Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus-to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

Amplicon Sequencing of the slpH Locus Permits Culture-Independent Strain Typing of Lactobacillus helveticus in Dairy Products.

The advent of massive parallel sequencing technologies has opened up possibilities for the study of the bacterial diversity of ecosystems without the need for enrichment or single strain isolation. By exploiting 78 genome data-sets from Lactobacillus helveticus strains, we found that the slpH locus that encodes a putative surface layer protein displays sufficient genetic heterogeneity to be a suitable target for strain typing. Based on high-throughput slpH gene sequencing and the detection of single-base DNA sequence variations, we established a culture-independent method to assess the biodiversity of the L. helveticus strains present in fermented dairy food. When we applied the method to study the L. helveticus strain composition in 15 natural whey cultures (NWCs) that were collected at different Gruyère, a protected designation of origin (PDO) production facilities, we detected a total of 10 sequence types (STs). In addition, we monitored the development of a three-strain mix in raclette cheese for 17 weeks.

Prevalence of antibiotic resistance in coagulase-negative staphylococci from spontaneously fermented meat products and safety assessment for new starters.

To provide new meat starter strains lacking antibiotic (AB) resistances, we explored the AB susceptibility in 116 coagulase-negative Staphylococcus (CNS) isolates from traditionally fermented sausages (n=40) manufactured with meat from conventional animal breeding, and from meat products (n=76) made from meat of animals raised in natural habitats under low- or no-antibiotic pressure. Less than 50% of these CNS isolates showed phenotypic resistances to at least one antibiotic (AB) by using microdilution assay. Resistances to penicillins and tetracycline were most often observed and could be traced back to blaZ and tet(K) genes. Prevalence of AB resistances was species-dependent and mainly found in isolates of Staphylococcus warneri (78%), Staphylococcus capitis (75%) and Staphylococcus epidermidis (67%), but only sporadically detected in Staphylococcus carnosus (27%) and Staphylococcus equorum (18%). AB resistances were more often observed in S. xylosus isolates originating from natural habitats compared to traditionally fermented sausages made from conventional meat. A selection of 101 isolates belonging to S. xylosus (n=63), S. carnosus (n=21) and S. equorum (n=17) were subsequently grouped by pulsed-field gel electrophoresis (PFGE) into strain clusters. No S. carnosus and only five S. xylosus strains were lacking AB resistances and exhibited a PFGE genotype different from commercial starters. These strains, together with 17 S. equorum strains, were further studied for safety and technological characteristics. The ability to produce biogenic amines was not detected in any strain. PCR amplifications for enterotoxin encoding genes seg-sej were detected in one, and for δ-hemolysin encoding gene hld in four S. equorum strains, but phenotypic hemolytic activity was visible for three S. xylosus and 15 S. equorum strains. Catalase and nitrate reductase activity was observed in all isolates tested; particularly S. equorum showed high nitrate reduction. In conclusion, we were able to select four new meat starter strains (two S. xylosus and two S. equorum strains) out of 116 investigated CNS, fulfilling all safety criteria including the absence of AB resistances, production of biogenic amines and genes encoding virulence factors but exhibiting high nitrate reductase and catalase activity as suitable technological characteristics. Thus, S. equorum isolates, often the dominant species in spontaneously fermented meat products, provided a prospective meat starter species exhibiting high nitrate reduction and low prevalence of AB resistances.

Identification of staphylococci and dominant lactic acid bacteria in spontaneously fermented Swiss meat products using PCR-RFLP.

Pathogenic, spoilage, and technologically important microorganisms were monitored in 21 spontaneously fermented Swiss meat products manufactured with meat from wildlife or animals grown in natural habitat. Thereby, PCR-restriction fragment length polymorphism (RFLP) on rpoB and 16S rRNA gene sequences provided a powerful tool for fast and accurate identification of the main microbial population. Lactobacillus sakei and Lactobacillus curvatus dominated in fermented meat products followed by Staphylococcus species, which constituted 88.2% of all Gram-positive, catalase-positive cocci (GCC(+)) with cell counts varying from 2.6 to 7.0 log cfu/g during maturation. Staphylococcus equorum was prevalent in frequency and cell counts during maturation (18.0%; 5.0-7.3 log cfu/g) and in the end products (28.4%; 1.8-6.2 log cfu/g) implicating a new presumptive starter species for meat fermentation. Nine out of 14 end products indicated safety risks to consumers due to the high incidence of Staphylococcus saprophyticus or Staphylococcus epidermidis combined with cell counts of 7.4 and 4.9 log cfu/g, respectively. This fact was supported by the detection of Staphylococcus aureus and Enterobacteriaceae in ready-to-eat products strongly exceeding the tolerable limit of 2 log cfu/g. Spontaneously fermented meat products produced from wildlife or animals grown in natural habitats not only gave rise to hygienic and safety concerns but also provided new presumptive starter strains.

Facultative anaerobic halophilic and alkaliphilic bacteria isolated from a natural smear ecosystem inhibit Listeria growth in early ripening stages.

In vitro and in situ anti-listerial properties of 3 strains of Facultative Anaerobic Halophilic and Alkaliphilic (FAHA) species, i.e. Alkalibacterium kapii ALK 6, Marinilactibacillus psychrotolerans ALK 9 and Facklamia tabacinasalis ALK 1, were investigated. The 3 strains were isolated from a smear ecosystem originating from a commercial Raclette type cheese and exhibiting strong anti-listerial activity in situ on cheese surface. In a first step, strains were tested in vitro for production of antimicrobial compounds against Listeria innocua 81000-1 and Listeria ivanovii HPB 28. M. psychrotolerans ALK 9 inhibited both indicator strains in spot-on-the-lawn tests while A. kapii ALK 6 showed no inhibiting effect. F. tabacinasalis ALK 1 exerted an in vitro inhibition on L. ivanovii HPB 28, but induced the formation of dense ball-shaped microcolonies of L. innocua 81000-1 in the soft agar, a typical biofilm microstructure. The extent of the biofilm zone was enhanced when F. tabacinasalis ALK 1 and M. psychrotolerans ALK 9 were tested together. In a second step, different combinations of strains were applied on Raclette cheeses ripened at pilot scale and contaminated with 50 cfu/cm(2)L. innocua at day 7. A control flora of 6 strains, isolated from ecosystem F and corresponding to species commonly found on smear cheeses, was applied on control and test cheeses. In test cheeses, we investigated the impact on Listeria growth of the addition of the 3 FAHA strains, applied as single or mixed cultures. A 1-log inhibition was obtained at day 15 on cheeses treated with FAHA strains applied either as single or mixed cultures. This 1-log inhibition was correlated with the development of FAHA species that reached their maximal count at day 15. This study suggests that the development of FAHA species in early ripening likely contributes to the initial part of the in situ inhibition exerted by the complex cheese surface ecosystem investigated.

Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis.

BACKGROUND: Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M). RESULTS: TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 x 10(2) CFU cm(-2), when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (10(5) CFU cm(-2) on cheeses smeared with a defined surface culture. CONCLUSIONS: This work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. TTGE revealed the presence of marine lactic acid bacteria that are likely related to the strong Listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in Listeria cell count.

Osmotic stress induced by salt increases cell yield, autolytic activity, and survival of lyophilization of Lactobacillus delbrueckii subsp. lactis.

Growth and stress adaptation of an autolytic strain of Lactobacillus delbrueckii subsp. lactis FAM-10991 was studied during pH-controlled batch fermentations. After an initial growth to an optical density at 650 nm of 0.8 under controlled optimal growth conditions (pH 5.5, 37 degrees C, no salt), exponentially growing cells were exposed to salt at concentrations from 1 to 3.5%, and temperatures between 48 and 53.5 degrees C, without pH control or with pH controlled at 5.5 or 4.5. Autolysis was induced by salt concentrations of 2.5 or 3.5% and suppressed at 53.5 degrees C or pH 4.5. Salt at concentrations of 2.5 or 3.5% or a temperature of 53.5 degrees C, without pH control or with pH controlled at 5.5, significantly enhanced (p<0.05) survival of lyophilization as compared with the survival of cells in control cultures or cultures with salt at concentration of 1 and 1.5%. The former conditions increased survival by 125- and 200-fold, respectively. However, no correlation was found between autolytic activity and survival of lyophilization. Cultures grown with salt at 2.5% gave high yields of viable cells in broths before and after lyophilization, with numbers being 27-fold higher than with control cultures, but with autolytic activity that was 2.5-fold higher than in cells from control cultures.