfer from four materials used in direct contact with apples.

Several materials such as plastic, wood, cardboard or stainless steel are used as working surfaces or packaging in direct contact with foodstuffs. In food industries, the hygienic surface status is one of the criteria to product conform packaging as described in the European regulation ECR 1935/2004. Today in European Union, it exists one harmonized regulation specific for Food Contact material made of plastic called EU N°10/2011 (Anonymous 2011a). This regulation specifies that materials intended for safe foodstuff contact must not modify food characteristics in terms of chemical, microbiological and sensorial properties. This study aims to compare the survival and transfer of Penicillium expansum conidia and Escherichia coli cells from several materials to apples. Poplar, cardboards, newly manufactured plastic and reusable plastic specimens were artificially inoculated with both microorganisms, subsequently put in contact with apples and stored under realistic storage conditions. After incubation for up to 1 week, apples and specimens were analysed to assess the survival of the microorganisms and their transfer from materials to apples. While P. expansum survived and did not grow on any of the materials, E. coli mortality was observed after 1 h on wood and cardboard and after 1 week on both plastics. The proportion of microorganisms transferred was different according to the considered material. This transfer was lower than 1% for wood.

Testing the Antimicrobial Characteristics of Wood Materials: A Review of Methods.

Some wood species have antimicrobial properties, making them a better choice over inert surfaces in certain circumstances. However, the organic and porous nature of wood raises questions regarding the use of this material in hygienically important places. Therefore, it is reasonable to investigate the microbial survival and the antimicrobial potential of wood via a variety of methods. Based on the available literature, this review classifies previously used methods into two broad categories: one category tests wood material by direct bacterial contact, and the other tests the action of molecules previously extracted from wood on bacteria and fungi. This article discusses the suitability of these methods to wood materials and exposes knowledge gaps that can be used to guide future research. This information is intended to help the researchers and field experts to select suitable methods for testing the hygienic safety and antimicrobial properties of wood materials.

Microbial Safety of Wood in Contact with Food: A Review.

Food packaging is multifunctional: it protects from harvest to table. Four main groups of materials for direct food contact are mentioned in the literature: wood, glass, plastic, and metal. In this review, the focus is on wooden packaging for direct contact with food. In Europe, wood as a food contact material is subject to European Regulation (EC) No 1935/2004 states that materials must not transfer their constituents to food. Today, wooden packaging, like other packaging materials, does not have a Europe-wide harmonized specific regulation, so member countries legislate at different levels. Wood has been safely used for centuries in contact with food but is usually questioned because of its microbiological behavior compared with smooth surfaces. Based on a review of published conclusions from scientific studies over the last 20 y and after a description of the general properties of wooden packaging, we focus on the microbiological status of natural wood. Then, we discuss the parameters influencing the survival of microorganisms on wood. Finally, we report on the transfer of microorganisms from wood to food and the factors influencing this phenomenon. This review demonstrates that the porous nature of wood, especially when compared with smooth surfaces, is not responsible for the limited hygiene of the material used in the food industry and that it may even be an advantage for its microbiological status. In fact, its rough or porous surface often generates unfavorable conditions for microorganisms. In addition, wood has the particular characteristic of producing antimicrobial components able to inhibit or limit the growth of pathogenic microorganisms.

Waterborne Leptospirosis: Survival and Preservation of the Virulence of Pathogenic Leptospira spp. in Fresh Water.

Many studies have implicated fresh water as a source of leptospirosis outbreaks. To estimate the survival and the preservation of the virulence of pathogenic Leptospira spp. maintained in water, we selected five still waters with various pH and mineral profiles. The water samples were artificially inoculated with a culture of a pathogenic strain belonging to serovar Icterohaemorrhagiae. Samples were stored for 20 months at 4, 20 or 30 °C. The survival and preservation of virulence of this pathogenic strain was estimated by subculturing these stored samples. After 14 and 20 months of storage, the strain Icterohaemorrhagiae was re-isolated, and its virulence was determined using an animal model. In these waters, the mean survival was 130 days for storage at 4 °C, 263 days at 20 °C, and 316 days at 30 °C. Unexpectedly, the mean survival was 344 days for a final pH < 7 and 129 days for pH ≥ 7. Moreover, the pathogenic strain remained fully virulent and was able to induce a lethal disease in gerbils even when the pH of the contaminated waters decreased to <6. These data showed that despite unfavourable storage conditions such as cold, nutrient-poor acidic waters, the survival and virulence of pathogenic Leptospira spp. was fully preserved over at least 20 months.

Undiagnosed leptospirosis cases in naïve and vaccinated dogs: properties of a serological test based on a synthetic peptide derived from Hap1/LipL32 (residues 154-178).

Leptospirosis is a common disease in dogs, despite having current vaccinations. However, leptospirosis diagnosis based on the routine Microscopic Agglutination Test (MAT) leads to confusing conclusions, especially for infected vaccinated dogs. Indeed, both bacterin and natural infection stimulate the production of agglutinating antibodies. In experimentally infected dogs, antibodies against the peptide PP derived from Hap1/Lipl32 were raised earlier than agglutinating antibodies. The background level of these antibodies was determined in a group of 109 healthy dogs, either vaccinated or not against leptospirosis, with a specificity for IgM of 96.4% and for IgG of 95.5%. PP ELISA was subsequently performed with 118 sera from dogs with suspected leptospirosis that was not confirmed by MAT. New leptospirosis cases based on the PP ELISA results were suspected in 14 out of 102 vaccinated dogs and in two out of 16 non-vaccinated dogs. These results highlight the importance of serological diagnosis corresponding to an interesting window when it is too late for PCR detection and too early to be confirmed by MAT.

Methods for recovering microorganisms from solid surfaces used in the food industry: a review of the literature.

Various types of surfaces are used today in the food industry, such as plastic, stainless steel, glass, and wood. These surfaces are subject to contamination by microorganisms responsible for the cross-contamination of food by contact with working surfaces. The HACCP-based processes are now widely used for the control of microbial hazards to prevent food safety issues. This preventive approach has resulted in the use of microbiological analyses of surfaces as one of the tools to control the hygiene of products. A method of recovering microorganisms from different solid surfaces is necessary as a means of health prevention. No regulation exists for surface microbial contamination, but food companies tend to establish technical specifications to add value to their products and limit contamination risks. The aim of this review is to present the most frequently used methods: swabbing, friction or scrubbing, printing, rinsing or immersion, sonication and scraping or grinding and describe their advantages and drawbacks. The choice of the recovery method has to be suitable for the type and size of the surface tested for microbiological analysis. Today, quick and cheap methods have to be standardized and especially easy to perform in the field.

Expanding the genetic toolbox for Leptospira species by generation of fluorescent bacteria.

Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-ß-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

Use of quantitative real-time PCR for studying the dissemination of Leptospira interrogans in the guinea pig infection model of leptospirosis.

The dynamics of leptospirosis infection have been poorly studied. The purpose of this study was to determine the LD(50), rate of bacterial dissemination, histopathology and antibody responses against leptospira following inoculation with the highly virulent Leptospira interrogans Fiocruz L1-130 strain in a guinea pig model of leptospirosis. Three routes of infection (intraperitoneal, conjunctival and subcutaneous inoculation) were used to establish disease in guinea pigs. The size and kinetics of leptospiral burdens in the blood and tissues of infected animals were determined over a 1 week course of infection using quantitative real-time PCR (qPCR). Bacteraemia peaked at day 5 post-infection reaching more than 5x10(4) leptospires ml(-1). The highest spirochaetal load was found in the liver and kidneys, and was associated with alterations in organ tissues and a decline in liver and kidney functions. In contrast, lesions and bacteria were not detected in guinea pigs infected with an avirulent strain derived from a high-passage-number in vitro-passaged variant of the Fiocruz L1-130 strain. The use of qPCR supports the findings of earlier studies and provides an easy and reliable method for the quantification of L. interrogans in the tissues of infected animals. qPCR will be used in future studies to evaluate the efficacy of vaccine candidates against leptospirosis and the virulence of selected L. interrogans mutants relative to the parental strain.

Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1.

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.