Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS.

In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Relationships among ensiling, nutritional, fermentative, microbiological traits and contamination in corn silages addressed with partial least squares regression.

The objective of this work was to reduce the predictor dimensionality and to develop a model able to forecast contamination in corn silages. A survey on 33 dairy farms was performed, and samples from core, lateral, and apical parts of the feed-out face of corn silage bunkers were analyzed for chemical, biological (digestible and indigestible NDF), fermentative (pH, ammonia nitrogen, lactic acid, VFA, and ethanol), and microbiological (yeasts and molds) traits. Corn silage samples were analyzed for cell and spore counts by adoption of a molecular DNA-based method. A partial least squares (PLS) regression with a leave-one-out cross-validation method was used to reduce the dimensionality of the original predictors ( = 30) by projecting the independent variables into latent constructs. In a first step of the model development, the importance of independent variables in predicting contamination was assessed by plotting factor loadings of both dependent and independent variables on the first 2 components and by verifying for each predictor the variable influence on projection values adopting the Wold's criterion as well as the entity of standardized regression coefficients. Three ensiling characteristics (bunker type, presence of lateral wrap plastic film, and penetration resistance as a measurement of the ensiled mass density), a chemical trait (DM), 9 characterizations of the fermentative profile (pH, ammonia nitrogen, acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, ethanol, and lactic acid), and 2 microbiological traits (yeasts and molds) were retained as important terms in the PLS model. Three reduced-variable PLS (rPLS) regressions-the first based on ensiling, chemical, fermentative, and microbiological retained important variables (rPLSecfm); the second based on chemical, fermentative, and microbiological retained important traits (rPLScfm); and the last based on only chemical and fermentative retained important variables (rPLScf)-were performed. The model that best fit the measurements was rPLSecfm. The rPLScfm and rPLScf models had similar regression performances but higher mean square errors of prediction than rPLSecfm. However, all tested models seemed adequate to rank corn silages for low, medium, and high risks of contamination. To avoid the visit on farm by trained people required to measure penetration resistance, the use of the rPLScf model is suggested as a useful tool to assess the risk of in corn silage.

Vaginal microbial communities from synchronized heifers and cows with reproductive disorders.

AIM: To evaluate changes in the resident microbial population in the cranial vaginal mucosa induced by a progesterone-releasing intravaginal device (PRID) compared to the vaginal microbiota of cows with reproductive disorders. METHODS AND RESULTS: Vaginal discharge was evaluated by clinical examination and a Vaginitis Diagnosis Score was performed by exfoliative cytology. All samples classified as positive and some classified as negative by clinical evaluation were later diagnosed as positive for vaginitis by cytological analysis. Bacterial diversity profiles were performed by PCR-DGGE and clustered according to the reproductive health status of the specimens, revealing a correspondence between the structures of the communities in the vagina and the clinical profile. Representative bands from each group were sequenced and identified as Ruminococcus sp., Dialister sp., Escherichia sp./Shigella sp., Virgibacillus sp., Campylobacter sp., Helcoccoccus sp., Staphylococcus sp., Bacillus sp., Actinopolymorpha sp., Exiguobacterium sp., Haemophilus sp./Histophilus sp., Aeribacillus sp., Porphyromonas sp., Lactobacillus sp. and Clostridium sp. CONCLUSION: Our results contribute to the knowledge of the vaginal microbiome in synchronized heifers showing positive or negative clinical vaginitis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of a dynamic vaginal colonization by bacterial consortiums during the synchronization with a widely used PRID protocol. Also, the results reveal the presence of well-known metritis-related pathogens as well as emerging uterine opportunistic pathogens. The provided information will allow to carry out further studies to elucidate functional roles of these native micro-organisms in the bovine reproductive tract.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

AIMS: To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. METHODS AND RESULTS: Fermentation was performed by daily backslopping at 30°C for 10 days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. CONCLUSION: Strains characterization led to the selection of Lact. plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. SIGNIFICANCE AND IMPACT OF THE STUDY: Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products.

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods.

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMß1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential anti-infective agent capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Nanomechanical analysis of Clostridium tyrobutyricum spores.

In this work we report on the measurement of the Young modulus of the external surface of Clostridium tyrobutyricum spores in air with an atomic force microscope. The Young modulus can be reliably measured despite the strong tip-spore adhesion forces and the need to immobilize the spores due to their slipping on most substrates. Moreover, we investigate the disturbing factors and consider some practical aspects that influence the measurements of elastic properties of biological objects with the atomic force microscopy indentation techniques.

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina.

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans, Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park.

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.

Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation.

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.

Identification of fungi from dairy products by means of 18S rRNA analysis.

The role of fungi as cause of spoilage of dairy products, such as cheese and yoghurt, has been clearly demonstrated. Despite of this, there is still a lack in rapid methods for the identification of food-associated fungi. In the course of the present work, molecular taxonomical techniques were developed and used to identify yeasts involved in the spoilage of yoghurt and moulds responsible for spoilage of vacuum-packaged hard cheese. Three methods for DNA extraction and purification were evaluated and the fungus-specific primers TR1 and TR2 were used to amplify a 581-bp fragment within the gene, coding for the small ribosomal subunit (18S rRNA) of fungi. The 18S rRNA sequence analysis of fungi isolated from yoghurt and packaged cheese allowed to identify yeast belonging to Zygosaccharomyces microellipsoides and moulds belonging to Penicillium chrysogenum and Cladosporium cladosporoides.

Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation.

The loss of microbial biodiversity due to the increase in large-scale industrial processes led to the study of the natural microflora present in a typical little known dairy product. The community of lactobacilli was studied in order to understand the natural fermentation of Ricotta forte cheese. The combined use of RAPD analysis, 16S rDNA sequencing and physiological tests allowed 33 different strains belonging to 10 species of Lactobacillus to be characterized. RAPD analysis revealed the heterogeneity of both the Lact. kefiri and Lact. paracasei species. The sequence analysis of the large 16S/23S rRNA spacer region enabled Lact. plantarum to be distinguished from Lact. paraplantarum, two closely related species belonging to the Lact. plantarum group. The recovery of strains endowed with interesting physiological characteristics, such as strong stress resistance, could improve technological and/or organoleptic characteristics of Ricotta forte cheese and other fermented foods.

Molecular characterization of the Lactobacillus community in traditional processing of Mozzarella cheese.

The natural Lactobacillus community involved in traditional Mozzarella cheese production has been investigated. The bacterial associations of whey, curd before stretching and Mozzarella were analyzed using randomly amplified polymorphic DNA (RAPD) to follow growth kinetics, and 16S rDNA sequencing to identify the taxonomical position of isolated strains. Analysis of RAPD fingerprints revealed that the Lactobacillus community was composed of 13 different biotypes and the sequence analysis of 16S rDNA demonstrated that the isolated strains belong to L. plantarum, L. fermentum, L. helveticus and L. casei subsp. casei. In addition, two strains of Weissella hellenica were isolated on selective media for lactobacilli. The four L. casei subsp. casei strains and W. hellenica contained sequences related to the prtP gene coding for proteinase, and the highest proteolytic activity in milk was found in one strain of L. casei subsp.casei.

Physiological and molecular techniques for the study of bacterial community development in sausage fermentation.

The development of the microbial community involved in the production process of Italian dry sausage was investigated using physiological analysis and molecular techniques for strain typing and taxonomical identification. A cycle of sausage production was followed collecting samples during the 2 months of ripening process. Microbiological analysis allowed the identification of the main bacterial groups responsible for the fermentation process as lactobacilli and coagulase-negative staphylococci. The use of a polymerase chain reaction-based technique of strain typing, RAPD fingerprinting, demonstrated that the environmental parameters interact to select a limited number of strains that dominate the fermentation process. The staphylococcal populations were characterized for their physiological properties and the two dominant strains were identified as Staphylococcus xylosus and Staph. sciuri. The use of 16S rDNA sequencing allowed the definition of the taxonomical position of the two dominant strains of lactic acid bacteria, as belonging to Lactobacillus sake and Lact. plantarum.

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture.

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.

Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22.

The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.

In vitro permeability evaluation and colonization of membranes for periodontal regeneration by Porphyromonas gingivalis.

The use of membranes for periodontal regeneration is well established. In clinical use, the exposure of membranes to the oral microflora may result in a pathway for periodontal infections. An important role in this process is played by Porphyromonas gingivalis. The purpose of the present study was to examine the colonization of 6 different bioresorbable and nonresorbable membranes for periodontal regeneration by the strain DSM 20709 of P. gingivalis and to determine the time needed by this microorganism to pass through the membranes. A device consisting of a tube sealed with the membranes and filled with a medium suitable for the growth of P. gingivalis was incubated in a bigger tube containing the same medium to study the process of colonization and the crossing of membranes. The outer tube was inoculated with 10(4) cells of P. gingivalis DSM 20709. The passage of bacteria through the membranes was monitored at 6, 24, and 48 hours by counting the number of cells in the inner tube. The colonized membranes were observed using a scanning electron microscope. Differences in the behavior of the 6 membranes analyzed were demonstrated.

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci.

A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation.

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains.

A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.