The Interior Surfaces of Wooden Barrels Are an Additional Microbial Inoculation Source for Lambic Beer Production.

Traditional lambic beer production takes place through wort inoculation with environmental air and fermentation and maturation in wooden barrels. These wooden casks or foeders are possible additional inoculation sources of microorganisms for lambic worts. To date, however, these lambic barrels have been examined only with culture-dependent techniques, thereby missing a portion of the microorganisms present. Moreover, the effects of the cleaning procedures (involving high-pressure water and/or fumigation) and the barrel type on the microbial community structures of the interior surfaces of wooden lambic barrels were unclear. The culture-dependent plating and culture-independent amplicon sequencing of swab samples obtained from the interior surfaces of different wooden casks and foeders used for traditional lambic beer production in Belgium revealed that the microbial compositions of these surfaces differed statistically throughout the barrel-cleaning procedures applied. At the end of the cleaning procedures, amplicon sequencing still detected fermentation- and maturation-related microorganisms, although only a few colonies were still detectable using culture-dependent methods. It is possible that some of the surviving microorganisms were missed due to the presence of many of these cells in a viable but not culturable state and/or engrained deeper in the wood. These surviving microorganisms could act as an additional inoculation source, besides brewery air and brewery equipment, thereby helping to establish a stable microbial community in the wort to diminish batch-to-batch variations in fermentation profiles. Furthermore, the microbial compositions of the interior barrel surfaces differed statistically based on the barrel type, possibly reflecting different characteristics of the lambic barrels in terms of age, wood thickness, and wood porosity.IMPORTANCE Although the coolship step is generally regarded as the main contributor to the spontaneous inoculation by environmental air of fresh worts for lambic beer production, it is known that microorganisms often associate with specific surfaces present in a brewery. However, knowledge about the association of microorganisms with the interior surfaces of wooden lambic barrels is limited. To clarify the role of casks and foeders as additional microbial inoculation sources, it was important to determine the influence of the barrel characteristics and the cleaning procedures on the microbial communities of the interior barrel surfaces. Moreover, this helped to elucidate the complex spontaneous lambic beer fermentation and maturation process. It will allow further optimization of the lambic beer production process, as well as the wooden-barrel-cleaning procedures applied.

Nonconventional starter cultures of coagulase-negative staphylococci to produce animal-derived fermented foods, a SWOT analysis.

Coagulase-negative staphylococci (CNS) are ubiquitous micro-organisms that are commonly present on animal skin and animal-derived foods. They are members of the beneficial microbial consortia of several fermented food products where they contribute to quality. Currently, only a few CNS species are included in commercial starter cultures, although many other ones with promising properties have been isolated from diverse food ecosystems. In the present study, a Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis of the potential use of unconventional CNS starter cultures for the fermentation of animal-derived foods is carried out. An overview of both their desirable and worrisome metabolic traits is given. In general, the application of innovative CNS-based starter cultures offers opportunities to modulate flavour, improve the safety and health aspects and develop novel colour development strategies for clean label products. Yet, their implementation is often not straightforward as nontrivial obstacles or threats are encountered, which relate to technological, food safety and legal concerns. As most of the desirable and undesirable characteristics of CNS species are strain dependent, a case-by-case evaluation is needed when evaluating specific strains for their potential use as novel starter cultures.

Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role.

Few data have been published on the occurrence and functional role of acetic acid bacteria (AAB) in lambic beer production processes, mainly due to their difficult recovery and possibly unknown role. Therefore, a novel aseptic sampling method, spanning both the spatial and temporal distributions of the AAB and their substrates and metabolites, was combined with a highly selective medium and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput dereplication method followed by comparative gene sequencing for their isolation and identification, respectively. The AAB (Acetobacter species more than Gluconobacter species) proliferated during two phases of the lambic beer production process, represented by Acetobacter orientalis during a few days in the beginning of the fermentation and Acetobacter pasteurianus from 7 weeks until 24 months of maturation. Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available disclosed possible reasons for this successive dominance. The spatial analysis revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) were produced at the tops of the casks, due to higher AAB counts and a higher metabolic activity of the AAB species at the air/liquid interface during the first 6 months of lambic beer production. In contrast, no differences in AAB species diversity occurred throughout the casks.IMPORTANCE Lambic beer is an acidic beer that is the result of a spontaneous fermentation and maturation process. Acidic beers are currently attracting attention worldwide. Part of the acidity of these beers is caused by acetic acid bacteria (AAB). However, due to their difficult recovery, they were never investigated extensively regarding their occurrence, species diversity, and functional role in lambic beer production. In the present study, a framework was developed for their isolation and identification using a novel aseptic sampling method in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry as a high-throughput dereplication technique followed by accurate molecular identification. The sampling method applied enabled us to take spatial differences into account regarding both enumerations and metabolite production. In this way, it was shown that more AAB were present and more acetic acid was produced at the air/liquid interface during a major part of the lambic beer production process. Also, two different AAB species were encountered, namely, Acetobacter orientalis at the beginning and Acetobacter pasteurianus in a later stage of the production process. This developed framework could also be applied for other fermentation processes.

Inulin-type fructan degradation capacity of Clostridium cluster IV and XIVa butyrate-producing colon bacteria and their associated metabolic outcomes.

Four selected butyrate-producing colon bacterial strains belonging to Clostridium cluster IV (Butyricicoccus pullicaecorum DSM 23266T and Faecalibacterium prausnitzii DSM 17677T) and XIVa (Eubacterium hallii DSM 17630 and Eubacterium rectale CIP 105953T) were studied as to their capacity to degrade inulin-type fructans and concomitant metabolite production. Cultivation of these strains was performed in bottles and fermentors containing a modified medium for colon bacteria, including acetate, supplemented with either fructose, oligofructose, or inulin as the sole energy source. Inulin-type fructan degradation was not a general characteristic among these strains. B. pullicaecorum DSM 23266T and E. hallii DSM 17630 could only ferment fructose and did not degrade oligofructose or inulin. E. rectale CIP 105953T and F. prausnitzii DSM 17677T fermented fructose and could degrade both oligofructose and inulin. All chain length fractions of oligofructose were degraded simultaneously (both strains) and both long and short chain length fractions of inulin were degraded either simultaneously (E. rectale CIP 105953T) or consecutively (F. prausnitzii DSM 17677T), indicating an extracellular polymer degradation mechanism. B. pullicaecorum DSM 23266T and E. hallii DSM 17630 produced high concentrations of butyrate, CO2, and H2 from fructose. E. rectale CIP 105953T produced lactate, butyrate, CO2, and H2, from fructose, oligofructose, and inulin, whereas F. prausnitzii DSM 17677T produced butyrate, formate, CO2, and traces of lactate from fructose, oligofructose, and inulin. Based on carbon recovery and theoretical metabolite production calculations, an adapted stoichiometrically balanced metabolic pathway for butyrate, formate, lactate, CO2, and H2 production by members of both Clostridium cluster IV and XIVa butyrate-producing bacteria was constructed.

The water kefir grain inoculum determines the characteristics of the resulting water kefir fermentation process.

AIMS: To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process. METHODS AND RESULTS: Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these micro-organisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii and Saccharomyces cerevisiae were always present and may be the key micro-organisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of micro-organisms, fast fermentation and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria. CONCLUSIONS: The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key micro-organisms was always present during fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a rational selection of a water kefir grain inoculum.

The cocoa bean fermentation process: from ecosystem analysis to starter culture development.

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.

Bifidobacterium longum D2 enhances microbial degradation of long-chain arabinoxylans in an in vitro model of the proximal colon.

Long-chain arabinoxylans (LC-AX) are degraded in the colon by intestinal bacteria possessing AX-degrading enzymes, such as bifidobacteria. Enzymatic activity of intestinal bacterial might vary depending on the composition of the gut microbiota. To compare the enzymatic activities of the bacterial gut communities of two healthy individuals (donors D1 and D2), these bacterial communities were inoculated into in vitro model M-SHIME(®). Differences in xylanase activities and denaturing gradient gel electrophoresis profiles, in particular a DNA-band corresponding with Bifidobacterium longum, were found in the proximal colon vessel. 16S rRNA gene sequencing analysis demonstrated the presence of two different B. longum species in these bacterial communities, showing 99% gene sequence similarity with B. longum NCC2705 and B. longum. subsp. longum KACC 91563, respectively, further referred to as B. longum D1 and B. longum D2. When grown on LC-AX as the sole added energy source, B. longum D2 displayed significantly higher activities of ß-xylanase (5.3-fold), ß-xylosidase (2.9-fold), and α-arabinofuranosidase (1.5-fold), respectively, compared to B. longum D1. When B. longum D2 was inoculated in the M-SHIME, inoculated with the bacterial gut communities of the individual with low AX-degrading enzyme activities, the ß-xylanase activity increased (1.5-fold) in the proximal vessel. We demonstrated the presence of differences in LC-AX degrading enzyme activities of the bacterial gut communities of two individuals in the in vitro M-SHIME model, which could be linked to the presence of a potent AX-degrading B. longum (D2) strain.

The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation.

The ability of coagulase-negative staphylococci (CNS) to use alternative energy sources in meat may partially explain their occurrence in fermented meats. Of 61 CNS strains tested, all metabolized adenosine and inosine in a meat simulation medium (MSM). The ability to catabolize arginine via the arginine deiminase (ADI) pathway varied between strains. All tested strains of Staphylococcus carnosus and Staphylococcus epidermidis possessed an arcA gene and showed ADI activity, whereas other species, such as Staphylococcus equorum and Staphylococcus succinus, did not. Arginine catabolic mobile elements (ACME), as in the positive control S. epidermidis ATCC 12228, were uncommon and only found in Staphylococcus xylosus 3PA6 (sausage isolate) and Staphylococcus chromogenes G222 (teat apex isolate). Monoculture experiments were performed in MSM with S. carnosus 833 and SS3-4, S. xylosus G211, and S. epidermidis ATCC 12228 and 2S7-4. At all pH values tested (5.3, 5.8, and 6.5), the strains of S. carnosus catabolized arginine faster than the strains of S. xylosus and S. epidermidis. Only at pH 6.5 could a low ADI activity be found for S. xylosus G211. Increased ADI activity occurred in the case of the ACME-positive S. epidermidis ATCC 12228, when compared to the ACME-negative S. epidermidis 2S7-4.

Antibacterial activities of coagulase-negative staphylococci from bovine teat apex skin and their inhibitory effect on mastitis-related pathogens.

AIMS: To explore antibacterial activities of coagulase-negative staphylococci (CoNS) from teat apices of dairy cows towards mastitis-causing pathogens. METHODS AND RESULTS: Of 254 CoNS, 38 displayed bacteriocin-like activity after a first screening. Seven of these strains displayed activity against at least one mastitis-related pathogen (Streptococcus uberis, Streptococcus dysgalactiae and Staphylococcus aureus). Staphylococcus chromogenes L217 displayed the strongest inhibitory effect, being active against all tested mastitis-related pathogens and most tested CoNS. Based on cation exchange and reversed-phase chromatography, in addition to N-terminal Edman degradation and PCR, the antibacterial peptide was identified as a nukacin-type bacteriocin and named nukacin L217. Although staphylococcal bacteriocins are generally found in the cell-free supernatants of liquid cultures, Staph. chromogenes L217 only led to detectable activity when grown on agar medium. CONCLUSIONS: Bacteriocin-like activities are not uncommon among CoNS from teat apices and may inhibit mastitis-causing pathogens, as found for nukacin L217 production by Staph. chromogenes L217. SIGNIFICANCE AND IMPACT OF THE STUDY: Nukacin L217 is the first identified bacteriocin of the species Staph. chromogenes and displays unusual production kinetics, that is, requiring surface growth of its producer. The fact that nukacins are produced by different CoNS species suggests a role in the teat skin ecosystem.

Summer Meeting 2013: growth and physiology of bifidobacteria.

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin-type fructans (ITF) (oligofructose and inulin) and arabinoxylan-oligosaccharides (AXOS). Beta-fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain-dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2-7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin-type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long-chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α-arabinofuranosidase and ß-xylosidase is strain-dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra- or intracellularly. The production of high amounts of acetate that accompanies inulin-type fructan degradation by bifidobacteria cross-feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain-dependent differences in prebiotic degradation indicate the existence of niche-specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.

Microbial ecology of sourdough fermentations: diverse or uniform?

Sourdough is a specific and stressful ecosystem inhabited by yeasts and lactic acid bacteria (LAB), mainly heterofermentative lactobacilli. On the basis of their inocula, three types of sourdough fermentation processes can be distinguished, namely backslopped ones, those initiated with starter cultures, and those initiated with a starter culture followed by backslopping. Typical sourdough LAB species are Lactobacillus fermentum, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus sanfranciscensis. Typical sourdough yeast species are Candida humilis, Kazachstania exigua, and Saccharomyces cerevisiae. Whereas region specificity is claimed in the case of artisan backslopped sourdoughs, no clear-cut relationship between a typical sourdough and its associated microbiota can be found, as this is dependent on the sampling, isolation, and identification procedures. Both simple and very complex consortia may occur. Moreover, a series of intrinsic and extrinsic factors may influence the composition of the sourdough microbiota. For instance, an influence of the flour (type, quality status, etc.) and the process parameters (temperature, pH, dough yield, backslopping practices, etc.) occurs. In this way, the presence of Lb. sanfranciscensis during sourdough fermentation depends on specific environmental and technological factors. Also, Triticum durum seems to select for obligately heterofermentative LAB species. Finally, there are indications that the sourdough LAB are of intestinal origin.

Community dynamics of coagulase-negative staphylococci during spontaneous artisan-type meat fermentations differ between smoking and moulding treatments.

Coagulase-negative staphylococci (CNS) that are naturally present in the raw meat batter of fermented sausages or that originate from the addition of a starter culture play a role in flavour development. A wide species diversity of CNS can be present in fermented meats, but it is not fully clear yet how specific process parameters select for specific CNS by affecting their community dynamics. Therefore, the influence of smoking and moulding treatments on the CNS community dynamics in spontaneously fermented, artisan-type sausages was investigated. During the fermentation stage, the meat batter was in all cases dominated by Staphylococcus saprophyticus, in addition to Lactobacillus sakei as governing lactic acid bacterium. Following fermentation, the bacterial communities were not perturbed by the smoking treatment, since both L. sakei and S. saprophyticus remained dominant throughout the ripening stage and prevailed in the end-products. Yet, when fermentation was followed by a moulding step with Penicillium nalgiovense, a shift of the CNS communities towards dominance by Staphyloccocus equorum was seen, despite a similar evolution of L. sakei. This effect was possibly due to a pH rise caused by the mould, a hypothesis which was reinforced by the finding that the isolated strain S. equorum DBX-S-17 was more sensitive to low pH than the isolated strain S. saprophyticus DFL-S-12 during growth experiments in brain heart infusion (BHI). Differences in CNS communities may affect sausage flavour, due to intraspecies variations in metabolic conversions of, for instance, amino acids. The fact that 3-methyl-butanal was only found in the moulded sausage, which was dominated by S. equorum, may be related to the finding that the isolated strain of this species was able to produce this compound in BHI medium, whereas the isolated strain of S. saprophyticus was not.

Application' and validation of autochthonous lactic acid bacteria starter cultures for controlled leek fermentations and their influence on the antioxidant properties of leek.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important outdoor vegetables, mainly cultivated for its white shaft. Fermentation of leek offers opportunities in view of biomass valorization and product diversification. This study deals with the implementation and validation of starter cultures to perform controlled leek fermentations and to ensure a high quality of the end-products. Therefore, a thorough study of the fermentation microbiology and the influence of three starter culture strains (Lactobacillus plantarum IMDO 788, Lactobacillus sakei IMDO 1358, and Leuconostoc mesenteroides IMDO 1347) on the metabolite kinetics of leek fermentation and antioxidant properties of leek was performed. Overall, the application of lactic acid bacteria starter cultures resulted in a fast prevalence of the species involved, coupled to an accelerated acidification. Of the three starter cultures tested, the mixed starter culture of L. plantarum IMDO 788 and L. mesenteroides IMDO 1347 was most promising, as its application resulted in fermented leek of good microbiological quality and in a more extensive carbohydrate consumption, whereby diverse end-metabolites were produced. However, high residual fructose concentrations allowed yeast outgrowth, resulting in increased ethanol and glycerol concentrations, and indicated the lack of a prevailing strictly heterofermentative LAB species. The antioxidant capacity of fermented leek samples, as measured with the oxygen radical absorbance capacity assay, increased when starter cultures were used, whereas with regard to 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity, only leek fermented with L. sakei IMDO 1358 scored higher than spontaneously fermented leek. The total phenolic content was not influenced by the use of starter cultures, while the S-alk(en)yl-L-cysteine sulfoxides content decreased strongly. A preliminary sensory analysis revealed that the spontaneously fermented leek and the one obtained with the mixed starter culture were preferred by consumers, emphasizing again the importance of microbial successions in vegetable fermentations.

Unraveling the microbiota of teat apices of clinically healthy lactating dairy cows, with special emphasis on coagulase-negative staphylococci.

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2.

Species diversity, community dynamics, and metabolite kinetics of spontaneous leek fermentations.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important vegetables. All or part of the green leek parts are often left on the fields because of their limited cooking applications compared to the white leek parts. Therefore, the possibility to perform leek fermentations in view of product valorization and diversification was investigated. This study deals with the community dynamics, species diversity, and metabolite kinetics of spontaneous leek fermentations, thereby studying the influence of added NaCl concentration, harvesting season, and duration of the fermentation. The combination of a culture-dependent and culture-independent approach revealed the prevalence of lactic acid bacteria (LAB) from the third day of fermentation onwards, which was not influenced by the fermentation conditions applied. Enterobacteriaceae, Pseudomonadaceae, and yeasts disappeared after one week of fermentation. Leuconostoc mesenteroides, Lactobacillus sakei, and Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus parabrevis were the most frequently isolated LAB species. Both added NaCl concentrations were suitable to perform successful fermentations within three weeks. By that time, glucose and fructose, the main leek carbohydrates, were metabolized into mainly lactic acid, acetic acid, ethanol, and mannitol. A sensory analysis revealed that the fermented white leek parts were generally more appreciated than the fermented green leek parts.

Characterization of coagulase-negative staphylococcus species from cows' milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips.

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n=366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMérieux, Marcy l'Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows' environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH.

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.

Intra-species diversity and epidemiology varies among coagulase-negative Staphylococcus species causing bovine intramammary infections.

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.

Species diversity and metabolic impact of the microbiota are low in spontaneously acidified Belgian sausages with an added starter culture of Staphylococcus carnosus.

Quality of fermented sausages is affected by acidifying lactic acid bacteria (LAB) and colour- and flavour-promoting coagulase-negative staphylococci (CNS), whether or not used as starter culture. Artisan fermented sausages are often perceived as superior to industrial variants, partially because of the specific microbiota due to spontaneous acidification, which may be considered as an artisan characteristic. Therefore, two kinds of spontaneously acidified Belgian sausages were prepared (Belgian-type salami and Boulogne sausage), but with addition of a Staphylococcus carnosus culture. The Belgian-type salami was made from pork and beef, whereas the Boulogne sausage contained pork and horse meat. In all cases, Lactobacillus sakei was the dominant LAB species present on the raw materials and during fermentation, whereas enterococci remained present in the background. Enterobacteriaceae vanished after fermentation. The CNS species diversity on the raw materials was large and differed between the pork, beef, and horse meat. Nevertheless, this species diversity was annihilated during fermentation by the added S. carnosus culture. The volatiles fraction was mainly composed of aldehydes that originated from lipid oxidation and spices-derived compounds. Aromatic compounds that are typically associated to CNS activity, such as end-products from the metabolism of branched-chain amino acids, were not present in the Belgian-type salami and only marginally present in the Boulogne sausage. In conclusion, spontaneous acidification of Belgian-type fermented sausages leads to dominance of L. sakei and is no guarantee for bacterial contribution to the aroma profile when S. carnosus is added as a starter culture.

The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494.

The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.