Genetic diversity and safety aspects of enterococci from slightly fermented sausages

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.     

Activation of fermentation by salts in Debaryomyces hansenii

The presence of 1.0 M KCl or NaCl during growth of Debaryomyces hansenii results in increased ethanol production. An additional increase of fermentation was observed when the salts were also present during incubation under nongrowing conditions. Extracts of cells grown in the presence of salt showed increased alcohol dehydrogenase and phosphofructokinase activities, indicating that these enzymes are responsible for the increased fermentation capacity. This is confirmed by measurements of the glycolytic intermediates. The increased fermentation capacity of the cells grown with salts seems to enable them to cope with the additional energy required for uptake and/or efflux of cations.     

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

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.     

Ecology of lactic acid bacteria in Italian fermented sausages: isolation, identification and molecular characterization

In this study, the ecology of the lactic acid bacteria (LAB) of three naturally fermented sausages produced in the Friuli-Venezia-Giulia region, in the North East of Italy, was investigated. A total of 465 strains isolated from three fermentations were identified by molecular methods and 12 different species of LAB were detected. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (67 and 353 strains isolated, respectively) and they were subjected to RAPD-PCR. Clusters containing strains isolated from different plants were observed, underlining a coherent population distribution in three different fermentations. However, we also observed clusters formed by strains isolated from a specific fermentation, only. This could be explained considering the different technologies and recipes used for the production in three plants. Ingredient composition, fermentation and maturation parameters could play an important role in the selection of specific populations adapted in a specific environment.     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate

Plants harvested in the Canary Islands Lanzarote and Fuerteventura were analyzed for the yeasts inhabiting their surface. Half of the isolates (22 out of 44) were identified as Debaryomyces hansenii. Black ascomycetes, viz. Hortaea werneckii and two Hormonema species were represented by 7 strains. Basidiomycetous yeasts, viz. Cryptococcus sp. (8 strains), Rhodotorula sp. (5 strains), Cerinosterus cyanescens (1 strain) and Pseudozyma sp. (1 strain) constituted a minority of 33%. Thirty strains were screened for their ability to assimilate various plant constituents including lipids of the cuticle and the cell membrane, hemicelluloses, nitrogenous compounds (protein, nucleic acids, amino acids) and benzene compounds. All strains were able to assimilate or to hydrolyze lipids, lecithin included. Many strains of D. hansenii, H. dematioides, H. werneckii, C. cyanescens, Cr. laurentii, Pseudozyma sp. and Rh. glutinis were proteolytic. Hemicelluloses like xylan and pectin were assimilated by black ascomycetous yeasts, Cryptococcus sp., Pseudozyma sp. and Rh. glutinis. Ferulic and hydroxycinnamic acids, gallic and tannic acids were assimilated by some strains of H. dematioides, C. cyanescens, Pseudozyma sp. and Rhodotorula sp.     

PCR-DGGE analysis of lactic acid bacteria and yeast dynamics during the production processes of three varieties of Panettone

Aims:  To study lactic acid bacteria (LAB) and yeast dynamics during the production processes of sweet-leavened goods manufactured with type I sourdoughs.
Methods and Results:  Fourteen sourdough and dough samples were taken from a baking company in central Italy during the production lines of three varieties of Panettone. The samples underwent pH measurements and plating analysis on three solid media. The microbial DNA was extracted from both the (sour)doughs and the viable LAB and yeast cells collected in bulk, and subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The molecular fingerprinting of the cultivable plus noncultivable microbial populations provide evidence of the dominance of Lactobacillus sanfranciscensis , Lactobacillus brevis and Candida humilis in the three fermentation processes. The DGGE profiles of the cultivable communities reveal a bacterial shift in the final stages of two of the production processes, suggesting an effect of technological parameters on the selection of the dough microflora.
Conclusions:  Our findings confirm the importance of using a combined analytical approach to explore microbial communities that develop during the leavening process of sweet-leavened goods.
Significance and Impact of the Study:  In-depth studies of sourdough biodiversity and population dynamics occurring during sourdough fermentation are fundamental for the control of the leavening process and the manufacture of standardized, high-quality products.     

Isolation of a psychrotolerant Debaryomyces hansenii strain from fermented tea plant (Camellia sinensis) leaves

Abstract

A psychrotolerant, halotolerant and alkalophilic yeast was isolated from fermented leaves of Camellia sinensis Kuntze, the tea plant. The yeast strain, named Tea-Y1, was both phenotypically and genotypically identified as belonging to the species Debaryomyces hansenii. This assignment was confirmed by scanning and transmission electron microscopy. The analysis of growth curves demonstrated the ability this yeast strain to grow in a temperature range between 4°C and 28°C, with an optimum of 23°C. The ecology of this yeast in the C. sinensis phyllosphere, as well as its possible role in tea fermentation and storage, with particular reference to iced tea, are discussed.     

Ecology and dynamics of coagulase-negative cocci isolated from naturally fermented Italian sausages

Coagulase-negative cocci (CNC) ecology in naturally fermented sausages from Friuli Venezia Giulia region, in the North East of Italy, was investigated. A total of 617 CNC strains, isolated from three different plants during the fermentation process, were identified by traditional methods (biochemical tests) and molecular methods based on species specific PCR, PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of the V3 region of the 16S rRNA gene. The identification, by using biochemical tests, was not successful for 130 strains. Moreover, incongruent results were observed comparing the traditional with the molecular identifications. The same species of CNC were found in all three processing plants, but their contribution to the fermentations was different. In two plants Staphylococcus xylosus was the main species involved in fermentation process, while in the third the maturation was carried out equally by three species: S. xylosus, Staphylococcus warneri and Staphylococcus pasteuri.     

Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. The molecular mechanisms underlying its extreme osmotolerance and halotolerance have drawn considerable attention in the recent past. However, progress in this regard has been limited due to lack of availability of a transformation system and molecular tools to study the functions of the genes in D. hansenii . Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain and the DhHIS4 gene as the selectable marker. By screening the D. hansenii genomic library, we have isolated several autonomous replication sequences that can be used for constructing a replicating vector. Moreover, our study is the first to demonstrate gene disruption in D. hansenii by homologous recombination.     

硫素对不同大豆品种成熟期AM真菌群落多样性的影响

研究硫素对不同大豆品种成熟期丛枝菌根（arbuscular mycorrhizal,AM）真菌群落多样性的影响,探索有利于提高3个特定大豆品种根围土壤和根系AM真菌多样性的最佳施硫量,为提高大豆产量和改善大豆品质提供理论依据。试验采用盆栽,选用黑农44（HN44）、黑农48（HN48）、黑农37（HN37）3个大豆品种作为试验材料,设4个硫素处理S1（对照）,S2（0.02g/kg）,S3（0.04g/kg）和S4（0.06g/kg）。采用PCR-DGGE技术分析3个大豆品种根围土壤和根系中AM真菌群落多样性。结果表明：在S2处理下HN37和HN44根围土壤和根系AM真菌多样性最高,而在S3处理下HN48根围土壤和根系AM真菌多样性最高;DGGE图谱中各样品优势种群变化显著,球囊霉属Glomus和柄囊霉属Funneliformis真菌为3个大豆品种根围土壤和根系中AM真菌的优势菌群。由此可见,硫素对不同大豆品种根围土壤和根系AM真菌群落多样性有显著影响,适量施硫能够提高大豆根围土壤和根系中AM真菌的多样性,不施或过高施硫反而抑制AM真菌的多样性。     

Development of compost maturity and Actinobacteria populations during full-scale composting of organic household waste

AIMS: This study investigates changes in microbiological and physicochemical parameters during large-scale, thermophilic composting of a single batch of municipal organic waste. The inter-relationships between the microbial biomass and community structure as well as several physicochemical parameters and estimates of maturation were evaluated. METHODS AND RESULTS: Analyses of signature fatty acids with the phospholipid fatty acid and ester-linked methods showed that the total microbial biomass was highest during the early thermophilic phase. The contribution of signature 10Me fatty acids from Actinobacteria indicated a relatively constant proportion around 10% of the microbial community. However, analyses of the Actinobacteria species composition with a PCR-denaturing gradient gel electrophoresis approach targeting 16S rRNA genes demonstrated clear shifts in the community structure. CONCLUSIONS: This study demonstrates that compost quality, particularly maturity, is linked to the composition of the microbial community structure, but further studies in other full-scale systems are needed to validate the generality of these findings. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of signature lipid and nucleic acid-based analyses greatly expands the specificity and the scope for assessing the microbial community composition in composts. The results presented in this study give new information on how the development of the compost microbial community is connected to curing and maturation in the later stages of composting, and emphasizes the role of Actinobacteria in this respect.     

Relationship between growth and pH gradients of individual cells of Debaryomyces hansenii as influenced by NaCl and solid substrate

AIMS: To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single-cell level. METHODS AND RESULTS: Using bioimaging techniques, the cell areas and early pH gradients (Delta pH(10)), i.e. the pH gradients determined 10 min after initiation of experiments, were determined for single cells of two D. hansenii strains in fluid and on solid (agar) substrate with and without 8% (w/v) NaCl. The combination of NaCl and solid substrate prolonged the growth initiation of both D. hansenii strains additively. In all our experiments, primarily two groups of cells existed; a vital group consisting of growing single cells with intact early pH gradients, and a group of dead cells without early pH gradients. CONCLUSIONS: Our results show that growth initiation of the D. hansenii cells is severely affected by NaCl and to a lesser extent by the type of substrate in an additive and strain dependent way. Moreover, the early pH gradient of a vital D. hansenii cell cannot be correlated with the rate of its subsequent growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the growth and pH gradients of D. hansenii on solid surfaces in the presence of NaCl.     

Isolation of a lactic acid bacterium and yeast consortium from a fermented material of Ulva spp. (Chlorophyta)

AIMS: Microbiota in a fermented culture of Ulva spp. was examined with the objective to characterize the type of fermentation and to obtain starter microbes for performing seaweed fermentation. METHOD AND RESULTS: Fermented Ulva spp. cultures which were obtained and transferred in a laboratory were examined for their microbiota. With phenotypic characterization and phylogenetic analysis based on rRNA gene nucleotide sequences, the predominant micro-organisms were identified as Lactobacillus brevis, Debaryomyces hanseni var. hansenii, and a Candida zeylanoides-related specimen, suggesting that the observed fermentation can be categorized to lactic acid and ethanol fermentation. Inoculating the individually cultured cell suspensions of the three kinds of micro-organisms with cellulase induced the fermentation in various kinds of seaweed. CONCLUSIONS: A microbial consortium composed of a lactic acid bacterium, L. brevis, and yeasts, D. hansenii and a C. zeylanoides-related specimen, were predominant in a fermented culture of Ulva spp. Lactic acid and ethanol fermentation could be induced in various kinds of seaweed by adding this microbial consortium along with cellulase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of lactic acid and ethanol fermentation in seaweed, which is expected to provide a new material for food and dietary applications.     

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.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Bacterial and fungal communities of wilted Italian ryegrass silage inoculated with and without Lactobacillus rhamnosus or Lactobacillus buchneri

Aims: To understand the effects of lactic acid bacteria (LAB) inoculation on fermentation products, aerobic stability and microbial communities of silage. Methods and Results: Wilted Italian ryegrass was stored in laboratory silos with and without inoculation of Lactobacillus rhamnosus and Lactobacillus buchneri. The silos were opened after 14, 56 and 120 days and then subjected to aerobic deterioration for 7 days. Intensive alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3‐butanediol content at day 14 was about 7 times higher than that of lactic and volatile fatty acids. Alcoholic fermentation was suppressed by L. rhamnosus and L. buchneri inoculation and lactic acid and acetic acid became the dominant fermentation products, respectively. Silages were deteriorated in untreated and L. rhamnosus‐inoculated silages, whereas no spoilage was found in L. buchneri‐inoculated silage. Enterobacteria such as Erwinia persicina, Pantoea agglomerans and Rahnella aquatilis were detected in untreated silage, whereas some of these bacteria disappeared or became faint with L. rhamnosus treatment. When silage was deteriorated, Lactobacillus brevis and Bacillus pumilus were observed in untreated and L. rhamnosus‐inoculated communities, respectively. The inoculated LAB species was detectable in addition to untreated bacterial communities. Saccharomyces cerevisiae and Pichia anomala were the main fungi in untreated and L. rhamnosus‐inoculated silages; however, P. anomala was not visibly seen in L. buchneri‐inoculated silage either at silo opening or after exposure to air. Conclusion: Inoculation with L. rhamnosus can suppress alcoholic fermentation of wilted grass silage with elimination of enterobacteria at the beginning of fermentation. Addition of L. buchneri may improve aerobic stability, with distinct inhibitory effect observed on P. anomala after silo opening. Significance and Impact of the Study: Bacterial and fungal community analyses help us to understand how inoculated LAB can function to improve the fermentation and aerobic stability of silage.