Oenococcus sicerae sp. nov., isolated from French cider.

Two Gram-stain-positive, small ellipsoidal cocci, non-motile, oxidase- and catalase-negative, and facultative anaerobic strains (UCMA15228T and UCMA17102) were isolated in France, from fermented apple juices (ciders). The 16S rRNA gene sequence was identical between the two isolates and showed 97 % similarity with respect to the closest related species Oenococcus oeni and O. kitaharae. Therefore, the two isolates were classified within the genus Oenococcus. The phylogeny based on the pheS gene sequences also confirmed the position of the new taxon. DNA-DNA hybridizations based on in silico genome-to-genome comparisons (GGDC) and Average Nucleotide Identity (ANI) values, as well as species-specific PCR, validated the novelty of the taxon. Various phenotypic characteristics such as the optimum temperature and pH for growth, the ability to metabolise sugars, the aptitude to perform the malolactic fermentation, and the resistance to ethanol and NaCl, revealed that the two strains are distinguishable from the other members of the Oenococcus genus. The combined genotypic and phenotypic data support the classification of strains UCMA15228T and UCMA17102 into a novel species of Oenococcus, for which the name O. sicerae sp. nov. is proposed. The type strain is UCMA15228T (=DSM107163T=CIRM-BIA2288T).

In vitro inhibition of avian pathogenic Enterococcus cecorum isolates by probiotic Bacillus strains.

Enterococcus cecorum is a commensal bacteria and opportunistic pathogen that can cause outbreaks of Enterococcal spondylitis ("kinky back") in poultry, with a growing concern worldwide. Numerous Bacillus-based probiotic strains are commercially available with proven effects in supporting gut health and growth performance, but efficacy against pathogenic E. cecorum is unknown. This study compared the in vitro inhibitory potential of cell-free supernatants (CFSs) of 18 Bacillus strains (14 commercial probiotic strains, 1 internal negative control and 3 type strains) on the growth of 9 clinical E. cecorum isolates. Standardized biomass cultures of live Bacillus were harvested and filtered to obtain CFSs. Inhibitory potential against E. cecorum isolates was assessed via a microdilution assay in which the final pathogen concentration was ∼ 104 CFU/mL. Absorbance (OD) was measured every 15 min for 15 h and used to calculate percentage growth inhibition at an OD equivalent to 0.4 in the positive control (PC) (pathogen but no CFS), and growth delay vs. PC. Growth kinetic responses of pathogen isolate-Bacillus strain combinations ranged from total pathogen inhibition to partial inhibition, lag in growth, no effect, or increased growth vs. PC. Percentage inhibition of individual isolates varied markedly among Bacillus strains, from 100% to -100% (growth promotion as recorded for the type strain) (B. amyloliquefaciens DSM7T). Five B. amyloliquefaciens CFSs produced higher average inhibition rates (>75%) than 2 out of 3 Bacillus licheniformis CFSs (-2.5, and -8.39% vs. PC, respectively) and 1 out of 2 Bacillus subtilis CFSs (7.3% vs. PC) (P < 0.05). Commercial strain 3AP4 exhibited the highest average percentage inhibition vs. PC (85.0% ± 7.9) and the most consistent inhibitory effect across pathogen isolates. The findings indicate that some commercially available poultry probiotic Bacillus strains are effective at inhibiting pathogenic E. cecorum in vitro, but effects are highly strain and pathogen isolate-dependent. Further work is required to confirm effects in vivo and isolate the inhibitory substances.

Multiplex PCR for rapid identification of major lactic acid bacteria genera in cider and other fermented foods.

Lactobacillus, Pediococcus, Oenococcus and Leuconostoc are the main Lactic Acid Bacteria (LAB) genera present in cider as they are able to survive this hostile environment. LAB play a significant role in cider quality, for example in the process of malolactic fermentation, even though they can also be involved in spoilage of cider (production of biogenic amines, exopolysaccharides, off-flavours…). In this context a better monitoring of the fermentation process is a matter of interest to guarantee cider quality. In the present study, we designed a genus-specific multiplex PCR for a rapid and simultaneous detection of the four main LAB genera involved in cider production. This multiplex PCR worked equally with purified genomic DNA of bacterial isolates and with colonies directly picked from agar plates. This new PCR method was also successfully extended to wine and dairy isolates, and thus constitutes an effective tool to quickly identify LAB associated with fermented foods. Moreover, many biodiversity studies would also benefit from this fast, cheap and reliable identification method.

Microorganisms in Fermented Apple Beverages: Current Knowledge and Future Directions.

Production of fermented apple beverages is spread all around the world with specificities in each country. 'French ciders' refer to fermented apple juice mainly produced in the northwest of France and often associated with short periods of consumption. Research articles on this kind of product are scarce compared to wine, especially on phenomena associated with microbial activities. The wine fermentation microbiome and its dynamics, organoleptic improvement for healthy and pleasant products and development of starters are now widely studied. Even if both beverages seem close in terms of microbiome and process (with both alcoholic and malolactic fermentations), the inherent properties of the raw materials and different production and environmental parameters make research on the specificities of apple fermentation beverages worthwhile. This review summarizes current knowledge on the cider microbial ecosystem, associated activities and the influence of process parameters. In addition, available data on cider quality and safety is reviewed. Finally, we focus on the future role of lactic acid bacteria and yeasts in the development of even better or new beverages made from apples.

Early adaptation to oxygen is key to the industrially important traits of Lactococcus lactis ssp. cremoris during milk fermentation.

BACKGROUND: Lactococcus lactis is the most used species in the dairy industry. Its ability to adapt to technological stresses, such as oxidative stress encountered during stirring in the first stages of the cheese-making process, is a key factor to measure its technological performance. This study aimed to understand the response to oxidative stress of Lactococcus lactis subsp. cremoris MG1363 at the transcriptional and metabolic levels in relation to acidification kinetics and growth conditions, especially at an early stage of growth. For those purposes, conditions of hyper-oxygenation were initially fixed for the fermentation. RESULTS: Kinetics of growth and acidification were not affected by the presence of oxygen, indicating a high resistance to oxygen of the L. lactis MG1363 strain. Its resistance was explained by an efficient consumption of oxygen within the first 4 hours of culture, leading to a drop of the redox potential. The efficient consumption of oxygen by the L. lactis MG1363 strain was supported by a coherent and early adaptation to oxygen after 1 hour of culture at both gene expression and metabolic levels. In oxygen metabolism, the over-expression of all the genes of the nrd (ribonucleotide reductases) operon or fhu (ferrichrome ABC transports) genes was particularly significant. In carbon metabolism, the presence of oxygen led to an early shift at the gene level in the pyruvate pathway towards the acetate/2,3-butanediol pathway confirmed by the kinetics of metabolite production. Finally, the MG1363 strain was no longer able to consume oxygen in the stationary growth phase, leading to a drastic loss of culturability as a consequence of cumulative stresses and the absence of gene adaptation at this stage. CONCLUSIONS: Combining metabolic and transcriptomic profiling, together with oxygen consumption kinetics, yielded new insights into the whole genome adaptation of L. lactis to initial oxidative stress. An early and transitional adaptation to oxidative stress was revealed for L. lactis subsp. cremoris MG1363 in the presence of initially high levels of oxygen. This enables the cells to maintain key traits that are of great importance for industry, such as rapid acidification and reduction of the redox potential of the growth media.

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm.

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.

Aromatic amino acids as precursors of antimicrobial metabolites in Geotrichum candidum.

Geotrichum candidum ATCC 204307 was previously found to generate phenyllactic acid (PLA) and indoleacetic acid (ILA) in complex culture media. In this study, a relationship between concentrations of PLA, ILA, and hydroxy PLA (OH-PLA) and initial concentrations of phenylalanine, tryptophan, and tyrosine, added respectively as unique sources of nitrogen in synthetic medium, was established. Phenylpyruvic acid (PPA), an intermediate compound of PLA metabolism, was able to induce not only PLA but also phenylethyl alcohol (PEA) production when used separately as initial substrate. Under pH, temperature, and salt concentrations used for cheese-making, phenylalanine was found to be the most efficient substrate for antimicrobial metabolite production. In excess of substrate, different yeast strains of Geotrichum candidum, Yarrowia lipolytica, Candida natalensis, and Candida catenulata were shown here to produce 1.6 ± 0.5-5.0 ± 0.2 mM of PLA from phenylalanine, 5.0 ± 0.1-10.9 ± 0.3 mM of ILA from tryptophan, and 1.3 ± 0.3-7.0 ± 0.02 of PLA and 0.1 ± 0.0-2.22 ± 0.09 mM of PEA from PPA. Geotrichum candidum ATCC 204307 was the highest producer. This is the first time these antimicrobial metabolites PLA, OH-PLA, ILA, and PEA are being reported as the reaction products of aromatic amino acids catabolism in G. candidum.

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix.

In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. Lactococcus lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression. Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.

Dynamic analysis of the Lactococcus lactis transcriptome in cheeses made from milk concentrated by ultrafiltration reveals multiple strategies of adaptation to stresses.

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.

Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures.

Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.