Microbial contamination pathways in a poultry abattoir provided clues on the distribution and persistence of Arcobacter spp.

The consumption of contaminated poultry meat is a significant threat for public health, as it implicates in foodborne pathogen infections, such as those caused by Arcobacter. The mitigation of clinical cases requires the understanding of contamination pathways in each food process and the characterization of resident microbiota in the productive environments, so that targeted sanitizing procedures can be effectively implemented. Nowadays these investigations can benefit from the complementary and thoughtful use of culture- and omics-based analyses, although their application in situ is still limited. Therefore, the 16S-rRNA gene-based sequencing of total DNA and the targeted isolation of Arcobacter spp. through enrichment were performed to reconstruct the environmental contamination pathways within a poultry abattoir, as well as the dynamics and distribution of this emerging pathogen. To that scope, broiler's neck skin and caeca have been sampled during processing, while environmental swabs were collected from surfaces after cleaning and sanitizing. Metataxonomic survey highlighted a negligible impact of fecal contamination and a major role of broiler's skin in determining the composition of the resident abattoir microbiota. The introduction of Arcobacter spp. in the environment was mainly conveyed by this source rather than the intestinal content. Arcobacter butzleri represented one of the most abundant species and was extensively detected in the abattoir by both metataxonomic and enrichment methods, showing higher prevalence than other more thermophilic Campylobacterota. In particular, Arcobacter spp. was recovered viable in the plucking sector with high frequency, despite the adequacy of the sanitizing procedure.IMPORTANCEOur findings have emphasized the persistence of Arcobacter spp. in a modern poultry abattoir and its establishment as part of the resident microbiota in specific environmental niches. Although the responses provided here are not conclusive for the identification of the primary source of contamination, this biogeographic assessment underscores the importance of monitoring Arcobacter spp. from the early stages of the production chain with the integrative support of metataxonomic analysis. Through such combined detection approaches, the presence of this pathogen could be soon regarded as hallmark indicator of food safety and quality in poultry slaughtering.

Selection of food cultures with protective properties for cooked ham.

Sliced cooked ham stored in modified atmosphere packaging (MAP) can be spoiled by lactic acid bacteria (LAB) which are dominating under psychrotrophic conditions. Depending on the strains, the colonization can result in a premature spoilage characterized by off-flavors, gas and slime production, discoloration, and acidification. The purpose of this study was the isolation, identification and characterization of potential food culture with protective properties, able to prevent or delay spoilage in cooked-ham. The first step was to identify by means of microbiological analysis, the microbial consortia both in unspoiled and in spoiled lots of sliced cooked ham by the use of media for the detection lactic acid bacteria and total viable count. Counts ranged from values lower than 1 Log CFU/g to 9 Log CFU/g in spoiled and unflawed samples. The interaction between consortia was then studied in order to screen for strains able to inhibit spoilage consortia. Strains showing antimicrobial activity were identified and characterized by molecular methods and tested for their physiological features. Among a total of 140 strains isolated, nine were selected for their ability to inhibit a large number of spoilage consortia, to grow and ferment at 4 °C and to produce bacteriocins. The effectiveness of the fermentation made by food culture was evaluated, through challenge tests in situ, analysing the microbial profiles of artificially inoculated cooked-ham slices during storage by high throughput 16 S rRNA gene sequencing. The native population in situ resulted competitive against the inoculated strains and only one strain was able to significantly reduce the native populations reaching about 46.7% of the relative abundance. The results obtained in this study provide information about the selection of autochthonous LAB on the base of their action against spoilage consortia, in order to select protective potential cultures able to improve the microbial quality of sliced cooked ham.

Functional pangenome analysis reveals high virulence plasticity of Aliarcobacter butzleri and affinity to human mucus.

Aliarcobacter butzleri is an emerging pathogen that may cause enteritis in humans, however, the incidence of disease caused by this member of the Campylobacteriaceae family is still underestimated. Furthermore, little is known about the precise virulence mechanism and behavior during infection. Therefore, in the present study, through complementary use of comparative genomics and physiological tests on human gut models, we sought to elucidate the genetic background of a set of 32 A. butzleri strains of diverse origin and to explore the correlation with the ability to colonize and invade human intestinal cells in vitro. The simulated infection of human intestinal models showed a higher colonization rate in presence of mucus-producing cells. For some strains, human mucus significantly improved the resistance to physical removal from the in vitro mucosa, while short time-frame growth was even observed. Pangenome analysis highlighted a hypervariable accessory genome, not strictly correlated to the isolation source. Likewise, the strain phylogeny was unrelated to their shared origin, despite a certain degree of segregation was observed among strains isolated from different segments of the intestinal tract of pigs. The putative virulence genes detected in all strains were mostly encompassed in the accessory fraction of the pangenome. The LPS biosynthesis and in particular the chain glycosylation of the O-antigen is harbored in a region of high plasticity of the pangenome, which would indicate frequent horizontal gene transfer phenomena, as well as the involvement of this hypervariable structure in the adaptive behavior and sympatric evolution of A. butzleri. Results of the present study deepen the current knowledge on A. butzleri pangenome by extending the pool of genes regarded as virulence markers and provide bases to develop new diagnostic approaches for the detection of those strains with a higher virulence potential.

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR < 0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR < 0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community. HIGHLIGHTS Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds. Differences were found between infected and healthy animals in gut microbiota. Candidatus Arthromitus was closely associated with infected birds. M. ornithogaster can affect intestinal microbiota composition of canaries.

A bioinformatics pipeline integrating predictive metagenomics profiling for the analysis of 16S rDNA/rRNA sequencing data originated from foods.

The recent advances in molecular biology, such as the advent of next-generation sequencing (NGS) platforms, have paved the way to new exciting tools which rapidly transform food microbiology. Nowadays, NGS methods such as 16S rDNA/rRNA metagenomics or amplicon sequencing are used for the taxonomic profiling of the food microbial communities. Although 16S rDNA/rRNA NGS-based microbial data are not suited for the investigation of the functional potential of the identified operational taxonomic units as compared to shotgun metagenomics, advances in the bioinformatics discipline allow now the performance of such studies. In this paper, a bioinformatics workflow is described integrating predictive metagenomics profiling with specific application to food microbiology data. Bioinformatics tools pertinent to each sub-module of the pipeline are suggested as well. The published 16S rDNA/rRNA amplicon data originated from an Italian Grana-type cheese, using an NGS platform, was employed to demonstrate the predictive metagenomics profiling approach. The pipeline identified the microbial community and the changes that occurred in the microbial profile during manufacture of the food product studied (taxonomic profiling). The workflow also indicated significant changes in the functional profiling of the community. The tool may help to investigate the functional potential, alterations, and interactions of a microbial community. The proposed workflow may also find an application in the investigation of the ecology of foodborne pathogens encountered in various food products.

Microbiota of an Italian Grana-Like Cheese during Manufacture and Ripening, Unraveled by 16S rRNA-Based Approaches.

UNLABELLED: The microbial ecology of cheese involves a rich and complex interaction between starter lactic acid bacteria and nonstarter lactic acid bacteria (NSLAB), mainly originating from raw milk and/or from the environment, that can contribute to the final characteristics of cheese. The aim of the present research was the exploration of the active microbiota by RNA-based approaches during the manufacturing and ripening of a Grana-like cheese. Reverse transcriptase PCR (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and RNA-based high-throughput sequencing were applied to profile microbial populations, while the enumeration of active bacteria was carried out by using quantitative PCR (qPCR). Three different cheese productions (named D, E, and F) collected in the same month from the same dairy plant were analyzed. The application of the qPCR protocol revealed the presence of 7 log CFU/ml of bacterial load in raw milk, while, during ripening, active bacterial populations ranged from <4 to 8 log CFU/ml. The natural whey starters used in the three productions showed the same microbiota composition, characterized by the presence of Lactobacillus helveticus and Lactobacillus delbrueckii Nevertheless, beta-diversity analysis of the 16S rRNA sequencing data and RT-PCR-DGGE showed a clear clustering of the samples according to the three productions, probably driven by the different milks used. Milk samples were found to be characterized by the presence of several contaminants, such as Propionibacterium acnes, Acidovorax, Acinetobacter, Pseudomonas, and NSLAB. The core genera of the starter tended to limit the development of the spoilage bacteria only in two of the three batches. This study underlines the influence of different factors that can affect the final microbiota composition of the artisanal cheese. IMPORTANCE: This study highlights the importance of the quality of the raw milk in the production of a hard cheese. Independent from the use of a starter culture, raw milk with low microbiological quality can negatively affect the populations of lactic acid bacteria and, as a consequence, impact the quality of the final product due to metabolic processes associated with spoilage bacteria.

Arcobacteraceae: An Exploration of Antibiotic Resistance Featuring the Latest Research Updates.

The Arcobacteraceae bacterial family includes species isolated from animals and related food products. Moreover, these species have been found in other ecological niches, including water. Some species, particularly Arcobacter butzleri and Arcobacter cryaerophilus, have been isolated from human clinical cases and linked to gastrointestinal symptoms. The presence of antibiotic-resistant strains is a concern for public health, considering the possible zoonoses and foodborne infections caused by contaminated food containing bacteria resistant to antibiotic treatments. This review aims to highlight the importance of antibiotic resistance in Arcobacter spp. isolates from several sources, including information about antibiotic classes to which this bacterium has shown resistance. Arcobacter spp. demonstrated a wide spectrum of antibiotic resistance, including several antibiotic resistance genes. Antibiotic resistance genomic traits include efflux pumps and mutations in antibiotic target proteins. The literature shows a high proportion of Arcobacter spp. that are multidrug-resistant. However, studies in the literature have primarily focused on the evaluation of antibiotic resistance in A. butzleri and A. cryaerophilus, as these species are frequently isolated from various sources. These aspects underline the necessity of studies focused on several Arcobacter species that could potentially be isolated from several sources.

Arcobacteraceae comparative genome analysis demonstrates genome heterogeneity and reduction in species isolated from animals and associated with human illness.

The Arcobacteraceae family groups Gram-negative bacterial species previously included in the family Campylobacteraceae. These species of which some are considered foodborne pathogens, have been isolated from different environmental niches and hosts. They have been isolated from various types of foods, though predominantly from food of animal origin, as well as from stool of humans with enteritis. Their different abilities to survive in different hosts and environments suggest an evolutionary pressure with consequent variation in their genome content. Moreover, their different physiological and genomic characteristics led to the recent proposal to create new genera within this family, which is however criticized due to the lack of discriminatory features and biological and clinical relevance. Aims of the present study were to assess the Arcobacteraceae pangenome, and to characterize existing similarities and differences in 20 validly described species. For this, analysis has been conducted on the genomes of the corresponding type strains obtained by Illumina sequencing, applying several bioinformatic tools. Results of the present study do not support the proposed division into different genera and revealed the presence of pangenome partitions with numbers comparable to other Gram-negative bacteria genera, such as Campylobacter. Different gene class compositions in animal and human-associated species are present, including a higher percentage of virulence-related gene classes such as cell motility genes. The adaptation to environmental and/or host conditions of some species was identified by the presence of specific genes. Furthermore, a division into pathogenic and non-pathogenic species is suggested, which can support future research on food safety and public health.

Transcriptome Analysis of Arcobacter butzleri Infection in a Mucus-Producing Human Intestinal In Vitro Model.

Arcobacter butzleri is a foodborne pathogen belonging to the Arcobacteraceae family. This Gram-negative bacterium is found in water, food, and various organisms, including farm animals, clams, and fish. Moreover, A. butzleri has been isolated from human stool samples, where it was associated with gastrointestinal symptoms such as diarrhea. The present study focused on the transcriptome analysis of three A. butzleri strains isolated from human stools and displaying variable virulence potential in vitro. We used a mucus-producing human intestinal in vitro model (Caco-2/HT29-MTX-E12) to study the colonization and invasion abilities of the three A. butzleri strains. The ability of all three A. butzleri strains to colonize our in vitro model system was subsequently confirmed. Moreover, transcriptomics showed the upregulation of putative virulence genes. Among these genes, tonB, exbB, and exbD, which belong to the same operon, were upregulated in strain LMG 11119, which also had the greatest colonization ability. Moreover, genes not currently considered A. butzleri virulence genes were differentially expressed during cell model colonization. The main functions of these genes were linked to organic acid metabolism and iron transport and particularly to the function of the TonB complex. IMPORTANCE Recent advancements in the genomic characterization of A. butzleri revealed putative virulence genes and highlighted the possible pathogenic mechanisms used by this foodborne pathogen. It is therefore possible to study the transcriptomes of these bacteria to explore possible virulence mechanisms under conditions that mimic the infection process. The transcriptome and colonization/invasion analyses that we performed in this study enabled the evaluation of A. butzleri-mediated infection of the mucus-producing human intestinal in vitro model. We confirmed the upregulation of previously proposed virulence genes in the A. butzleri strains. In addition, we identified the differential expression of a number of other genes, which are not currently thought to be associated with virulence, in three A. butzleri strains during infection of mucus-producing human epithelial cells. Changes in the concentration of acetic acid and the upregulation of genes associated with organic acid metabolism during host-pathogen contact were also observed. These findings highlight the importance of previously unreported genes in the virulence mechanisms of A. butzleri.

Metataxonomic signature of beef burger perishability depends on the meat origin prior grinding.

Spoilage dynamics of two beef burger batches from different beef origins were followed from their shared processing run until the use-by date and beyond. Amplicon based sequencing of bacterial and fungal communities were compared with microbial counts and volatilome profile in order to determine whether and at which extent their perishability was related to the batch origin. Microbiological counts did not differ between batch A and B, whereas Volatile Organic Compounds (VOCs) profiles were only distinguishable after the use-by date. Metataxonomic analysis showed that both batches shared the same initial fungal and bacterial community, which however represented a transient signature of the processing run. Indeed, it was rapidly replaced by batch-autochthonous species of fungi and bacteria. Different temporal succession patterns of psychotropic lactic acid bacteria (LAB) were observed between the batches from the fourth day of vacuum storage. In particular, the sequential dominance of Carnobacterium divergens and Leuconostoc piscium in batch B was correlated with a more heterogeneous volatilome and greater production of VOCs linked to off-odours, such as the acetoin. The metataxonomic survey was able to discriminate between the two batches of hamburgers in relation to their origin and regardless of the initially shared processing-derived contamination.

comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence.

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Functional Biodiversity of Yeasts Isolated from Colombian Fermented and Dry Cocoa Beans.

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

Effect of Atmospheric Pressure Plasma on Listeria monocytogenes Attached to Abiotic Surfaces.

Listeria monocytogenes can be introduced into food processing plants via raw material of animal or plant origin and can establish endemic populations through formation of biofilms. Biofilms are a continuous source of contamination for food products, and L. monocytogenes cells in biofilms are more resistant to stress and sanitizing agents than are planktonic cells. The use of gas-discharge plasmas may offer a feasible alternative to conventional sanitization methods. Plasmas are a mixture of charged particles, chemically reactive species, and UV radiation and can be used to destroy microorganisms. The purpose of this study was to measure the effectiveness of cold atmospheric pressure plasma (APP) treatments against bacteria attached to a solid surface and to evaluate the individual susceptibility of various L. monocytogenes strains. Attention was focused on the state of the cells after treatment, combining detection by viable counts and quantitative PCR (qPCR). Most of the culturable cells were inactivated after APP treatment, but the qPCR assay targeting the 16S rRNA revealed the presence of injured cells or their entrance into the viable but nonculturable state. These results were at least partly confirmed by a resuscitation experiment. After APP treatment, L. monocytogenes cell suspensions were incubated in brain heart infusion broth; some cells grew in the medium and therefore had survived the treatment. An understanding of the effects of APP on L. monocytogenes can inform the development of sanitation programs incorporating APP for pathogen removal. Methods other than those based of the culturability of the cells should be used to monitor pathogens in food processing plants because cultivation alone may underestimate the actual microbial load.

Antilisterial Effect and Influence on Listeria monocytogenes Gene Expression of Enterocin or Enterococcus faecalis in Sliced Dry-Cured Ham Stored at 7°C.

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Detection, quantification and vitality of Listeria monocytogenes in food as determined by quantitative PCR.

In this paper we describe the development of a quantitative PCR (qPCR) technique to detect, quantify and determine the vitality of Listeria monocytogenes in foods. The method was based on the amplification of the intergenic region spacer (IGS) between the 16S and 23S rRNA genes. A panel of more than 100 strains of Listeria spp. and non-Listeria was used in order to verify the specificity of the primers and Taqman probe and amplification signals were obtained only when L. monocytogenes DNA and RNA were loaded in the qPCR mix. Standard curves were constructed in several food matrices (milk, meat, soft cheese, fermented sausage, cured ham and ready-to-eat salad). The quantification limit was of 10(3)-10(4) cfu/g or ml, while for the determination of vitality it was 10(4)-10(5) cfu/g or ml. After an overnight enrichment in BHI at 37 degrees C also 10 cfu/g or ml could be detected in all the matrices used in this study. When we applied the protocol to food samples collected from the market or from small food processing plants, on a total number of 66 samples, 4 fresh cheeses from raw milk gave positive results prior to the overnight incubation, while 9 samples, of which only one represented by fresh meat and the others by cheeses from raw milk, were positive after the enrichment. Out of the 4 positive samples, only one could be quantified and it was determined to contain 4x10(3) cfu/g.

Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology.

The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.

Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria.

The aim of this research was to study the bacterial populations involved in the production of artisanal Raschera PDO cheese (Italian Maritime Alps, northwest Italy) in order to collect preliminary knowledge on indigenous lactic acid bacteria (LAB). A total of 21 samples of Raschera PDO cheese, collected from six dairy farms located in the production area, were submitted to microbiological analysis. LAB were randomly isolated from M17 agar, MRS agar and KAA plates and identified by combining PCR 16S-23S rRNA gene spacer analysis, species-specific primers and 16S rRNA gene sequencing. Biodiversity of Lactococcus lactis subsp. lactis isolates was investigated by RAPD-PCR. LAB microflora showed the highest count values among all microbial groups targeted. They reached counts of 10(9) colony forming unit (cfu)/g in cheese samples after 3 days of salting and 15 days of ripening. Yeast population also showed considerable count values, while enterococci and coagulase-negative cocci (CNC) did not overcome 10(7)cfu/g. L. lactis subsp. lactis was the species most frequently isolated from Raschera PDO samples at all different production stages while in aged cheeses Lactobacillus paracasei was frequently isolated. RAPD-PCR highlighted that isolates of L. lactis subsp. lactis isolated from Raschera PDO were highly homogeneous.

Sausage fermentation and starter cultures in the era of molecular biology methods.

Fermented sausages have a long tradition originating from Europe and they constitute a significant part of the Mediterranean diet. This kind of products has a specific microbiota that is typical of the region or area where they are produced. Therefore, in order to protect the traditional aspect of these products, it is essential to understand the microbial ecology during fermentation by studying the dynamic changes that occur and to select autochthonous starter cultures that can be used in the production. In this paper we summarize the state of the art concerning the selection and use of starter cultures and ecology aspects of naturally fermented sausages. We pay particular attention to the application of bacteriocinogenic strains as they could provide an additional tool in the prevention of foodborne pathogens as well as enhancing the competitiveness of the starter organisms. Microbial ecology of fermented sausages has been determined by traditional microbiological methods, but the introduction in food microbiology of new molecular techniques complements the studies carried out so far and allows scientists to overcome the limitations of traditional methods. Next Generation Sequencing (NGS) techniques represent a change in the way microbiologists address ecology and diversity in foods. Indeed the application of metataxonomics and metagenomics will permit a detailed understanding of microbial ecology. A thorough knowledge of the mechanisms behind the biological processes will enhance meat fermentation control and modulation to obtain products with desired organoleptic properties.

Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

Genetic Characterization of Lactic Acid Bacteria Isolated from Tunisian Milk Waste and their Antimicrobial Activity Against some Bacteria Implicated in Nosocomial Infections.

BACKGROUND: A total of 94 lactic acid bacteria (LAB) were isolated from Tunisian artisanal (Ricotta cheese's whey) and industrial (bactofugate) milk waste, identified and then screened for their antimicrobial activity against some bacteria implicated on nosocomial infections. OBJECTIVE: Bacterial genera and species identification was performed using molecular tools. The antimicrobial activity was tested against 7 strains of Gram-negative bacteria and 4 strains of Gram-positive bacteria as well as 6 yeasts. METHOD: The Crude extract was found to have a narrow antimicrobial spectrum on Gram-positive bacteria mainly Listeria monocytogenes. Among the strains which showed antibacterial activity, four were determined to be bacteriocins-producers. They were identified as Lactococcus lactis. RESULTS: Brain Heart Infusion (BHI) Agar was found more adapted than Man, Rogosa and Sharpe (MRS) to investigate the antimicrobial activity of L. actococcus lactis against L. isteria monocytogenes. The genetic determinants encoding the antimicrobial peptides were targeted by specific PCR. CONCLUSION: All L. lactis bacteriocin producing strains possessed the Nisine Z gene (nisZ) except for one, which contained both Nisine A and Nisine Z genes (nisA and nisZ). They have been identified as antilisterial agentS.