Coaggregation of oral bacteria. A physicochemical study based on microcalorimetry

Coaggregation is defined as the specific recognition and interaction between bacteria in suspension. It was first reported in 1970 by Gibbons & Nygaard and has been mostly described between bacteria isolated from human dental plaque. Since the last ten years, coaggregation has been recognized amongst bacteria isolated from freshwater ecosystems, human and animal gastrointestinal and urogenital tracts. ... Zie: Summary.

Screening of fast biofilm formation on stainless steel by thermophilic sporeformers originated from dairy powder and their resistance against CIP

International audienceScreening of fast biofilm formation on stainless steel by thermophilic sporeformers originated from dairy powder and their resistance against CIPLouis DELAUNAYa; Florence POSTOLLECb; Ivan LEGUERINELa; Anne-Gabrielle MATHOTaa Université de Brest, EA3882, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, UMT14.01 SPORE-RISK, IBSAM, 6 rue de l’Université, 29334, Quimper, Franceb ADRIA Développement, UMT14.01 SPORE-RISK, Z.A. de Creac’h Gwen, F-29196, Quimper Cedex, FranceIntroduction: Thermophilic sporeformers are present in raw milk at very low concentration and resist to pasteurisation applicated to destruct vegetative and pathogenic cells. Those spores can adhere to stainless steel due to their hydrophobicity and can form biofilms. Early stage biofilms are important because it can increase the matrix and the adhesion of other cells. Because of those biofilms, the three main species: Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis can resists to Cleaning In Place (CIP) procedure, and contaminate a new process.Material and Methods: Early stage adhesion was conducted on stainless steel submerged by milk inoculated with a fresh culture of bacteria (G. stearothermophilus (N=15), A. flavithermus (N=32) and B. licheniformis (N=15)) for 6h of growth at 55°C under agitation. The ability of sporeformers to form biofilms under those conditions were measured by image analysis after a fluorescent coloration (acridine orange) and random photography. A coverage percentage was calculated by ImageJ ; and a positive threshold was set up at 5% of covering.The efficiency of CIP procedures were obtained after a caustic soda and nitric acid treatment during different duration and temperature of treatment. Tested biofilms were formed in milk during 12h at 55°C, in stainless steel microplates (96 wells) on the same species (3 strains for each) under agitation. Surviving spores were enumerated by the microcolony method.Results:Early stage adhesion shows that 62.5 % (N=20) of A. flavithermus strains can form biofilm within 6h, whereas only 6.7% (N=1) of G. stearothermophilus and 0% (N=0) of B. licheniformis biofilm in 6h at 55°C on submerged stainless steel. However, the maximum covering % on A. flavithermus was 35%; while on the only biofilm forming strain of G. stearothermophilus, this percentage reach 75%. Image analysis also shows biofilm structure from 2D to 3D.The presence and the resistance of spores to chemical cleaning was highly variable within strains. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains.Significance: Those results shows that strong biofilms are mainly composed of spore and are very resistant to CIP used in dairy industries. That is why a better understanding of control methods can lead to a finer and suitableness use of cleaning products

Use of stainless steel chips for the recovery of adhering bacteria in the pipelines of 7 french egg breaking companies

The adhesion of microorganisms to industrial surfaces is frequently observed in the food industry. Adhering bacteria can colonize the surfaces and organize into biofilms that provide cell protection against many stresses such as those due to disinfectants. Poorly controlled, they can be the source of food product contamination and/or food spoilage, and therefore may have important economical and sanitary consequences.The aim of this study was to use stainless steel chips for the identification of the type of adhering bacteria inside the pipelines of 7 French egg breaking companies.Sterile stainless steel chips were suspended inside the pipelines at two levels, before and after the pasteurizer, and at two seasons (summer and winter). After two months, the adhering flora was harvested by sonication and scrapping in tryptone salt, followed by plating onto BHI-YE and incubation for 1 to 7 day(s) at 30°C, with or without oxygen. After incubation, the colonies were identified by sequencing.The harvested cells were mainly facultative aerobic-anaerobic and Gram positive bacteria. However, a broad range of diversity was highlighted, at the location level (7 companies), at the process level (before and after pasteurization), and at the season level (summer and winter).This study, using a simple system of recovery of adhering cells, highlights the flora present on the surface of the equipments of egg breaking companies and represents the first step to better understand and control cell adhesion and biofilm development in this type of food industry

The use of a pilot scale loop system to reproduce microflora adhering to industrial stainless steel pipelines

Persistent bacteria in processing sites are of great concern in food industry, causing continuous recontamination and thus yielding compromised food quality and safety, lost productivity and environmental issues. The aim of this study was to develop a tool to reproduce bioadhesion under industrial conditions in order to investigate the impact of successive stress exposure on adhering microflora and further optimize food formulation, industrial process and surface sanitation.A pilot scale loop system was used to reproduce adhering flora after circulation of liquid egg in stainless steel pipeline at refrigerated temperature. Inoculation of the system was performed either by the circulation of non-pasteurized liquid egg or by the introduction of contaminated probe surface or insert. After 9 days of continuous flow, stainless steel inserts and sections were removed and analyzed with various conditions of incubation. The identification of culturable (ana)aerobe psychrotrophic and mesophilic isolates was performed by 16SrDNA sequencing. Moreover, the detection of Bacillus species was also performed using the PCR based GeneDisc® plateform.After loop circulation of non pasteurized liquid egg, bacterial isolates recovered from stainless steel surfaces were mainly composed of Gram-negative bacteria such as Pseudomonas, Myroides and Aeromonas. Enterobacteria and lactic acid bacteria were as well identified but with a lower extend, while Bacillus species were only detected by GeneDisc® plate. When the loop system was inoculated with an insert preliminary left for 2 months in an industrial pipeline, the proportion of recovered Gram-positive flora was higher. Indeed, Corynebacterium, Enterococcus and Staphylococcus were identified from industrial surfaces.Even though this system might favour fast growing colonizers, correlation was found between bacterial diversity recovered from pilot scale system and industrial pipeline surfaces. It is interesting to note a shift of diversity recovered from surfaces when industrial surfaces were used to inoculate the loop, suggesting the survival and persistence of given species after consecutive steps of production/sanitation. This system is promising to further study bacterial removal and inactivation upon exposure to acid or biocide taking into account protection due to embedded structure or food residues

Problématiques liées à la bioadhésion en IAA. Gestion des biofilms

La formation de biofilm est un phénomène naturel et universel, i.e. l’adhésion microbienne aux surfaces est un phénomène progressif dans lequel les bactéries passent d’un mode planctonique à un mode sessile. Ce phénomène s’accompagne de modifications phénotypiques importantes comme l’expression de facteurs de virulence des cellules adhérées ou la sécrétion massive d’exopolysubstances qui confère à la communauté une résistance accrue aux biocides.Au niveau des industries agroalimentaires, les problématiques liées à la bio-adhésion peuvent être à l’origine de la persistance des contaminations et ne peuvent être dissociées de la maitrise des écoulements et encrassement des surfaces. En effet, les surfaces étant régulièrement soumises à des procédures de nettoyage et désinfection, le phénomène de bio-adhésion est souvent associé aux surfaces difficilement accessibles.La maîtrise des contaminations dues à l’adhésion de flore d’altération au niveau des surfaces se trouvant en contact avec des denrées agroalimentaires constitue donc un enjeu crucial et de nombreux centres d’expertise interviennent sur cette problématique dans le cadre de réseaux au niveau régional ou national.Au travers de quelques cas pratiques, cette intervention présentera l’étude d’écosystèmes complexes analysés par des méthodes microbiologiques et moléculaires, après prélèvement sur sites industriels ou après reconstitution en laboratoire. En effet, pour la recherche de moyens de contrôle ciblés, il reste important d’évaluer la composition, diversité, variabilité saisonnière et localisation des biocontaminations mais aussi de déterminer des cinétiques d’adhésion/décrochage aux surfaces et d’évaluer l’efficacité des principes actifs

Assessing resistance of Anoxybacillus biofilm to optimise sanitation process in dairy powder processing

International audienceIntroduction:Thermophilic sporeforming bacteria (Anoxybacillus flavithermus and Geobacillus stearothermophilus) are the main contaminants of dairy powder, reaching a concentration up to 5 log CFU/g. Spores in raw milk can resist pasteurisation, grow during the process due to favourable conditions and persist on stainless steel surfaces by forming biofilm, giving a resistance against Cleaning In Place (CIP) procedures (caustic soda and nitric acid).Purpose:The study first aimed at I) assessing A. flavithermus biofilm formation in skimmed milk and II) evaluating the resistance of these biofilms to caustic soda and nitric acid for various conditions needed to inactivate adhering vegetative cells and spores.Methods:Firstly, 31 strains of A. flavithermus originated from French dairy powders, characterized by molecular method, were tested for their biofilm coverage on submerged stainless steel coupons after 6 hours of growth in agitated skimmed milk using fluorescent microscopy coupled with an acridine orange stain by random photography analysis.Secondly, to assess the effect of different concentrations of caustic soda and nitric acid at different temperatures and durations of treatment (22 conditions per strains), A. flavithermus biofilm formed on stainless steel microplates (6h at 55°C) were brought into contact with sodium hydroxide and nitric acid solutions. Residual adhered bacteria were enumerated with a spot-plating counting method quantifying resistant vegetative and spores cells.Results:Of the 31 A. flavithermus isolates tested, 20 were biofilm producers (> 5% covering) with a maximum median coverage of 35% and 12 strains were 3D biofilm producer. High amounts of spores (up to 5.9 log spores/cm²) were obtained in 6h in biofilms. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains. A. flavithermus seems more resistant to high caustic soda and low nitric acid concentration as compared to G. stearothermophilus in similar conditions (data not shown). The effect of temperature and time of treatment have also been measured.Significance:Data acquisition on the impact of biocides on biofilms destruction in “real life conditions” is essential to optimize CIP treatment. This study show different behaviour among thermophilic sporeforming bacteria contaminating dairy powders

Recent advances in quantitative PCR (qPCR) applications in food microbiology

 Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported

Preface: spoilers, wonder spores and diehard microorganisms: new insights to integrate these super foes in food spoilage risk management

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