Foodborne Microbial Communities as Potential Reservoirs of Antimicrobial Resistance Genes for Pathogens: A Critical Review of the Recent Literature.

Antimicrobial resistance (AMR) is a global and increasing threat to human health. Several genetic determinants of AMR are found in environmental reservoirs, including bacteria naturally associated with widely consumed fermented foods. Through the food chain, these bacteria can reach the gut, where horizontal gene transfer (HGT) can occur within the complex and populated microbial environment. Numerous studies on this topic have been published over the past decades, but a conclusive picture of the potential impact of the non-pathogenic foodborne microbial reservoir on the spread of AMR to human pathogens has not yet emerged. This review critically evaluates a comprehensive list of recent experimental studies reporting the isolation of AMR bacteria associated with fermented foods, focusing on those reporting HGT events, which represent the main driver of AMR spread within and between different bacterial communities. Overall, our analysis points to the methodological heterogeneity as a major weakness impairing determination or a causal relation between the presence of AMR determinants within the foodborne microbial reservoir and their transmission to human pathogens. The aim is therefore to highlight the main gaps and needs to better standardize future studies addressing the potential role of non-pathogenic bacteria in the spread of AMR.

FDF-DB: A Database of Traditional Fermented Dairy Foods and Their Associated Microbiota.

BACKGROUND: Fermented foods are attracting increasing interest due to their nutritional and health benefits, including a positive impact on gut microbiota exerted by their associated microbes. However, information relative to traditional fermented dairy products, along with their autochthonous microbiota, is still fragmented and poorly standardized. Therefore, our aim was to collect and aggregate data useful for obtaining a comprehensive overview translated in a classical database interface that can be easily handled by users. METHODS: a preliminary inventory was built up by systematically collecting data from publicly available resources for the creation of a list of traditional dairy foods produced worldwide, including additional metadata useful for stratifying, and collapsing subgroups. RESULTS: we developed the Fermented Dairy Food Database (FDF-DB), a feasible resource comprising 1852 traditional dairy foods (cheeses, fermented milks, and yogurt) for which microbial content and other associated metadata such as geographical indication label, country/region of origin, technological aspects were gathered. CONCLUSIONS: FDF-DB is a useful and user-friendly resource where taxonomic information and processing production details converge. This resource will be of great aid for researchers, food industries, stakeholders and any user interested in the identification of technological and microbiological features characterizing traditional fermented dairy products.

Semantics of Dairy Fermented Foods: A Microbiologist's Perspective.

Food ontologies are acquiring a central role in human nutrition, providing a standardized terminology for a proper description of intervention and observational trials. In addition to bioactive molecules, several fermented foods, particularly dairy products, provide the host with live microorganisms, thus carrying potential "genetic/functional" nutrients. To date, a proper ontology to structure and formalize the concepts used to describe fermented foods is lacking. Here we describe a semantic representation of concepts revolving around what consuming fermented foods entails, both from a technological and health point of view, focusing actions on kefir and Parmigiano Reggiano, as representatives of fresh and ripened dairy products. We included concepts related to the connection of specific microbial taxa to the dairy fermentation process, demonstrating the potential of ontologies to formalize the various gene pathways involved in raw ingredient transformation, connect them to resulting metabolites, and finally to their consequences on the fermented product, including technological, health and sensory aspects. Our work marks an improvement in the ambition of creating a harmonized semantic model for integrating different aspects of modern nutritional science. Such a model, besides formalizing a multifaceted knowledge, will be pivotal for a rich annotation of data in public repositories, as a prerequisite to generalized meta-analysis.

Colonization Ability and Impact on Human Gut Microbiota of Foodborne Microbes From Traditional or Probiotic-Added Fermented Foods: A Systematic Review.

A large subset of fermented foods act as vehicles of live environmental microbes, which often contribute food quality assets to the overall diet, such as health-associated microbial metabolites. Foodborne microorganisms also carry the potential to interact with the human gut microbiome via the food chain. However, scientific results describing the microbial flow connecting such different microbiomes as well as their impact on human health, are still fragmented. The aim of this systematic review is to provide a knowledge-base about the scientific literature addressing the connection between foodborne and gut microbiomes, as well as to identify gaps where more research is needed to clarify and map gut microorganisms originating from fermented foods, either traditional or added with probiotics, their possible impact on human gut microbiota composition and to which extent foodborne microbes might be able to colonize the gut environment. An additional aim was also to highlight experimental approaches and study designs which could be better standardized to improve comparative analysis of published datasets. Overall, the results presented in this systematic review suggest that a complex interplay between food and gut microbiota is indeed occurring, although the possible mechanisms for this interaction, as well as how it can impact human health, still remain a puzzling picture. Further research employing standardized and trans-disciplinary approaches aimed at understanding how fermented foods can be tailored to positively influence human gut microbiota and, in turn, host health, are therefore of pivotal importance.

A Comprehensive Evaluation of the Impact of Bovine Milk Containing Different Beta-Casein Profiles on Gut Health of Ageing Mice.

Ageing is often characterised by nutritional deficiencies and functional alterations of the digestive and immune system. The aim of the present study was to analyse the impact of consumption of conventional milk with A1/A2 beta-casein, compared to milk containing only the A2 beta-casein variant, characterised by a protein profile favouring gut health. Twenty-four ageing Balb-c mice (20 months old) were fed for 4 weeks, with either a control diet (CTRL), a diet supplemented with bovine milk containing A1/A2 beta-casein (A1A2) or a diet containing A2/A2 beta-casein (A2A2). Lymphocyte subpopulations, enzymatic activities, cytokine secretion, gut morphology and histopathological alterations were measured in different gut segments, while short-chain fatty acids (SCFAs) content and microbiota composition were evaluated in faecal samples. The A2A2 group showed higher content of faecal SCFAs (in particular, isobutyrate) of intestinal CD4+ and CD19+ lymphocytes in the intraepithelial compartment and improved villi tropism. The A1A2 group showed higher percentages of intestinal TCRγδ+ lymphocytes. Faecal microbiota identified Deferribacteriaceae and Desulfovibrionaceae as the most discriminant families for the A2A2 group, while Ruminococcaceae were associated to the A1A2 group. Taken together, these results suggest a positive role of milk, in particular when containing exclusively A2 beta-casein, on gut immunology and morphology of an ageing mice model.

Supplementation with dairy matrices impacts on homocysteine levels and gut microbiota composition of hyperhomocysteinemic mice.

PURPOSE: Several studies highlighted a correlation between folic acid deficiency and high plasma homocysteine concentration, considered a risk factor for multifactorial diseases. Natural folates represent an emerging alternative strategy to supplementation with synthetic folic acid, whose effects are controversial. The present work was, therefore, performed in hyperhomocysteinemic mice to study the impact of supplementation with dairy matrices containing natural folates on plasma homocysteine levels and faecal microbiota composition. METHODS: Forty mice were divided into six groups, two of which fed control or folic acid deficient (FD) diets for 10 weeks. The remaining four groups were fed FD diet for the first 5 weeks and then shifted to a standard control diet containing synthetic folic acid (R) or a FD diet supplemented with folate-enriched fermented milk (FFM) produced by selected lactic acid bacteria, fermented milk (FM), or milk (M), for additional 5 weeks. RESULTS: Supplementation with dairy matrices restored homocysteine levels in FD mice, although impacting differently on hepatic S-adenosyl-methionine levels. In particular, FFM restored both homocysteine and S-adenosyl-methionine levels to the control conditions, in comparison with FM and M. Next generation sequencing analysis revealed that faecal microbiota of mice supplemented with FFM, FM and M were characterised by a higher richness of bacterial species in comparison with C, FD and R groups. Analysis of beta diversity highlighted that the three dairy matrices determined specific, significant variations of faecal microbiota composition, while hyperhomocysteinemia was not associated with significant changes. CONCLUSIONS: Overall, the results represent a promising starting point for the applicability of food matrices enriched in natural folates to manage hyperhomocysteinemia.

Caenorhabditis Elegans and Probiotics Interactions from a Prolongevity Perspective.

Probiotics exert beneficial effects on host health through different mechanisms of action, such as production of antimicrobial substances, competition with pathogens, enhancement of host mucosal barrier integrity and immunomodulation. In the context of ageing, which is characterized by several physiological alterations leading to a low grade inflammatory status called inflammageing, evidences suggest a potential prolongevity role of probiotics. Unraveling the mechanisms underlying anti-ageing effects requires the use of simple model systems. To this respect, the nematode Caenorhabditis elegans represents a suitable model organism for the study of both host-microbe interactions and for ageing studies, because of conserved signaling pathways and host defense mechanisms involved in the regulation of its lifespan. Therefore, this review analyses the impact of probiotics on C. elegans age-related parameters, with particular emphasis on oxidative stress, immunity, inflammation and protection from pathogen infections. The picture emerging from our analysis highlights that several probiotic strains are able to exert anti-ageing effects in nematodes by acting on common molecular pathways, such as insulin/insulin-like growth factor-1 (IIS) and p38 mitogen-activated protein kinase (p38 MAPK). In this perspective, C. elegans appears to be advantageous for shedding light on key mechanisms involved in host prolongevity in response to probiotics supplementation.

The Foodborne Strain Lactobacillus fermentum MBC2 Triggers pept-1-Dependent Pro-Longevity Effects in Caenorhabditis elegans.

Lactic acid bacteria (LAB) are involved in several food fermentations and many of them provide strain-specific health benefits. Herein, the probiotic potential of the foodborne strain Lactobacillus fermentum MBC2 was investigated through in vitro and in vivo approaches. Caenorhabditis elegans was used as an in vivo model to analyze pro-longevity and anti-aging effects. L. fermentum MBC2 showed a high gut colonization capability compared to E. coli OP50 (OP50) or L. rhamnosus GG (LGG). Moreover, analysis of pumping rate, lipofuscin accumulation, and body bending showed anti-aging effects in L. fermentum MBC2-fed worms. Studies on PEPT-1 mutants demonstrated that pept-1 gene was involved in the anti-aging processes mediated by this bacterial strain through DAF-16, whereas the oxidative stress protection was PEPT-1 independent. Moreover, analysis of acid tolerance, bile tolerance, and antibiotic susceptibility were evaluated. L. fermentum MBC2 exerted beneficial effects on nematode lifespan, influencing energy metabolism and oxidative stress resistance, resulted in being tolerant to acidic pH and able to adhere to Caco-2 cells. Overall, these findings provide new insight for application of this strain in the food industry as a newly isolated functional starter. Furthermore, these results will also shed light on C. elegans molecular players involved in host-microbe interactions.

In Vitro and in Vivo Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives.

Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB) strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 Enterococcus gallinarum, 3 E. casseliflavus, 14 Leuconostoc mesenteroides, 19 Lactobacillus pentosus, 7 L. coryniformis, and 2 L. oligofermentans. The L. pentosus and L. coryniformis strains were subjected to further screening to evaluate their probiotic potential, using a combination of in vitro and in vivo approaches. The majority of them showed high survival rates under in vitro simulated gastro-intestinal conditions, and positive antimicrobial activity against Salmonella enterica serovar Typhimurium, Listeria monocytogenes and enterotoxigenic Escherichia coli (ETEC) pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three L. coryniformis strains were selected as very good performers in the initial in vitro testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth in vitro. Parallel screening employing the simplified model organism Caenorhabditis elegans, fed the Lactobacillus strains as a food source, revealed that one L. pentosus and one L. coryniformis strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial features for novel LAB strains, which renders them promising candidates as starters for the manufacturing of fermented table olives with probiotic added value.

Impact of supplementation with a food-derived microbial community on obesity-associated inflammation and gut microbiota composition.

BACKGROUND: Obesity is a complex pathology associated with dysbiosis, metabolic alterations, and low-grade chronic inflammation promoted by immune cells, infiltrating and populating the adipose tissue. Probiotic supplementation was suggested to be capable of counteracting obesity-associated immune and microbial alterations, based on its proven immunomodulatory activity and positive effect on gut microbial balance. Traditional fermented foods represent a natural source of live microbes, including environmental strains with probiotic features, which could transiently colonise the gut. The aim of our work was to evaluate the impact of supplementation with a complex foodborne bacterial consortium on obesity-associated inflammation and gut microbiota composition in a mouse model. METHODS: C57BL/6J mice fed a 45% high fat diet (HFD) for 90 days were supplemented with a mixture of foodborne lactic acid bacteria derived from the traditional fermented dairy product "Mozzarella di Bufala Campana" (MBC) or with the commercial probiotic GG strain of Lactobacillus rhamnosus (LGG). Inflammation was assessed in epididymal white adipose tissue (WAT) following HFD. Faecal microbiota composition was studied by next-generation sequencing. RESULTS: Significant reduction of epididymal WAT weight was observed in MBC-treated, as compared to LGG and control, animals. Serum metabolic profiling showed correspondingly reduced levels of triglycerides and higher levels of HDL cholesterol, as well as a trend toward reduction of LDL-cholesterol levels. Analysis of the principal leucocyte subpopulations in epididymal WAT revealed increased regulatory T cells and CD4+ cells in MBC microbiota-supplemented mice, as well as decreased macrophage and CD8+ cell numbers, suggesting anti-inflammatory effects. These results were associated with lower levels of pro-inflammatory cytokines and chemokines in WAT explants. Faecal bacterial profiling demonstrated increased Firmicutes/Bacteroidetes ratio in all mice groups following HFD. CONCLUSIONS: Taken together, these results indicate a protective effect of MBC microbiota supplementation toward HFD-induced fat accumulation and triglyceride and cholesterol levels, as well as inflammation, suggesting a stronger effect of a mixed microbial consortium vs single-strain probiotic supplementation. The immunomodulatory activity exerted by the MBC microbiota could be due to synergistic interactions within the microbial consortium, highlighting the important role of dietary microbes with yet uncharacterised probiotic effect.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Impact of NaCl reduction on lactic acid bacteria during fermentation of Nocellara del Belice table olives.

Table olives are widely consumed worldwide but, due to the presence of NaCl in fermenting brines, they contain high levels of sodium. A promising strategy to lower sodium content is the reduction or substitution of NaCl in brines with other chlorides. However, these procedures may impact safety, spoilage, as well as quality and technological properties, including the evolution and final composition of the fermenting microbiota. In the present work the effects of partially replacing NaCl with KCl in fermenting brines on the microbiological quality of Nocellara del Belice olives produced by Spanish style (Sivigliano) or Castelvetrano methods have been analyzed. In both cases, the fermentation steps were performed in parallel, in brines containing either NaCl alone, or partially replaced with different proportions of KCl (25, 50 and 75%), while maintaining a final saline concentration of 9% (Sivigliano method) or 7% (Castelvetrano). To compare microbial dynamics in the experimental brines, changes in bacterial ecology were monitored during fermentation with a polyphasic approach, including both microbiological methods and culture-independent techniques based on DGGE and NGS analysis. The main microbial groups detected in the olive microbiota from both production procedures were LAB and yeasts. Overall, the data demonstrate that partial replacement of NaCl with KCl does not increase the risk of contamination, nor the overgrowth of pathogens or spoiler microbes.

Impact of a Complex Food Microbiota on Energy Metabolism in the Model Organism Caenorhabditis elegans.

The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism.

Functional screening of antibiotic resistance genes from a representative metagenomic library of food fermenting microbiota.

Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.

Bacteriophage P22 to challenge Salmonella in foods.

In this study we considered the influence of phage addition on the fate of Salmonella enterica serovar Typhimurium in different foods. Phage P22 was applied to the following: liquid eggs, energy drinks, whole and skimmed milk, apple juice, chicken breast and chicken mince all spiked with its host, whose growth was monitored for 24 and 48 h at 4 °C. Appreciable host inactivation, generally in the order of 2 log cycles, was achieved compared to phage-free controls in all food matrices when 10(4) UFC/g host inoculum was used. Furthermore, wild food strains belonging to the serotypes Typhimurium, Enteritidis, Derby Give, Newport, Muenchen and Muenster were assayed towards phage P22. Only isolates of Salmonella Typhimurium as well as Salmonella Derby and Salmonella Enteritidis was inhibited by the presence of P22 phage. Additional challenge experiments were carried out by spiking liquid-eggs, chicken breast and chicken mince with mixes of wild Salmonella Typhimurium (at concentration of about 10(4) UFC/g) strains along with their relative phage P22. The results showed a reduction of 2-3 log cycles after 48 h at 4 °C depending on both mix of strains and the specific food. Overall, the results indicate that phages may be useful in the control of food-borne pathogens. The food matrices considered, the liquid more than the solid, do not seem to affect the phage ability of infection compared to similar tests performed in vitro.

Update on antibiotic resistance in foodborne Lactobacillus and Lactococcus species.

Lactobacilli represent a major Lactic Acid Bacteria (LAB) component within the complex microbiota of fermented foods obtained from meat, dairy, and vegetable sources. Lactococci, on the other hand, are typical of milk and fermented dairy products, which in turn represent the vast majority of fermented foods. As is the case for all species originating from the environment, foodborne lactobacilli and lactococci consist of natural, uncharacterized strains, whose biodiversity depends on geographical origin, seasonality, animal feeding/plant growth conditions. Although a few species of opportunistic pathogens have been described, lactobacilli and lactococci are mostly non-pathogenic, Gram-positive bacteria displaying probiotic features. Since antibiotic resistant (AR) strains do not constitute an immediate threat to human health, scientific interest for detailed studies on AR genes in these species has been greatly hindered. However, increasing evidence points at a crucial role for foodborne LAB as reservoir of potentially transmissible AR genes, underlining the need for further, more detailed studies aimed at identifying possible strategies to avoid AR spread to pathogens through fermented food consumption. The availability of a growing number of sequenced bacterial genomes has been very helpful in identifying the presence/distribution of mobile elements associated with AR genes, but open questions and knowledge gaps still need to be filled, highlighting the need for systematic and datasharing approaches to implement both surveillance and mechanistic studies on transferability of AR genes. In the present review we report an update of the recent literature on AR in lactobacilli and lactococci following the 2006 EU-wide ban of the use of antibiotics as feed additives in animal farming, and we discuss the limits of the present knowledge in evaluating possible risks for human health.

Molecular characterization of a novel mosaic tet(S/M) gene encoding tetracycline resistance in foodborne strains of Streptococcus bovis.

The presence of antibiotic-resistance (AR) genes in foodborne bacteria of enteric origin represents a relevant threat to human health in the case of opportunistic pathogens, which can reach the human gut through the food chain. Streptococcus bovis is a human opportunistic pathogen often associated with infections in immune-compromised or cancer patients, and it can also be detected in the environment, including fermented foods. We have focused on the molecular characterization of a tetracycline (Tet)-resistance gene present in 39 foodborne isolates of S. bovis phenotypically resistant to this drug. The gene was identified as a novel tet(S/M) fusion, encoding a mosaic protein composed of the N-terminal 33 amino acids of Tet(S), in-frame with the Tet(M) coding sequence. Heterologous expression of the mosaic gene was found to confer Tet resistance upon Escherichia coli recipients. Moreover, the tet(S/M) gene was found to be transcriptionally inducible by Tet under the endogenous tet(S) promoter in both S. bovis and E. coli. Nucleotide sequencing of the surrounding genomic region of 16.2 kb revealed large blocks of homology with the genomes of Streptococcus infantarius and Lactococcus lactis. A subregion of about 4 kb containing mosaic tet(S/M) was flanked by two copies of the IS1216 mobile element. PCR amplification with primers directed outwards from the tet(S/M) gene identified the presence of a 4.3 kb circular form corresponding to the intervening chromosomal region between the two IS1216 elements, but lacking a replication origin. The circular element shared extensive overall homology with a region of the multidrug-resistance plasmid pK214 from Lc. lactis, containing tet(S), as well as the IS1216 transposase-containing element and intervening non-coding sequences. Linear reconstruction of the insertion events likely to have occurred within this genomic region, inferred from sequence homology, provides further evidence of the chromosomal rearrangements that drive genomic evolution in complex bacterial communities such as the gut and food microbiota.

Antibiotic resistance determinants in the interplay between food and gut microbiota.

A complex and heterogeneous microflora performs sugar and lactic acid fermentations in food products. Depending on the fermentable food matrix (dairy, meat, vegetable etc.) as well as on the species composition of the microbiota, specific combinations of molecules are produced that confer unique flavor, texture, and taste to each product. Bacterial populations within such "fermented food microbiota" are often of environmental origin, they persist alive in foods ready for consumption, eventually reaching the gastro-intestinal tract where they can interact with the resident gut microbiota of the host. Although this interaction is mostly of transient nature, it can greatly contribute to human health, as several species within the food microbiota also display probiotic properties. Such an interplay between food and gut microbiota underlines the importance of the microbiological quality of fermented foods, as the crowded environment of the gut is also an ideal site for genetic exchanges among bacteria. Selection and spreading of antibiotic resistance genes in foodborne bacteria has gained increasing interest in the past decade, especially in light of the potential transferability of antibiotic resistance determinants to opportunistic pathogens, natural inhabitants of the human gut but capable of acquiring virulence in immunocompromised individuals. This review aims at describing major findings and future prospects in the field, especially after the use of antibiotics as growth promoters was totally banned in Europe, with special emphasis on the application of genomic technologies to improve quality and safety of fermented foods.

Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods.

The aim of this study was to evaluate the susceptibility of 197 isolates of Lactobacillus paracasei, isolated from Italian fermented products coming from different geographical areas, to tetracycline and erythromycin, two antimicrobials widely used in clinical and animal therapy. Isolation media were supplemented with antibiotics according to the microbiological breakpoints (BPs) defined by European Food Safety Authority (EFSA). Isolates were identified at the species level and were typed by rep-PCR using the (GTG)(5) primer. A total of 121 genotypically different strains were detected and their phenotypic antimicrobial resistance to tetracycline and erythromycin was determined as the minimum inhibitory concentration (MIC) using the broth microdilution method. The presence of the genes ermB, ermC and tetL, tetM, tetS, tetW, in the phenotypically resistant isolates was investigated by PCR. Tetracycline induction of tetM expression on representative resistant strains, grown in medium either lacking or containing the antibiotic, was also analyzed by RT-PCR. Among the 121 tested strains, 77.7% were susceptible to tetracycline (MICor=1024 microg/ml) (Erm(R)). The tetM and ermB genes were the most frequently detected in the Tet(R) and/or Erm(R) strains. The tetM expression was induced by antibiotic addition to the growth medium. Our study confirmed that L. paracasei is quite sensitive to tetracycline and erythromycin, but the high level of resistance of Erm(R) strains suggested that acquired resistance took place. Further investigations are required to analyze whether the genes identified in L. paracasei isolates might be horizontally transferred to other species. Since "commensal" bacteria, which L. paracasei belongs to, may play an active role in the spreading of antibiotic resistance, a series of measures inspired from a principle of precaution should be taken before they are used as commercial starters or probiotic cultures in food products, complemented by a more prudent use of antibiotics in agriculture, veterinary, and human medicine.

Characterization of the Tn916 conjugative transposon in a food-borne strain of Lactobacillus paracasei.

Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.