Omics-based monitoring of microbial dynamics across the food chain for the improvement of food safety and quality.

The diffusion of high-throughput sequencing has dramatically changed the study of food microbial ecology. Amplicon-based description of the microbial community may be routinary implemented in the food industry to understand how the processing parameters and the raw material quality may affect the microbial community of the final product, as well as how the community changes during the shelf-life. In addition, application of shotgun metagenomics may represent an invaluable resource to understand the functional potential of the microbial community, identifying the presence of spoilage-associated activities or genes related to pathogenesis. Finally, retrieving Metagenome-Assembled Genomes (MAGs) of relevant species may be useful for strain-tracking along the food chain and in case of food poisoning outbreaks. This review gives an overview of the possible applications of sequencing-based approaches in the study of food microbial ecology, highlighting limitations that still prevent the spreading of these techniques to the food industry.

Strain-Level Diversity Analysis of Pseudomonas fragi after In Situ Pangenome Reconstruction Shows Distinctive Spoilage-Associated Metabolic Traits Clearly Selected by Different Storage Conditions.

Microbial spoilage of raw meat causes huge economic losses every year. An understanding of the microbial ecology associated with the spoilage and its dynamics during the refrigerated storage of meat can help in preventing and delaying the spoilage-related activities. The raw meat microbiota is usually complex, but only a few members will develop during storage and cause spoilage upon the pressure from several external factors, such as temperature and oxygen availability. We characterized the metagenome of beef packed aerobically or under vacuum during refrigerated storage to explore how different packaging conditions may influence the microbial composition and potential spoilage-associated activities. Different population dynamics and spoilage-associated genomic repertoires occurred in beef stored aerobically or in vacuum packaging. Moreover, the pangenomes of Pseudomonas fragi strains were extracted from metagenomes. We demonstrated the presence of specific, storage-driven strain-level profiles of Pseudomonas fragi, characterized by different gene repertoires and thus potentially able to act differently during meat spoilage. The results provide new knowledge on strain-level microbial ecology associated with meat spoilage and may be of value for future strategies of spoilage prevention and food waste reduction.IMPORTANCE This work provides insights on the mechanisms involved in raw beef spoilage during refrigerated storage and on the selective pressure exerted by the packaging conditions. We highlighted the presence of different microbial metagenomes during the spoilage of beef packaged aerobically or under vacuum. The packaging condition was able to select specific Pseudomonas fragi strains with distinctive genomic repertoires. This study may help in deciphering the behavior of different biomes directly in situ in food and in understanding the specific contribution of different strains to food spoilage.

Impact of Lactobacillus curvatus 54M16 on microbiota composition and growth of Listeria monocytogenes in fermented sausages.

Lactobacillus curvatus 54M16 produced bacteriocins sak X, sak Tα, sak Tß and sak P. The aim of this study was to investigate the anti-listerial activity of the bacteriocins-producing strain against Listeria monocytogenes in vitro co-culture experiments and during the manufacture of fermented sausages. In MRS broth, Lb. curvatus 54M16 was able to inhibit L. monocytogenes to undetectable levels after 48 h at 20 °C or 5 days at 15 °C. Anti-listerial activity was lower during the production of fermented sausages with pathogen inoculation at levels of approximately 4 Log CFU g-1. However, total inhibition of L. monocytogenes native to the raw ingredients was achieved over the course of the fermentation. Moreover, 16S rRNA-based analysis revealed the ability of Lb. curvatus 54M16 to dominate and affect the bacterial ecosystem, whereas spoilage-associated bacterial genera, such as Brochothrix, Psychrobacter, Pseudomonas and some Enterobacteriaceae, were found until the end of ripening in sausages without Lb. curvatus 54M16. The use of the bacteriocins-producing Lb. curvatus 54M16 in fermented sausages could be an important contribution to product safety, provided that eco-physiological factors and other preservation methods are maintained at levels required for the inhibition of pathogens in controlled conditions.

Dynamics of bacterial communities during manufacture and ripening of traditional Caciocavallo of Castelfranco cheese in relation to cows' feeding.

Traditional Caciocavallo of Castelfranco is a semi-hard "pasta-filata" cheese produced from raw cows' milk in Campania region. The aim of the present research is mainly focused on the study, by 16S rRNA gene pyrosequencing and viable counts, of the dynamics of bacterial communities during manufacture and ripening of traditional Caciocavallo cheese. Moreover, the possible correlation between cheese microbiota and cows' feeding based on silage or hay was also evaluated. In general, except for enterococci, the technological process significantly affected all the microbial groups. According to 16S rRNA, raw cows' milk was dominated by Streptococcus thermophilus, L. lactis and Pseudomonas sp. in hay cheese production, whereas Lactococcus lactis and Acinetobacter sp. dominated silage production. Differences in the taxonomic structure of the milk's microbiota within diet groups were not related to silage and hay cows' feeding. Moreover, S. thermophilus was the unique species that dominate from raw milks to fermented intermediates and cheese in both hay and silage cheese productions. Feeding and ripening time influenced significantly sensory characteristics of the cheeses.

Overlap of Spoilage-Associated Microbiota between Meat and the Meat Processing Environment in Small-Scale and Large-Scale Retail Distributions.

UNLABELLED: Microbial contamination in food processing plants can play a fundamental role in food quality and safety. The aims of this study were to learn more about the possible influence of the meat processing environment on initial fresh meat contamination and to investigate the differences between small-scale retail distribution (SD) and large-scale retail distribution (LD) facilities. Samples were collected from butcheries (n = 20), including LD (n = 10) and SD (n = 10) facilities, over two sampling campaigns. Samples included fresh beef and pork cuts and swab samples from the knife, the chopping board, and the butcher's hand. The microbiota of both meat samples and environmental swabs were very complex, including more than 800 operational taxonomic units (OTUs) collapsed at the species level. The 16S rRNA sequencing analysis showed that core microbiota were shared by 80% of the samples and included Pseudomonas spp., Streptococcus spp., Brochothrix spp., Psychrobacter spp., and Acinetobacter spp. Hierarchical clustering of the samples based on the microbiota showed a certain separation between meat and environmental samples, with higher levels of Proteobacteria in meat. In particular, levels of Pseudomonas and several Enterobacteriaceae members were significantly higher in meat samples, while Brochothrix, Staphylococcus, lactic acid bacteria, and Psychrobacter prevailed in environmental swab samples. Consistent clustering was also observed when metabolic activities were considered by predictive metagenomic analysis of the samples. An increase in carbohydrate metabolism was predicted for the environmental swabs and was consistently linked to Firmicutes, while increases in pathways related to amino acid and lipid metabolism were predicted for the meat samples and were positively correlated with Proteobacteria Our results highlighted the importance of the processing environment in contributing to the initial microbial levels of meat and clearly showed that the type of retail facility (LD or SD) did not apparently affect the contamination. IMPORTANCE: The study provides an in-depth description of the microbiota of meat and meat processing environments. It highlights the importance of the environment as a contamination source of spoilage bacteria, and it shows that the size of the retail facility does not affect the level and type of contamination.

Lactic acid bacteria and their controversial role in fresh meat spoilage.

Lactic acid bacteria (LAB) constitute a heterogeneous group that has been widely associated with fresh meat and cooked meat products. They represent a controversial cohort of microbial species that either contribute to spoilage through generation of offensive metabolites and the subsequent organoleptic downgrading of meat or serve as bioprotective agents with strains of certain species causing unperceivable or no alterations. Therefore, significant distinction among biotypes is substantiated by studies determining spoilage potential as a strain-specific trait corroborating the need to revisit the concept of spoilage.

Bacterial populations and the volatilome associated to meat spoilage.

Microbial spoilage of meat is a complex event to which many different bacterial populations can contribute depending on the temperature of storage and packaging conditions. The spoilage can derive from microbial development and consumption of meat nutrients by bacteria with a consequent release of undesired metabolites. The volatile organic compounds (VOCs) that are generated during meat storage can have an olfactory impact and can lead to rejection of the product when their concentration increase significantly as a result of microbial development. The VOCs most commonly identified in meat during storage include alcohols, aldehydes, ketones, fatty acids, esters and sulfur compounds. In this review, the VOCs found in fresh meat during storage in specific conditions are described together with the possible bacterial populations responsible of their production. In addition, on the basis of the data available in the literature, the sensory impact of the VOCs and their dynamics during storage is discussed to highlight their possible contribution to the spoilage of meat.

Exploring the sources of bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing.

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.

Decarboxylase gene expression and cadaverine and putrescine production by Serratia proteamaculans in vitro and in beef.

Studies of the molecular basis of microbial metabolic activities that are important for the changes in food quality are valuable in order to help in understanding the behavior of spoiling bacteria in food. The growth of a psychrotrophic Serratia proteamaculans strain was monitored in vitro and in artificially inoculated raw beef. Two growth temperatures (25°C and 4°C) were tested in vitro, while growth at 15°C and 4°C was monitored in beef. During growth, the expression of inducible lysine and ornithine-decarboxylase genes was evaluated by quantitative reverse transcription-PCR (qRT-PCR), while the presence of cadaverine and putrescine was quantified by LC-ESI-MS/MS. The expression of the decarboxylase genes, and the consequent production of cadaverine and putrescine were shown to be influenced by the temperature, as well as by the complexity of the growth medium. Generally, the maximum gene expression and amine production took place during the exponential and early stationary phase, respectively. In addition, lower temperatures caused slower growth and gene downregulation. Higher amounts of cadaverine compared to putrescine were found during growth in beef with the highest concentrations corresponding to microbial loads of ca. 9CFU/g. The differences found in gene expression evaluated in vitro and in beef suggested that such activities are more reliably investigated in situ in specific food matrices.

Antimicrobial packaging to retard the growth of spoilage bacteria and to reduce the release of volatile metabolites in meat stored under vacuum at 1°C.

A nisin-EDTA solution was used for activation of the internal surface of plastic bags that were used to store beef chops at 1°C after vacuum packaging. The aim of the work was to evaluate the effect of the antimicrobial packaging on beef during storage. Volatile compounds and microbial populations were monitored after 0, 9, 20, 36, and 46 days of storage. The active packaging retarded the growth of lactic acid bacteria. Brochothrix thermosphacta was unable to grow for the whole storage time in treated samples, while the levels of Carnobacterium spp. in treated samples were below the detection limit for the first 9 days and reached loads below 5 Log CFU/cm(2) after 46 days. On the other hand, Enterobacteriaceae and Pseudomonas spp. were not affected by the use of the antimicrobial packaging and grew in all of the samples, with final populations of about 4 Log CFU/cm(2). Carnobacterium divergens was identified by PCR-denaturing gradient gel electrophoresis analysis of DNA extracted from beef after 36 days of storage. During beef storage, alcohols, aldehydes, ketones, and carboxylic acids were detected in the headspace of beef samples by solid-phase microextraction-gas chromatography-mass spectrometry analysis. The microbial metabolic activity was affected by the use of the antimicrobial film from the beginning up to 36 days with a maximum in the differences of volatile metabolites in samples analyzed at 20 days. The volatiles were also determined by electronic nose, allowing differentiation based on the time of storage and not on the type of packaging. The active packaging reduces the loads of spoilage microbial populations and the release of metabolites in the headspace of beef with a probable positive impact on meat quality.

A combination of modified atmosphere and antimicrobial packaging to extend the shelf-life of beefsteaks stored at chill temperature.

An antimicrobial polyethylene (PE) film was obtained by coating a nisin-based antimicrobial solution. PE sheets were coated on both sides and were used for the packaging of beefsteaks to be stored in air or modified atmosphere packaging (MAP, 60% O2-40% CO2). Microbial populations, species diversity, headspace volatile organic compounds, colour and sensory properties were monitored after 0, 1, 7 and 12 days of storage at 4 °C. The viable counts showed that there was an effect of MAP and antimicrobial film on the development of all the spoilage associated microbial populations. Carnobacterium spp., Brochothrix thermosphacta, Pseudomonas fragi and Rhanella aquatilis were found in most of the samples. C. maltaromaticum was identified in MRS bulk cells from samples stored in air as well as MAP. Quantitative data of headspace-SPME-GC/MS analysis showed that during storage the production of volatile organic compounds (VOCs) was affected by the use of the treated film and the MAP storage. Compounds such as phenylethylalcohol, nonanal, decanal and ethylbutanoate were produced only from 7 to 12 day of storage and only in the samples stored in air. In agreement with the microbiological and VOCs data, the meat stored in active packaging scored the best rankings in the sensory evaluation. Principal component analysis of microbial, sensory and instrumental data showed that beefsteaks stored with the combination of MAP and active packaging for 12 days at 4 °C differed from the other samples that were more associated to high microbial loads, VOCs concentration and meat off odour perception. In conclusion, the antimicrobial sheets in combination with MAP storage at 4 °C were effective for the storage of beefsteaks by retarding the growth of spoilage bacteria, determining lower concentration of VOCs and keeping acceptable levels of colour and other sensory parameters for more than 10 days.

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.

Characterization in the archaeological excavation site of heterotrophic bacteria and fungi of deteriorated wall painting of Herculaneum in Italy.

Microbiological characterization of frescos in four different locations (Collegio degli Augustali, Casa del Colonnato Tuscanico, Casa dello Scheletro and Casa del Gran Portale) of excavation sites of Herculaneum was carried out. The use of infrared thermography allowed detecting sample points on frescos with greatest moisture not visible to the naked eye, resulting in structural damage. The microclimatic conditions provided perfect habitat for bacteria and fungi, particularly of spore forming and mould. In fact, heterotrophic bacteria were prevalent in all wall paintings monitored (ranging from 18 +/- 2 CFU 100 cm(-2) to 68 +/- 4 CFU 100 cm(-2)), whereas fungi were also detected but at lower levels (ranging from 9 +/- 2 CFU 100 cm(-2) to 45 +/- 3 CFU 100 cm(-2)). Cultural-based method allow us to identify by 16S and 26S rRNA partial sequence analysis heterotrophic microorganisms belonging to different genera of Bacillus and Aspergillus, Penicillium and Fusarium together with the unusual genera as Microascus and Coprinus. By using this approach, Bacillus-related species (B. cereus/B. thuringiensis group, B. simplex/B. muralis group, B. megaterium and B. subtilis) were isolated in all sample points analysed with the exception of the Casa dello Scheletro in which Micrococcus luteus/Arthrobactersp. group and Streptomyces fragilis were found. DGGE analysis of PCR amplified V3 region of rDNA from DNA directly recovered from frescos samples, enabled identification of bacterial species not identified using culturable technology asthose closest related to Microbacterium group, often associated with Brevibacterium, Streptomyces and Stenotrophomonas. Combination of culture-dependent and independent methods provided better microbiology characterization of heterotrophic microbiota present on the surface of ancient frescos of this important archaeological site.

Monitoring of microbial metabolites and bacterial diversity in beef stored under different packaging conditions.

Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [(1)H NMR]), and microbial diversity (by PCR-denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that Brochothrix thermosphacta dominated during the early stages of storage in A and MAP, while Pseudomonas spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and Carnobacterium divergens was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions. (1)H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.

Spoilage-related activity of Carnobacterium maltaromaticum strains in air-stored and vacuum-packed meat.

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.

Atomic force microscopy analysis shows surface structure changes in carvacrol-treated bacterial cells.

Carvacrol is a major component in some essential oils such as oregano and thyme and its inhibitory effect on the growth of various microorganisms is well documented. However, the active mechanism of carvacrol, as well as that of other essential oil components, has not yet been fully established and has generally not been well investigated. In this study, the antimicrobial activity of carvacrol against some Gram-positive and Gram-negative food-related bacterial strains was preliminarily verified and the effect of carvacrol on their cell envelope was further investigated by atomic force microscopy analysis. The atomic force microscopy images of the cells treated with carvacrol 3.3 mM for 1 h were analyzed by an appropriate software in order to visualize the effect of the treatment and to determine the values of cell surface roughness and some biometric parameters (cell length and width). The results showed that all microorganisms tested were sensitive to carvacrol both in solid and liquid media. Furthermore, images of cells of all strains treated with carvacrol exhibited appreciable modifications, indicating a change in cell surface structure. Finally, both length and diameter of the microorganisms decreased after contact with carvacrol.

Different molecular types of Pseudomonas fragi have the same overall behaviour as meat spoilers.

The functional diversity of a population of sixty-five different strains of P. fragi isolated from fresh and spoiled meat was studied in order to evaluate the population heterogeneity related to meat spoilage potential. The strains were characterized for the proteolytic activity at 4 degrees C on beef sarcoplasmic proteins and only 9 strains were found to be proteolytic. An iron-dependent growth behaviour was shown when each strain was grown in citrate medium containing either myoglobin, haemoglobin or iron chloride as iron sources. Increase of maximum population and mu(max) in presence of different iron sources was registered. The release of volatile organic compounds (VOC) by each strain in beef during aerobic storage at 4 degrees C was evaluated by GC-MS. A considerable variability of occurrence of each molecule in the GC-MS profiles obtained by the different strains was observed ranging from 3% to 79% although the strains showed a high degree of similarity. In particular, ethylhexanoate, ethyloctanoate, ethylnonenoate, ethyldecanoate, 1-octen-3-ol, 3-octanone, 4-methylthiophenol, and 2-pentylfurane were produced by more than 50% of the strains. Representative strains were used to spoil meat in the same conditions used for the VOC analysis and the samples were evaluated by a sensory panel. The results of the sensory analysis indicated that the different strains could significantly affect the odour of meat and strains characterized by production of esters gave fruity odours to the spoiled meat. However, the similarity of strains based on the sensory profiles does not necessarily match the similarity shown in VOC profiles. P. fragi has a significant role in the microbial ecology of meat and the influence of meat-related sources of iron on the growth behaviour of many different strains suggests that meat can be an ecological niche for P. fragi. Regardless of the proteolytic and lipolytic capacities shown in vitro, different molecular types of P. fragi can release odour active volatile molecules and play a similar overall role as spoilage agents of meat.

Selection and use of phytate-degrading LAB to improve cereal-based products by mineral solubilization during dough fermentation.

New producers of phytate-degrading enzymes, especially lactic acid bacteria (LAB), were used to improve mineral solubilization during dough fermentation. In all, among strains from different sources by microorganisms (150 lactic acid bacteria, 36 yeasts), 38 (24%) exhibited a clear zone around the colonies by hydrolyzing hexacalcium phytate contained in solid medium. When phytase-positive strains from plate assay were tested for phytase activity in liquid medium, 6 of the strains (37%) exhibited phytate-degrading activity in at least one of the 3 different media used. Of the LAB, the highest phytase values were found for Enterococcus faecium A86 (0.74 U/mL) and Lactobacillus plantarum H5 (0.71 U/mL). Two different starter cultures obtained by combinations of phytase-positive (phy+: L. plantarum H5 and L3, Leuconostoc gelidum A16, and E. faecium A86) or phytase-negative (phy-: L. gelidum LM249, L. plantarum H19, and L. plantarum L8) selected LAB strains, were used to measure mineral concentrations of iron, zinc, and manganese during dough fermentation. Although the 2 kinds of starter showed similar acidic values, the presence of phytate-degrading LAB strains increased mineral solubilization in comparison to the starter phy-.

Development of spoilage microbiota in beef stored in nisin activated packaging.

The aim of this study was to assess the microbial populations causing the spoilage of chilled beef during storage and to evaluate the effect of the use of an antimicrobial packaging for the meat storage. A nisin activated antimicrobial packaging was developed by using a nisin, HCL and EDTA solution and used for the storage of beef cuts at 1 degrees C. The common spoilage related microbial groups were monitored during the storage of beef in activated and non activated plastic bags by using selective media. The use of the antimicrobial packaging caused an overall significant reduction of viable counts of Gram positive bacteria such as carnobacteria, lactic acid bacteria and Brochotrix thermosphacta whose development was inhibited for at least 11 days of storage compared to the control. Moreover, a 1-3 log cycles reduction of enterobacteria was also registered between 22 and 32 days of storage. The microbiota was assessed at species level by using Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA gene of DNA extracted directly from meat and from bulk cells from selective media plates and showed that the species occurring within the targeted microbial groups did not change according to storage conditions. In conclusion, the use of the nisin activated packaging reduced the number of spoilage populations but did not affect the species diversity. Improved antimicrobial packaging is needed, possibly coupled with vacuum storage, to possibly achieve a simultaneous inhibition of more spoilage microbial groups and to preserve the microbiological quality of beef during chilled storage.

Pseudomonas fragi strains isolated from meat do not produce N-acyl homoserine lactones as signal molecules.

Quorum sensing (QS) is a signalling mechanism through which bacteria cellular functions are modified to promote access to nutrients and more favorable environmental niches. The frequent occurrence of Pseudomonas spp. in fresh and spoiled meat may involve enhanced gene expression regulated by QS. Several Pseudomonas spp. produce different N-acyl homoserine lactone (AHL) signal molecules. Meat spoilage during aerobic, refrigerated storage is often associated with the presence of Pseudomonas fragi. As with other Pseudomonas species in natural habitats, the dominance and activities of P. fragi in meat may be regulated by QS. In this study, five biosensor strains were used to detect AHL production on three different media by 72 different P. fragi strains isolated from fresh and spoiled meat. Positive and negative AHL-producing strains were used to verify the assays. None of the strains produced detectable quantities of AHLs, even when concentrated cell-free culture supernatants were assayed, nor did exogenous lactones increase biofilm formation in P. fragi strains. However, all isolates produced furanosyl borate diesters (type II autoinducers; AI-2) when tested using the bioluminescent biosensor strain of Vibrio harveyi (BB170). The production of AI-2 was presumed to be of metabolic origin even though Pseudomonas spp. have not been shown to harbor the luxS gene. Thus, the efficient development of P. fragi in fresh meat is not regulated by an AHL-mediated QS system. The mechanism of AI-2 production and its possible role in spoilage dynamics needs further study.