Agapi I. Doulgeraki

Spoilage microbiota associated to the storage of raw meat in different conditions

The spoilage of raw meat is mainly due to undesired microbial development in meat during storage. The type of bacteria and their loads depend on the initial meat contamination and on the specific storage conditions that can influence the development of different spoilage-related microbial populations thus affecting the type and rate of the spoilage process. This review focuses on the composition of raw meat spoilage microbiota and the influence of storage conditions such as temperature, packaging atmosphere and use of different preservatives on the bacterial diversity developing in raw meat. In addition, the most recent tools used for the detection and identification of meat microbiota are also reviewed.

Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens.

A community-based sessile life style is the normal mode of growth and survival for many bacterial species. Under such conditions, cell-to-cell interactions are inevitable and ultimately lead to the establishment of dense, complex and highly structured biofilm populations encapsulated in a self-produced extracellular matrix and capable of coordinated and collective behavior. Remarkably, in food processing environments, a variety of different bacteria may attach to surfaces, survive, grow, and form biofilms. Salmonella enterica, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus are important bacterial pathogens commonly implicated in outbreaks of foodborne diseases, while all are known to be able to create biofilms on both abiotic and biotic surfaces. Particularly challenging is the attempt to understand the complexity of inter-bacterial interactions that can be encountered in such unwanted consortia, such as competitive and cooperative ones, together with their impact on the final outcome of these communities (e.g., maturation, physiology, antimicrobial resistance, virulence, dispersal). In this review, up-to-date data on both the intra- and inter-species interactions encountered in biofilms of these pathogens are presented. A better understanding of these interactions, both at molecular and biophysical levels, could lead to novel intervention strategies for controlling pathogenic biofilm formation in food processing environments and thus improve food safety.

Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions

The whole cell protein and macrorestriction analysis of DNA of Enterobacteriaceae isolates recovered from minced beef stored at 0, 5, 10 and 15 °C aerobically and under modified atmosphere packaging consisting of 40% CO(2)-30% O(2)-30% N(2) in the presence (MAP+) and absence (MAP-) of oregano essential oil were studied. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) profiles obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups. Moreover, application of a modified PFGE protocol with XbaI restriction, resulted into 19 different fingerprints. The Enterobacteriaceae community of fresh meat consisted of Serratia liquefaciens and Serratia proteamaculans. S. liquefaciens strain VK23 was the dominant isolate of Enterobacteriaceae for the most conditions adopted, except 10 °C and 15 °C under MAP + and 10 °C under MAP-. In the latter cases, Hafnia alvei represented the dominant fingerprint. Citrobacter freundii was recovered from minced beef stored aerobically, while H. alvei and Proteus vulgaris were recovered under MAP. Storage conditions affected the Enterobacteriaceae community; modified atmosphere packaging increased both species and strain diversity.

Co-Culture with Listeria monocytogenes within a Dual- Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.

Potential of a simple HPLC-based approach for the identification of the spoilage status of minced beef stored at various temperatures and packaging systems

The shelf life of minced beef stored (i) aerobically, (ii) under modified atmosphere packaging (MAP), and (iii) under MAP with oregano essential oil (MAP/OEO) at 0, 5, 10, and 15°C was investigated. The microbial association of meat and the temporal biochemical changes were monitored. Microbiological analyses, including total viable counts (TVC), Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae, and yeasts/moulds, were undertaken, in parallel with sensory assessment, pH measurement and HPLC analysis of the organic acid profiles. Spectral data collected by HPLC were subjected to statistical analysis, including principal component analysis (PCA) and factorial discriminant analysis (FDA). This revealed qualitative discrimination of the samples based on their spoilage status. Partial least squares regression (PLS-R) was used to evaluate quantitative predictions of TVC, Pseudomonas spp., Br. thermosphacta, lactic acid bacteria, Enterobacteriaceae, and yeasts/moulds. Overall, the HPLC analysis of organic acids, was found to be a potential method to evaluate the spoilage and microbial status of a meat sample regardless of the storage conditions. This could be a very useful tool for monitoring the quality of meat batches during transportation and storage in the meat food chain.

Genotypic characterization of Brochothrix thermosphacta isolated during storage of minced pork under aerobic or modified atmosphere packaging conditions.

A total of 306 colonies were isolated from the selective medium for Brochothrix spp., during the spoilage of minced pork stored at 0, 5, 10 and 15 °C and packed aerobically and under modified atmosphere packaging conditions (MAP). Brochothrix biodiversity was assessed by Pulsed Field Gel Electrophoresis (PFGE), and representative strains were further analysed by Rep-PCR using primer (GTG)₅ and Sau-PCR with primers SAG₁ and SAG₂. Although, different results were obtained from the different methods, a significant diversity among isolates recovered from aerobic conditions was observed. On the contrary, isolates from MAP showed a lower degree of heterogeneity. The storage conditions affected the Brochothrix diversity, the strains isolated in the initial stage being different from the ones present at the final stage of storage at chill temperatures. A representative number of isolates, based on the results of the clustering by molecular methods, were subjected to 16S rRNA gene sequencing, revealing that all belonged to Brochothrix thermosphacta.

Autoinducer-2-like activity in lactic acid bacteria isolated from minced beef packaged under modified atmospheres.

Fifteen fingerprints (assigned to Leuconostoc spp., Leuconostoc mesenteroides, Weissella viridescens, Leuconostoc citreum, and Lactobacillus sakei) of 89 lactic acid bacteria (LAB) isolated from minced beef stored under modified atmospheres at various temperatures were screened for their ability to exhibit autoinducer-2 (AI-2)-like activity under certain growth conditions. Cellfree meat extracts (CFME) were collected at the same time as the LAB isolates and tested for the presence of AI-2-like molecules. All bioassays were conducted using the Vibrio harveyi BAA-1117 (sensor 1(-), sensor 2(+)) biosensor strain. The possible inhibitory effect of meat extracts on the activity of the biosensor strain was also evaluated. AI-2-like activity was observed for Leuconostoc spp. isolates, but none of the L. sakei strains produced detectable AI-2-like activity. The AI-2-like activity was evident mainly associated with the Leuconostoc sp. B 233 strain, which was the dominant isolate recovered from storage at 10 and 15°C and at the initial and middle stages of storage at chill temperatures (0 and 5°C). The tested CFME samples displayed low AI-2-like activity and inhibited AI-2 activity regardless of the indigenous bacterial populations. The LAB isolated during meat spoilage exhibited AI-2-like activity, whereas the LAB strains retrieved depended on storage time and temperature. The production of AI-2-like molecules may affect the dominance of different bacterial strains during storage. The results provide a basis for further research concerning the effect of storage temperature on the expression of genes encoding AI-2 activity and on the diversity of the ephemeral bacterial population.

Methicillin-resistant food-related Staphylococcus aureus: a review of current knowledge and biofilm formation for future studies and applications.

There is increasing concern about the public health impact of methicillin-resistant Staphylococcus aureus. Food and animal are vectors of transmission, but the contribution of a contaminated environment is not well characterized. With regard to this, staphylococcal biofilms serve as a virulence factor, allowing MRSA strains to adhere to surfaces and other materials used in the food industry. Methicillin resistance and biofilm-forming capacity may contribute to the success of S. aureus as a human pathogen in both health care and community settings and the food production chain. This review summarizes current knowledge about the significance of food- and animal-derived MRSA strains and provides data on attachment and biofilm formation of MRSA. In addition, the impact of quorum sensing on MRSA gene expression and biofilm formation is examined.

Quantification and characterization of microbial biofilm community attached on the surface of fermentation vessels used in green table olive processing

The aim of the present study was the quantification of biofilm formed on the surface of plastic vessels used in Spanish-style green olive fermentation and the characterization of the biofilm community by means of molecular fingerprinting. Fermentation vessels previously used in green olive processing were subjected to sampling at three different locations, two on the side and one on the bottom of the vessel. Prior to sampling, two cleaning treatments were applied to the containers, including (a) washing with hot tap water (60 °C) and household detergent (treatment A) and (b) washing with hot tap water, household detergent and bleach (treatment B). Population (expressed as log CFU/cm(2)) of total viable counts (TVC), lactic acid bacteria (LAB) and yeasts were enumerated by standard plating. Bulk cells (whole colonies) from agar plates were isolated for further characterization by PCR-DGGE. Results showed that regardless of the cleaning treatment no significant differences were observed between the different sampling locations in the vessel. The initial microbial population before cleaning ranged between 3.0-4.5 log CFU/cm(2) for LAB and 4.0-4.6 log CFU/cm(2) for yeasts. Cleaning treatments exhibited the highest effect on LAB that were recovered at 1.5 log CFU/cm(2) after treatment A and 0.2 log CFU/cm(2) after treatment B, whereas yeasts were recovered at approximately 1.9 log CFU/cm(2) even after treatment B. High diversity of yeasts was observed between the different treatments and sampling spots. The most abundant species recovered belonged to Candida genus, while Wickerhamomyces anomalus, Debaryomyces hansenii and Pichia guilliermondii were frequently detected. Among LAB, Lactobacillus pentosus was the most abundant species present on the abiotic surface of the vessels.

Next generation microbiological risk assessment meta-omics: The next need for integration

The development of a multi-omics approach has provided a new approach to the investigation of microbial communities allowing an integration of data, which can be used to better understand the behaviour of and interactions between community members. Metagenomics, metatranscriptomics, metaproteomics and metabolomics have the potential of producing a large amount of data in a very short time, however an important challenge is how to exploit and interpret these data to assist risk managers in food safety and quality decisions. This can be achieved by integrating multi-omics data in microbiological risk assessment. In this paper we identify limitations and challenges of the multi-omics approach, underlining promising potentials, but also identifying gaps, which should be addressed for its full exploitation. A view on how this new way of investigation will impact the traditional microbiology schemes in the food industry is also presented.