Search

In this work, the synergistic/antagonistic impact of glucose and mixed starter cultures, namely Latilactobacillus sakei, Pediococcus pentosaceus, and Staphylococcus xylosus, was evaluated in Italian salami in terms of metab-olomics and sensory profiles. As expected, Salami manufactured with 0.5 % glucose exhibited a substantial pH drop, showing values close to 5 at 12 days of ripening. Metabolomics revealed 1841 metabolites, mainly belonging to amino acids, peptides, glycerolipids, and nucleic acids, showing a greater hierarchical role of glucose addition when compared with inoculated microbial starters. Distinct metabolomic fingerprints could be observed across treatments, mainly concerning glutamyl peptides like gamma-glutamyl-glutamate (related to the kokumi taste), biogenic amines (spermine), and lipid oxidation products (i.e., the oxylipin 13S-hydroperoxylino-lenic acid). Such differences may drive the differences in sensory profiles recorded among treatments. These findings indicate the need to select ad-hoc starter cultures to improve the safety, quality, and sensory traits of salami.

The impact of duckweed extracts (DEs) on the shelf-life of packaged beef burgers was evaluated through classical assays and untargeted metabolomics. Beef burgers were formulated with an antioxidants-free control (CON), 1 g/kg sodium ascorbate (ASC), and increasing levels of a DEs, namely 1 (DE1), 5 (DE5), and 10 (DE10) g/kg, packaged under modified atmosphere and stored at 4 °C for 19 days. The DEs, abundant in phytochemicals, determined no issues with the hygienic status of the product. DEs modulated the redox status, being ineffective in preserving linolenic acid from peroxidation. However, the oxidation marker 2-nonenoic acid was down-accumulated in the DE10 sample following 19 days of storage, recording a lower glutathione:glutathione disulfide ratio. The accumulation of adipate semialdehyde revealed the inefficiency of DEs in coping with protein oxidation, while DEs prevented the accumulation of biogenic amines. Therefore, this work suggests a potential pro-oxidant role of the formulated DEs.

Llama sausage is still produced following artisanal procedures, with the autochthonous microbiota being mainly responsible for the fermentation process. In this work, the taxonomical identification and technological-safety criteria of coagulase-negative staphylococci (CNS) isolated from two dierent productions of llama sausages (P: pilot and A: artisanal) were investigated. Staphylococcus (S) equorum and S. saprophyticus were the species most frequently found in P production, followed by S. succinis and S. warneri; a wider species variability was observed in A factory being S. equorum, S. capitis, S. xylosus, S. pasteuri, S. epidermidis and S. saprophyticus as the main identified species. The technological characterization of 28 CNS strains showed their ability to hydrolyze gelatin and tributyrin together with a relevant nitrate reductase activity. Phenotypic and genotypic approaches were conducted to investigate the main safety traits. Llama’s CNS strains exhibited weak decarboxylase and hemolytic activity and low biofilm production; additionally, no enterotoxin genes were detected. Correlation analysis between phenotypic and genotypic data showed low values for the biofilm parameters, while high correlation was observed for oxacillin, ampicillin, tetracycline and aminoglycosides resistance and their genetic determinants. Data obtained may contribute to broaden knowledge about the autochthonous strains of this poorly studied fermented product, thus helping to select an appropriate combination of potential starter cultures to improve llama sausage safety and quality.

In this work, quinoa and buckwheat cooked seeds were fermented by two autochthonous strains of lactic acid bacteria isolated from the corresponding seeds, namely Lactobacillus paracasei A1 2.6 and Pediococcus pentosaceus GS·B, with lactic acid chemically acidified seeds as control. The impact of cooking and fermentation on the comprehensive phenolic profile of quinoa and buckwheat seeds was evaluated through untargeted ultra-highpressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). Samples were analyzed also for in vitro antioxidant capacity (as FRAP and ORAC assays) and total phenolic content (TPC). The in vitro spectrophotometric assays highlighted that the microbial fermentation was more efficient in increasing (p < .05) the TPC and in vitro antioxidant potential in quinoa cooked seeds. However, an increase (p < .05) in TPC and ORAC radical scavenging was observed in both pseudocereals after the different cooking processes (i.e., boiling or toasting). The untargeted phenolic profiling depicted the comprehensive phenolic composition in these matrices. Raw seeds of both pseudocereals possessed a similar phenolic content (4.4 g kg−1 equivalents; considering free and bound fractions). Besides, the metabolomics-based approach showed that all treatments (i.e., cooking and fermentation) induced the release of specific classes, namely phenolic acids and tyrosols. The PLS-DA multivariate approach identified in flavonoids the best markers allowing to discriminate the different treatments considered (i.e., cooking, chemical acidification and microbial fermentation). These findings support the use of cooking and microbial fermentation to ensure the health-promoting properties of non-wheat grains, such as buckwheat and quinoa.

Pseudocereals as Quinoa (Chenopodium quinoa), Chia (Salvia hispanica), Amaranth (Amaranthus caudatus), Buckwheat (Polygonum fagopyrum) and Hemp (Canapa sativa) produce seeds with high protein and essential amino-acid content. Moreover they are considered a good source of fiber, minerals, polyphenolic compounds, omega-3 and omega-6 fatty acids. In addition, the lack of gluten makes these seeds suitable for new consumers and celiac subjects. The use of these flours is limited due to the low baking and sensory quality of the final products, however strategies such as fermentation could improve both characteristics. The aim of this study was to identify and characterize lactic acid bacteria (LAB) communities present in these seeds in view to design novel functional starter cultures for these flours. Identification and intra-species differentiation allowed to group 66 isolates within five LAB species: Pediococcus pentosaceus (dominant in Buckwheat, Chia and Hemp) Lactobacillus paracasei (prevalent in Amaranth), Leuconostoc mesenteroides and Enterococcus mundtii (common in Quinoa) and Enterococcus faecium. The characterization of 19 representative LAB strains was focused on the technological properties (acidification, proteolytic and amylolytic activities) and the safety characteristics (biogenic amines production, antimicrobial activity against Listeria innocua and Listeria monocytogenes and antibiotic susceptibility) by both traditional and molecular approaches. All strains were able to acidify the medium to pH 4.2 - 4.5 after 24 h except for enterococci. No strain showed amylolytic and amino-acid decarboxylase activities, whereas some proteolytic activity was found in all lactobacilli strains. Three strains of L. paracasei were susceptible to all assayed antibiotics, while most of the strains displayed a double resistance pattern (Kanamycin and Tetracycline). Interestingly, two strains of pediococci exhibited antagonistic activity against Listeria. All these data allow

Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10-22 years of age, and compared the findings with age-matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non-delF508 mutations. We used PCR-DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate-reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate-reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H-2-consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non-microbial routes.

Llama represents for the Andean regions a valid alternative to bovine and pork meat and thanks to the high proteins and low fat content; it can constitute a good product for the novel food market. In this study, culture-dependent and independent methods were applied to investigate the microbial ecology of naturally fermented llama sausages produced in Northwest Argentina. Two different production technologies of llama sausage were investigated: a pilot-plant scale (P) and an artisanal one (A). Results obtained by High-Throughput Sequencing (HTS) of 16S rRNA amplicons showed that the production technologies influenced the development of microbial communities with a different composition throughout the entire fermentation process. Both sequencing and microbiological counts demonstrated that Lactic Acid Bacteria (LAB) contributed largely to the dominant microbiota. When a total of 230 isolates were approached by RAPD-PCR, presumptive LAB strains from P production exhibited an initial variability in RAPD fingerprints switching to a single profile at the final of ripening, while A production revealed a more heterogeneous RAPD pattern during the whole fermentation process. The constant presence of Lactobacillus sakei along the fermentation in both productions was revealed by HTS and confirmed by species-specific PCR from isolated strains. The technological characterization of Lb. sakei isolates evidenced their ability to grow at 15 °C, pH 4.5 and 5% NaCl (95%). Most strains hydrolyzed myofibrillar and sarcoplasmic proteins. Bacteriocins encoding genes and antimicrobial resistance were found in 35% and 42.5% of the strains, respectively. An appropriate choice of a combination of autochthonous strains in a starter formulation is fundamental to improve and standardize llama sausages safety and quality.

Introduction Typical dry fermented sausages have been produced for centuries using natural casings, which are portions of animal intestines derived from slaughtering. Natural casings are very important components of many traditional meat fermented products, and they represent a peculiar case study of gut-derived environments that are used for the production of edible fermented foods. Materials & Methods In the present study we investigated by means of culture-dependent methods and Illumina high-throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. Results Several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted in significant amounts, isolated and characterized at phylogenetic level. High-throughput sequencing analyses revealed a highly diverse bacterial diversity, which differed strongly between casings of different animal species. The technological processes involved in the preparation for casing had also a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Conclusions Overall results indicate that natural casings are an important source of several bacterial species whose role both in the fermentation and in the microbiological properties of the final products has been underestimated.

Methanogens commonly inhabit swine intestine. We analyzed the gut archaeal population by extracting DNA from the feces of nine piglets. We performed PCR to target the V6–V8 region of the 16S rRNA gene. Subsequent denaturing gradient gel electrophoresis (DGGE) revealed the presence of Methanobrevibacter boviskoreani, which has not previously been identified in pigs. We confirmed these data with a PCR-DGGE analysis of the mcrA gene, and subsequent sequencing. At 63 days old, the only band in fecal samples corresponded to M. boviskoreani. The DGGE analysis also showed that Methanobrevibacter smithii, which was abundant at 28 days, was dramatically reduced at 42 days, and it completely disappeared at 63 days. To confirm these data, we quantified M. smithii and the total archaeal population by quantitative PCR (qPCR); moreover, we designed a new set of species-specific primers based on the 16S rRNA gene of M. boviskoreani. The qPCR results confirmed the reduction in M. smithii over time and a simultaneous increase in M. boviskoreani. At 63 days, the total numbers of archaea and M. boviskoreani genomes were comparable, which suggested that M. boviskoreani represented the dominant archaea. This work showed that the archaeal population shifted during weaning, and M. boviskoreani replaced M. smithii

Natural casings derived from different intestine portions have been used for centuries in the production of fresh and dry-fermented sausages. Here we analysed by means of culture-dependent methods and Illumina high-throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. Several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted, isolated and characterised at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. The technological processes involved in the preparation for casing had also a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Natural casings were demonstrated to be complex ecological environments, whose role as microbiological inoculants in the production of sausages should not be underestimated.

In this study the bacterial biodiversity, during the maturation process of traditional sausages (Salame Mantovano), produced with two different kinds of casing (hog middle or "Crespone" and hog bung or "Gentile"), was investigated by means of culture-dependent and -independent methods. In order to assess the natural variability linked to the type of casing used in production, the ingredients, as well as ripening conditions, were identical in both productions. The aim of the study was to understand the contribution of casing microflora during sausage ripening by identifying the dominant species and strains. The bacterial ecology of casings and salami at different ripening stages, as determined by plating, revealed higher staphylococci and enterococci counts for Gentile casing and for the entire ripening period of the salami studied. After molecular identification of 219 Lactobacilli and 225 cocci gram positive catalase positive (GPCP) isolates, the species most frequently isolated were Lactobacillus sakei, Lactobacillus curvatus, Staphylococcus xylosus, and Staphylococcus saprophyticus. Some L. sakei and S. saprophyticus strains, coming from casing, were also found in the salami at different times of ripening. A richer biodiversity was only detected at the beginning of maturation. We also report the first detection, by PCR-DGGE method, of Arcobacter marinus and Brochothrix thermosphacta species in casings and Kokuria salsicia in fresh sausage. Results suggesting that casing can be an important source of bacteria during natural fermentation when starter cultures are not used.

Bacteria carrying antibiotic resistance (AR) genes may represent a hazard for human health. The objective of this study was to evaluate the relationship between the origin, cell number, biotype, phenotypic and genotypic AR profile of coagulase-negative staphylococci (CNS) isolated from the production chain of Salame Piacentino. The 390 isolates were genetically ascribed to 98 different strains belonging to 13 Staphylococcus species: Staphylococcus xylosus and Staphylococcus pasteuri were the predominant followed by Staphylococcus hominis, Staphylococcus cohnii and Staphylococcus epidermidis. Eight antibiotic resistant strains, isolated from skin and salami samples, were detected in different steps along the manufacturing process. Determination of the minimum inhibition concentrations of all CNS strains showed a high frequency of phenotypic tetracycline and erythromycin (83.7% and 68.4% respectively) and a lower kanamycin (40.8%) resistant. The most frequently detected AR genes were ermC (37.8%), tet(L) and tet(K) (31.6% and 34.7% respectively) mainly in skin before washing with water at 65 degrees C and in faeces samples, but only S. xylosus harbouring tet(K) gene was detected during the salami ripening. Our findings show that, the production chain of Salame Piacentino is a source of antibiotic resistant CNS, but the salami manufacturing process leads a strong reduction of the antibiotic resistant strains and the hazard for human health.

Llama meat fermented sausages are traditional products consumed in the Andean region of South America. During sausage fermentation, lactic acid bacteria (LAB) and coagulase negative staphylococci (CNS) play an important role in meat preservation and fermentation processes and for this reason are the most microorganisms used as starter cultures in meat fermentation. The aim of the present study was to identify at species level and characterize for technological and safety characteristics, CNS isolated from fermented llama sausage (without addition of starter cultures), produced in a pilot-plant (P) and on artisanal (A) scale, to select potential starter cultures. A total of 140 colonies were isolated from Baird Parker medium, but only 55 isolates were genetically ascribed to Staphylococcus genera belonging to 6 Staphylococcus species: Staphylococcus equorum and Staphylococcus saprophyticus were the predominant followed by Staphylococcus capitis, Staphylococcus pasteuri and Staphylococcus xylosus and Staphylococcus warneri. Both productions exhibited a high variability in RAPD fingerprints: 14 and 15 strains were clustered in P and A production respectively. The CNS strains were studied to determine technological properties (nitrate reductase, proteolytic and lipolytic activities; and the ability to grow at the temperature and pH values of fermenting sausage, and at high NaCl concentrations) and safety characteristics (amino acid decarboxylase, enterotoxigenic activities and antibiotic susceptibility) by traditional methods and molecular approach. We evaluated the minimum inhibition concentrations (MIC) for eight antibiotics: ampicillin, ofloxacin, oxacyllin, kanamycin, erythromycin, tetracycline, cephaloridine and vancomycin antimicrobial. The MIC results showed high resistance for the first three antibiotics and susceptibility for the last two. Nitrate reductase activity was observed in 87% of S. equorum, whereas was absent in all S. saprophyticus and S. warneri str

Natural casings are portions of animal intestines that have been used for centuries in the manufacturing of many typical dry-fermented sausages. Because of their intestinal origin, casings have a high microbial load, but their possible role as inoculants of microorganisms that can play a role in the ripening of dry-fermented sausages has not been investigated yet in detail. Here, we analyzed by means of culture-dependent methods and Illumina high- throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. We also compared the bacterial ecology of casings, meat mixtures and sausages at different ripening stages in the production chains of Salame Mantovano, a typical Italian dry-fermented sausage. Culture-based methods relied on the isolation and characterization of strains on different typical media, while for the molecular methods multi-million reads were originated and analyzed after amplification and Illumina MiSeq sequencing of 16S rRNA amplicons. From the animal casings, several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted in significant amounts, isolated and characterized at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. It was also found that the technological processes had a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Finally, the analyses on the production chain of Salame Mantovano revealed that several strains found in the casings before stuffing were also detected in the final ripened products, thus confirming our main hypothesis. This work shows the importance of the use of natural casings in the manufacturing of typical dry-fermented products, and highlights

The final characteristics of fermented meats are strongly determined by the microbial species involved in ripening. Here we applied high-throughput sequencing Illumina technology (HTS) coupled to the PCR amplification of the 16s rRNA subunit to assess at fine scale level the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were also carried out in order to be compared with HTS results. A total of 722,196 high quality sequences targeting the two bacterial 16S hypervariable regions V3 and V4 were obtained; manual curation of 16S database allowed a correct taxonomical classification at the species level for 95 % of these reads. HTS results confirmed the presence of main bacterial species involved in the fermentation of salami, but the resolution and the number of identified species were much higher as compared to PCR-DGGE. Thirty-two different Staphylococcus and 33 Lactobacillus species were identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the diversity observed at DNA level. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating a convergence in the overall diversity in different batches of the same local fermented dry sausage.

The final characteristics of fermented meats are strongly determined by the microbial species involved in ripening. Here we applied high-throughput sequencing Illumina technology (HTS) coupled to the PCR amplification of the 16s rRNA subunit to assess at fine scale level the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were also carried out in order to be compared with HTS results. A total of 722,196 high quality sequences targeting the two bacterial 16S hypervariable regions V3 and V4 were obtained; manual curation of 16S database allowed a correct taxonomical classification at the species level for 95 % of these reads. HTS results confirmed the presence of main bacterial species involved in the fermentation of salami, but the resolution and the number of identified species were much higher as compared to PCR-DGGE. Thirty-two different Staphylococcus and 33 Lactobacillus species were identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the diversity observed at DNA level. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating a convergence in the overall diversity in different batches of the same local fermented dry sausage.

The microbiological composition plays a pivotal role in the production, final characteristics and safety of fermented foods. Such composition can be assessed by means of culture-based and molecular methods, where the latter are necessary to analyze non-culturable species. PCR-DGGE has been for several years the most applied technique in molecular microbiology: advantages include the relatively low cost and ease of use and the possibility of processing samples altogether; the method is however only semi-quantitative and the resolution power is limited to a few dominant species. Recent advancements in next generation sequencing technologies (NGS) are revolutionizing our comprehension and quantitative measurements of microbial communities. The initial approach is the same as for PCR-DGGE, i.e., the extraction of microbial DNA from food and the PCR amplification of 16s rRNA hypervariable regions. The PCR amplicons instead of being analyzed on a denaturating gel can be instead sequenced on NGS platforms such as Illumina, which provide multi-million reads of hundreds of base pairs. Here we present the validation of this approach on two case studies, one dealing with typical Italian fermented dry-sausages (salami) from different producers and ripening stages, the second with Grana Padano cheese produced with and without the use of lysozyme. A total of 722,196 high-quality reads where obtained for the salami, 1,240,592 for the cheese case study. More than 95% of these sequences could be classified up to the species level, and the number of species identified was very high: in the case of salami, 33 different species of lactobacilli where quantified, whereas on the same samples PCR-DGGE identified much less. Comparisons with RT-PCR measurements also showed that NGS has the potential for a quantitative assessment of hundreds of species altogether: further studies will be useful in order to validate and standardize the method for metrological measurements in food science.

Contamination of some varieties of soft smear cheeses with Listeria monocytogenes is an important problem for the consumer's health, leading to industrial substantial financial losses. Especially smear and soft traditional Italian cheeses such as Taleggio and Gorgonzola, may be risk products from a listeriosis point of view since they constitute a suitable medium for growth of L. monocytogenes. For this reason there is considerable interest in developing microbial ripening consortia which display anti-listerial activity. In this study, 42 Lactobacillus strain isolated from different traditional Italian cheeses surface (Taleggio, Gorgonzola, Casera Valtellina, Scimudin and Formaggio di Fossa) were analyzed for their ability to produce anti-listeria substances. Lactobacillus sakei, L. curvatus, L. plantarum, L. rhamnosus, L. paracasei, L. casei, L. acidophillus and L. acidipiscis, were preliminary screening by an agar spot test and the well diffusion assays. PCR was also performed to search the presence of bacteriocin encoding genes. Although inhibitory activity against L. monocytogenes was detected for all isolates by the agar stop test, the number of positive strains was severely reduced, when the neutralized cell-free supernatants (CFS) were subjected to the well-diffusion assay resulting much less inhibitory against L. monocytogenes. The preliminary characterization of the antimicrobial substances present on the CFS revealed high thermo stability while the sensibility to the treatment with trypsin, proteinase K and papain was different and strain dependent. Based on our results we found 14 news bacteriocin-producing Lactobacillus strains, belong to the genera L. curvatus (8), L. plantarum (2), L. rhamnosus (1), L. casei (1), L. acidophillus (1) and L. acidipiscis (1). According to trycine–SDS–PAGE, these inhibitory actives molecules are approximately 2.5-7 kDa in size. PCR- based analysis demonstrated a great diversity among bacteriocin genes presents in different

Andean regions are centre of origin and domestication of a large number of plant species such as quinoa and amaranth. Because of their nutritional valua (Balanced composition of essential amino acids, minerals and vitamins) and the absence of gluten, new consumer demands have emerged for these pseudo cereals and their flours. Although the use of such alternative flours is restricted due to their low baking quality, fermentation may improve both the sensory and baking qualities.

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 (MIC ≤ 4 µg/ml; BP = 4 µg/ml) and 94.2% to erythromycin (MIC ≤ 1 µg/ml; BP = 1 µg/ml). Overall, 27 strains appeared to be resistant to tetracycline (16 ≤ MIC ≤ 128 µg/ml ) (TetR), and 7 of them were also resistant to erythromycin (MIC ≥ 1024 µg/ml) (ErmR). The tetM and ermB genes were the most frequently detected in the TetR and/or ErmR 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 ErmR 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 spreadi

Aims: To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages. Methods and Results: We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei, Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet(M) and erm(B). Conclusions: This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy. Significance and Impact of the Study: Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation.