Food grade disinfectants based on hydrogen peroxide/peracetic acid and sodium hypochlorite interfere with the adhesion of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes to stainless steel of differing surface roughness.

This study aimed to evaluate the potential of the bacterium Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes to adhere to stainless steel discs with differing degrees of surface roughness (Ra = 25.20-961.90 nm). Stainless steel is a material commonly used in the food industry for processing equipment, which is regularly exposed to cleaning procedures. The investigation included the commercial disinfectants hydrogen peroxide/peracetic acid and sodium hypochlorite which were evaluated for their antibacterial and anti-adhesion activity. The adhesion was assessed by the standard plate count method, while the broth microdilution method CLSI M07-A10 was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the disinfectants. Based on the MIC values, both disinfectants exerted significant inhibitory effects with MIC values for hydrogen peroxide/peracetic acid and sodium hypochlorite of 250 µg ml-1 and 500 µg ml-1, respectively. Whereas the MBC values were equal to the MIC for all bacteria except for E. coli with values 2-fold higher than the MIC. Obtained results also revealed that all tested bacteria were able to adhere to stainless steel surfaces, although differences were found for strains and surface roughness. The lowest adhesion rate of each strain was recorded on the roughest stainless steel disc at a Ra of 961.90 nm. Further, at a concentration of 1 MIC, the disinfectant sodium hypochlorite reduced initial bacterial adhesion to stainless steel surfaces to a significantly greater extent than the disinfectant hydrogen peroxide/peracetic acid. These findings are consistent with the results obtained by Scanning Electron Microscopy (SEM) analysis, which indicates the great applicability of the tested disinfectants for the control of bacterial adhesion in the food industry.

Influence of culture conditions on co-aggregation of probiotic yeast Saccharomyces boulardii with Candida spp. and their auto-aggregation.

Systemic infections caused by pathogenic Candida species pose a significant threat to public health in the past decades due to increasing resistance to existing antifungal drugs. Given this scenario, probiotics have been suggested as an alternative approach for managing Candida infections. Hence, the purpose of this study was to evaluate whether probiotic yeast Saccharomyces boulardii co-aggregate with Candida spp. as well as to determine their auto-aggregation ability in dependence on temperature (28 °C, 37 °C, 42 °C) and pH (4.5, 7.0, 8.5) after 5 h and 24 h. Our results revealed that the aggregation of tested yeasts was lower in the first 5 h but increased significantly after 24 h. All strains were able to auto-aggregate in different degrees ranging from 47.46 to 95.95% assessed at 24 h of incubation. Among them the highest auto-aggregation values had C. albicans and C. krusei strains followed by probiotic strain S. boulardii, while the less were observed in C. glabrata strains. In addition, co-aggregation between probiotic and Candida strains was strain-specific. It was evident that S. boulardii significantly inhibited the aggregation of C. albicans ATCC 10261, C. krusei ATCC 6258, and C. glabrata ZIM 2369. However, in C. glabrata ZIM 2382, the aggregation was even enhanced. Temperature and pH also affected the ability to aggregate in a different way only after 5 h of incubation, with the highest cell aggregation evidenced at temperature 37 °C in most cases and pH 4.5. These findings may be of importance when trying to establish probiotic use against pathogenic Candida species.

Bacterial Adhesion of Streptococcus mutans to Dental Material Surfaces.

The aim of this study was to investigate and understand bacterial adhesion to different dental material surfaces like amalgam, Chromasit, an Co-Cr alloy, an IPS InLine ceramic, yttrium stabilized tetragonal polycrystalline zirconia (TPZ), a resin-based composite, an Au-Pt alloy, and a tooth. For all materials, the surface roughness was assessed by profilometry, the surface hydrophobicity was determined by tensiometry, and the zeta potential was measured by electrokinetic phenomena. The arithmetic average roughness was the lowest for the TPZ ceramic (Ra = 0.23 µm ± 0.02 µm), while the highest value was observed for the Au-Pt alloy (Ra = 0.356 µm ± 0.075 µm). The hydrophobicity was the lowest on the TPZ ceramic and the highest on the Co-Cr alloy. All measured streaming potentials were negative. The most important cause of tooth caries is the bacterium Streptococcus mutans, which was chosen for this study. The bacterial adhesion to all material surfaces was determined by scanning electron microscopy. We showed that the lowest bacterial extent was on the amalgam, whereas the greatest extent was on tooth surfaces. In general, measurements showed that surface properties like roughness, hydrophobicity and charge have a significant influence on bacterial adhesion extent. Therefore, dental material development should focus on improving surface characteristics to reduce the risk of secondary caries.

Bacterial adhesion rate on food grade ceramics and Teflon as kitchen worktop surfaces.

Here we present a research of bacterial adhesion to two most often used materials in modern kitchens, namely food grade ceramics and Teflon. To test the bacterial adhesion on kitchen worktops Escherichia coli, Pseudomonas aeruginosa and Campylobacter jejuni were used as the most common foodborne contaminants. Contact angle, roughness and streaming potential measurements were used for surface characterization. Crystal violet staining and scanning electron microscopy were applied for bacterial adhesion analysis. We showed that the adhesion of tested bacteria strains was lower on the Teflon surface compared to the ceramics. The hydrophobicity of the surface substantially contributed to the bacterial adhesion rate. On the other hand, the surface roughness and charge did not play a crucial role in the adhesion process.

Challenges of teaching food microbiology in Brazil.

Food Microbiology is included in majors such as Food Engineering, Food Science and Technology, Nutrition, Veterinary Medicine, Gastronomy, Pharmaceutical Sciences, among others. Food safety and hygiene are usually the focus, but the technological applications of microbes through fermentations are also covered. During an education symposium at the Brazilian Congress of Microbiology in 2017, a group of professors expressed their difficulties associated with teaching to new generations, the use of technology in the classroom, and the application of new learning tools. The objective of this study was to gather information about the educational practices among Brazilian professors who teach Food Microbiology throughout the country. The results indeed confirmed the diversity of careers in which food microbiology is taught. We verified that professors mixed traditional teaching strategies with modern active learning methods, even though some difficulties associated with lack of time, pedagogical training, and low adherence of students for adopting these modern methods were commonly highlighted. The preferred teaching approaches were dialogued or discussed lectures, seminars, homework, case studies, and field visits. It is noteworthy that most professors still use traditional teaching methods. It is crucial that awareness concerning the educational needs in different careers and the challenges and dilemmas facing education for the new generations should be dealt with by using effective teaching approaches in food microbiology education. We suggest that a more permanent discussion forum among faculty members dealing with food microbiology in the country should be launched and this work is a step towards this goal.

Biofouling of stainless steel surfaces by four common pathogens: the effects of glucose concentration, temperature and surface roughness.

There is a wide range of factors affecting bacterial adhesion and biofilm formation. However, in both food processing and medical settings, it is very hard to obtain suitably controlled conditions so that the factors that reduce surface colonisation and biofouling can be studied. The aim of this study was to evaluate the effect of glucose concentration, temperature and stainless steel (SS) surface roughness on biofouling by four common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and L. monocytogenes). Among the tested variables, the untreated SS surface (3C) was shown to be fouled more than 3D polished, brushed or electropolished SS surfaces. Although an array of parameters influenced biofouling, the most promising control measure was the influence of low temperature (4 °C) that reduced biofouling even in the case of the psychrophilic Listeria monocytogenes. The study findings could significantly contribute to the prevention of SS surface contamination and consequential biofouling by food and healthcare associated pathogens.

Synergistic and antagonistic effects of immunomodulatory drugs on the action of antifungals against Candida glabrata and Saccharomyces cerevisiae.

Candidemia and other forms of invasive fungal infections caused by Candida glabrata and to a lesser extent Saccharomyces cerevisiae are a serious health problem, especially if their steadily rising resistance to the limited range of antifungal drugs is taken into consideration. Various drug combinations are an attractive solution to the resistance problem, and some drug combinations are already common in the clinical environment due to the nature of diseases or therapies. We tested a few of the common antifungal-immunomodulatory drug combinations and evaluated their effect on selected strains of C. glabrata and S. cerevisiae. The combinations were performed using the checkerboard microdilution assay and interpreted using the Loewe additivity model and a model based on the Bliss independence criterion. A synergistic interaction was confirmed between calcineurin inhibitors (Fk506 and cyclosporine A) and antifungals (fluconazole, itraconazole, and amphotericin B). A new antagonistic interaction between mycophenolic acid (MPA) and azole antifungals was discovered in non-resistant strains. A possible mechanism that explains this is induction of the Cdr1 efflux pump by MPA in C. glabrata ATCC 2001. The Pdr1 regulatory cascade plays a role in overall resistance to fluconazole, but it is not essential for the antagonistic interaction. This was confirmed by the Cgpdr1Δ mutant still displaying the antagonistic interaction between the drugs, although at lower concentrations of fluconazole. This antagonism calls into question the use of simultaneous therapy with MPA and azoles in the clinical environment.

Adhesion of Candida spp. and Pichia spp. to Wooden Surfaces.

Yeast adhesion to and biofilm formation on surfaces is present in many different environments. In food industry, biofilms may be a source of contaminations, causing food spoilage and reducing quality of products. Candida and Pichia are two common yeast genera involved in the spoilage of some food products. The aim of this study is to assess the potential of Candida and Pichia species to adhere to two types of wooden surfaces (smooth and rough), one of the materials typical for the food processing industry, and investigate the influence of surface roughness of wood on the degree of yeast adhesion. The adhesion of the cells to the wooden surfaces was determined by rinsing them from the surface, followed by methylene blue staining, and quantification after imaging under microscope by automatic counting of viable cells. The results showed that all Candida and Pichia strains were able to adhere to the wooden surfaces in a species- and strain-dependent manner. On the other hand, our data indicated that adhesion by these yeasts was not significantly affected by the roughness of the wood surfaces.

The influence of Dekkera bruxellensis on the transcriptome of Saccharomyces cerevisiae and on the aromatic profile of synthetic wine must.

A double compartment membrane system was constructed in order to systematically study possible microbial interactions between yeasts Saccharomyces cerevisiae and Dekkera bruxellensis and their impact on wine aroma. The presence of D. bruxellensis induced 77 transcripts of S. cerevisiae. These were mostly of unknown function; however, some were involved in thiamine biosynthesis and in amino acid and polyamine transport, suggesting a competitive relationship between the two yeast species. Among the transcripts with no biological function, 14 of them were found to be the members of the PAU gene family that is associated with response to anaerobiosis stress. In separated cultures, S. cerevisiae produced glycerol which was subsequently consumed by D. bruxellensis. The concentration of ethylphenols was reduced and we assume that they were absorbed onto the surfaces of S. cerevisiae yeast walls. Also in separated cultures, D. bruxellensis formed a typical profile of aromatic esters with decreased levels of acetate esters and increased level of ethyl esters.

Influence of growth conditions on adhesion of yeast Candida spp. and Pichia spp. to stainless steel surfaces.

An understanding of adhesion behavior of Candida and Pichia yeast under different environmental conditions is key to the development of effective preventive measures against biofilm-associated infection. Hence in this study we investigated the impact of growth medium and temperature on Candida and Pichia adherence using stainless steel (AISI 304) discs with different degrees of surface roughness (Ra = 25.20-961.9 nm), material typical for the food processing industry as well as medical devices. The adhesion of the yeast strains to stainless steel surfaces grown in Malt Extract broth (MEB) or YPD broth at three temperatures (7 °C, 37 °C, 43 °C for Candida strains and 7 °C, 27 °C, 32 °C for Pichia strains) was assessed by crystal violet staining. The results showed that the nutrient content of medium significantly influenced the quantity of adhered cells by the tested yeasts. Adhesion of C. albicans and C. glabrata on stainless steel surfaces were significantly higher in MEB, whereas for C. parapsilosis and C. krusei it was YPD broth. In the case with P. pijperi and P. membranifaciens, YPD broth was more effective in promoting adhesion than MEB. On the other hand, our data indicated that temperature is a very important factor which considerably affects the adhesion of these yeast. There was also significant difference in cell adhesion on all types of stainless steel surfaces for all tested yeast.

Effect of saliva on physical food properties in fat texture perception.

Sensory properties of food drive our food choices and it is generally accepted that lipids greatly contribute to the sensory properties of many foods and consequently to eating pleasure. Many studies have investigated the mechanisms of the fat perception. Unfortunately they used a variety of methods and products, thereby making generalization very difficult. The mechanism of fat perception in oral cavity is combined of several processes. Lipid composition and its properties strongly influence food structure. During consumption food is exposed to a range of in-mouth processing steps. Oral sensation of fat texture changes with time, from a first bite to chewing, while mixing with saliva, up to swallowing and even after swallowing. The present work reviews many aspects of fat texture perception from physical chemistry to physiology. Understanding the underlying mechanisms of in-mouth lipid processing would provide new concepts to produce low-fat food products with full-fat perception.

Quorum-sensing in yeast and its potential in wine making.

This mini-review synthesises the present knowledge of microbial quorum-sensing, with a specific focus on quorum-sensing in yeast, and especially in wine yeast. In vine and wine ecosystems, yeast co-interact with a large variety of microorganisms, thereby affecting the fermentation process and, consequently, the flavour of the wine. The precise connections between microbial interactions and quorum-sensing remain unclear, but we describe here how and when some species start to produce quorum-sensing molecules to synchronously adapt their collective behaviour to new conditions. In Saccharomyces cerevisiae, the quorum-sensing molecules were identified as 2-phenylethanol and tryptophol. However, it was recently shown that also a quorum-sensing molecule formerly identified only in Candida albicans, tyrosol, appears to be regulated in S. cerevisiae according to cell density. This review describes the methods for detection and quantification of those quorum-sensing molecules, their underlying mechanisms of action, and their genetic background. It also examines the external stimuli that evoke the quorum-sensing mechanism in the wine-processing environment. The review closes with insight into the biotechnological applications that are already making use of the advantages of quorum-sensing systems and indicates the important questions that still need to be addressed in future research into quorum-sensing.

Polyphenol, antioxidant and antimicrobial potential of six different white and red wine grape processing leftovers.

BACKGROUND: During winemaking, grape polyphenols are only partly extracted, and consequently unexploited. The main aim was to characterize the phenolic content of freeze-dried grape skin and seed (FDSS) extracts obtained from Slovenian and international grape varieties and to evaluate their antioxidant, antimicrobial and anti-adhesive activities. RESULTS: FDSS of six Vitis vinifera L. grapevine cultivars from Vipava Valley region (Slovenia) underwent extraction and sonification under different conditions. Flavonols were the predominant content of extracts from white 'Zelen' and 'Sauvignon Blanc' grape varieties, with strong antimicrobial activities against Gram-negative bacteria. 'Pinot Noir' FDSS extracted with 50% aqueous ethanol extraction produced a high phenolic content in the final extract, which was further associated with strong antioxidant and antimicrobial activities against all tested bacteria. Bacterial adhesion to stainless steel surfaces with minimal and maximal surface roughness was significantly inhibited (up to 60%) across a wide FDSS concentration range, with lower concentrations also effective with two types of stainless steel surfaces. CONCLUSION: FDSS extracts from winery by-products show interesting phenolic profiles that include flavonols, catechins, anthocyanins and hydroxycinnamic acids, with yields influenced by grapevine cultivar and extraction conditions. The antioxidant, antimicrobial and anti-adhesive activities of 50% aqueous ethanol 'Pinot Noir' FDSS extract reveals potential applications in food, pharmaceutical and cosmetic industries for these bioactive residues. © 2016 Society of Chemical Industry.

Probiotic yeast Saccharomyces boulardii (nom. nud.) modulates adhesive properties of Candida glabrata.

Following the widespread use of immunosuppressive therapy together with broad-spectrum antimycotic therapy, the frequency of mucosal and systemic infections caused by the pathogenic yeast Candida glabrata has increased in the past decades. Due to the resistance of C. glabrata to existing azole drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. In this study, we investigated the effect of the probiotic yeast Saccharomyces boulardii (nom. nud.) on C. glabrata adhesion at different temperatures, pH values, and in the presence of fluconazole, itraconazole and amphotericin B. We also studied the adhesion of C. glabrata co-culture with Candida krusei, Saccharomyces cerevisiae, two bacterial probiotics Lactobacillus rhamnosus and Lactobacillus casei The method used to assess adhesion was crystal violet staining. Our results showed that despite the nonadhesiveness of S. boulardii cells, this probiotic significantly affected the adherence ability of C. glabrata This effect was highly dependent on C. glabrata strain and was either antagonistic or synergistic. Regarding the extrinsic factors, temperature did not indicate any significant influence on this S. boulardii modulatory effect, while at high pH and at increased concentrations of antimycotics, S. boulardii did not manage to repress the adhesion of C. glabrata strains. The experiments of C. glabrata co-cultures with other species showed that the adhesiveness of two separate cultures could not be used to predict the adhesiveness of their co-culture.

Attenuation of Adhesion, Biofilm Formation and Quorum Sensing of Campylobacter jejuni by Euodia ruticarpa.

Thermophilic campylobacters are a major cause of bacterial food-borne diarrhoeal disease. Adherence and biofilm formation are key elements of Campylobacter jejuni persistence in unfavourable environmental conditions. The phytochemical analysis of Euodia ruticarpa fruit ethanol solution extract (EREE) indicated that the major compounds were evodiamine (1), rutaecarpine (2) and evocarpine (9). E. ruticarpa fruit ethanol solution extract, compounds 1 and 2 as well as a mixture of quinolinone alkaloids with 41.7% of 9 were tested for antibacterial, antibiofilm and antiquorum sensing activities against C. jejuni. Minimal inhibitory concentrations varied from 64 to 1024 µg/mL. A mutant strain that lacks the functional gene coding for the CmeB efflux pump protein was the most susceptible. Interestingly, in addition to the wild-type (NCTC 11168) and cmeB mutant, also a mutant that lacks autoinducer-2 production (luxS) was able to adhere (1 h) and to produce a biofilm (24, 48 and 72 h). The subinhibitory concentrations of all preparations at least partly inhibited C. jejuni adhesion and biofilm formation with the most visible effect of the quinolinone alkaloid fraction. Using a Vibrio harveyi luminescence assay, the inhibition of autoinducer-2 production was observed in the wild-type and cmeB mutant after 48 h with the most visible effect of EREE and its fraction Q. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of natural antimicrobials on bacterial cell hydrophobicity, adhesion, and zeta potential.

Interactions between bacterial cells and contact materials play an important role in food safety and technology. As bacterial strains become ever more resistant to antibiotics, the aim of this study was to analyse adhesion of selected foodborne bacterial strains on polystyrene surface and to evaluate the effects of natural antimicrobials on bacterial cell hydrophobicity, adhesion, and zeta potential as strategies of adhesion prevention. The results showed strain-specific adhesion rate on polystyrene. The lowest and the highest adhesion were found for two B. cereus lines. Natural antimicrobials ferulic and rosmarinic acid substantially decreased adhesion, whereas the effect of epigallocatechin gallate was neglectful. Similar results were found for the zeta potential, indicating that natural antimicrobials reduce bacterial adhesion. Targeting bacterial adhesion using natural extracts we can eliminate potential infection at an early stage. Future experimental studies should focus on situations that are as close to industrial conditions as possible.

Characterisation of the yeast and mould biota in traditional white pickled cheeses by culture-dependent and independent molecular techniques.

Artisanal white pickled cheese of Western Serbia is a product of complex microbial community which detection by culture-dependent method only is hampered by its limitations. Thus, in the present study, we used a culture-independent, semi-quantitative technique based on construction of an internal transcribed spacer (ITS)-clone library from metagenomic DNA. This approach, based on direct DNA extraction followed by amplification of fungal internal transcribed regions (ITS) cloned into plasmid and restricted by endonucleases, revealed greater species richness in analysed cheeses and their by-products (17 species in total) compared to the more commonly used techniques of the culture-dependent method (8 species) and LSU-DGGE (10 species). The most frequently occurring yeast species which are commonly associated with cheeses production were Debaryomyces hansenii, Kluyveromyces lactis and Candida zeylanoides. On the other hand, Yarrowia lipolytica and Galactomyces geotrichum were detected only in one cheese sample. Moreover, some species, mainly moulds (Filobasidium globisporum, Cladosporium sp., Aspergillus sp. or Alternaria sp.) were identified only by culture-independent methods. The discrepancies between the techniques were confirmed by low correlation factor and by different indices of general biodiversity and dominance of species. The ITS-clone library approach provides the opportunity to analyse complex fungal communities associated with food products.

High reactive oxygen species levels are detected at the end of the chronological life span of translocant yeast cells.

Chromosome translocation is a major genomic event for a cell, affecting almost every of its life aspects ranging from metabolism, organelle maintenance and homeostasis to gene maintenance and expression. By using the bridge-induced translocation system, we defined the effects of induced chromosome translocation on the chronological life span (CLS) of yeast with particular interest to the oxidative stress condition. The results demonstrate that every translocant strain has a different CLS, but all have a high increase in reactive oxygen species and in lipid peroxides levels at the end of the life span. This could be due to the very unique and strong deregulation of the oxidative stress network. Furthermore, the loss of the translocated chromosome occurs at the end of the life span and is locus dependent. Additionally, the RDH54 gene may play a role in the correct segregation of the translocant chromosome, since in its absence there is an increase in loss of the bridge-induced translocated chromosome.

Quorum-Sensing Kinetics in Saccharomyces cerevisiae: A Symphony of ARO Genes and Aromatic Alcohols.

The kinetics of quorum sensing in Saccharomyces cerevisiae were studied using a mini-fermentation platform. The quorum-sensing molecules were monitored using our previous HPLC approach that is here supported by quantitative real-time PCR analysis of the quorum-sensing genes. We thus initially confirm correlations between peak production rates of the monitored quorum-sensing molecules 2-phenylethanol, tryptophol, and tyrosol and peak expression of the genes responsible for their synthesis: ARO8, ARO9, and ARO10. This confirms the accuracy of our previously implemented kinetic model, thus favoring its use in further studies in this field. We also show that the quorum-sensing kinetics are precisely dependent on the population growth phase and that tyrosol production is also regulated by cell concentration, which has not been reported previously. Additionally, we show that during wine fermentation, ethanol stress reduces the production of 2-phenylethanol, tryptophol, and tyrosol, which opens new challenges in the control of wine fermentation.