Co-culture with Tetragenococcus halophilus improved the ethanol tolerance of Zygosaccharomyces rouxii by maintaining cell surface properties.

The accumulation of ethanol has a negative effect on the viability and fermentation performance of microorganisms during the production of fermented foods because of its toxicity. In this study, we investigated the effect of co-culture with Tetragenococcus halophilus on ethanol stress resistance of Zygosaccharomyces rouxii. The result showed that co-culture with T. halophilus promoted cell survival of Z. rouxii under ethanol stress, and the tolerance improved with increasing co-culture time when ethanol content was 8%. Physiological analysis showed that the co-cultured Z. rouxii cells maintained higher intracellular content of trehalose and amino acids including tyrosine, tryptophan, arginine and proline after 8% ethanol stress for 90 min. The membrane integrity analysis and biophysical analysis of the cell surface indicated that the presence of ethanol resulted in cell membrane damage and changes of Young's modulus value and roughness of cell surface. While the co-cultured Z. rouxii cells exhibited better membrane integrity, stiffer and smoother cell surface than single-cultured cells under ethanol stress. As for transcriptomic analyses, the genes involved in unsaturated fatty acid biosynthesis, trehalose biosynthesis, various types of N-glycan biosynthesis, inositol phosphate metabolism, MAPK signaling pathway and tight junction had higher expression in co-cultured Z. rouxii cells with down-regulation of majority of gene expression after stress. And these genes may function in the improvement of ethanol tolerance of Z. rouxii in co-culture.

Effects of Tetragenococcus halophilus and Candida versatilis on the production of aroma-active and umami-taste compounds during soy sauce fermentation.

BACKGROUND: Soy sauce is a well-known condiment worldwide. However, the high salt content in soy sauce contributes to the high intake of sodium salt, which usually results in high blood pressure. High salt soy sauce usually has the better quality (aroma and taste) than low salt. Tetragenococcus halophilus and Candida versatilis are important starters for soy sauce fermentation. It is of urgent need to explore what the effect of these two strains on the aroma- and taste-attributes of soy sauce to achieve high quality fermentation with low salt. RESULTS: In this study, aroma-active and taste compounds in soy sauce were reviewed and listed. Then, soy sauce fermentation inoculated with different combinations of T. halophilus (at different inoculated time) and C. versatilis were completed. Aroma-active and taste compounds in different samples were quantified. Multivariate analysis was used to analyze these data. The aroma-active compounds which were highly related to the inoculation of T. halophilus and C. versatilis were found. Meanwhile, the addition time of T. halophilus could also be highly related to the production of aroma-active compounds. More importantly, T. halophilus was highly correlated with the production of umami-taste compounds in soy sauce, including aspartic acid, glutamic acid, alanine and N -succinyl-glutamic acid. CONCLUSION: These results will provide a better understanding of the effects of T. halophilus and C. versatilis on the formation of significant aroma-active and umami-taste constituents in soy sauce. Furthermore, it will be helpful to realize fermentative control of soy sauce with high quality at low salt. © 2020 Society of Chemical Industry.

Tetragenococcus halophilus MJ4 as a starter culture for repressing biogenic amine (cadaverine) formation during saeu-jeot (salted shrimp) fermentation.

Biogenic amines (BAs) are frequently present in traditionally fermented salted foods. In this study, a Tetragenococcus halophilus strain (MJ4) with no BA-producing ability was isolated from a fish (anchovy) sauce. Strain MJ4 did not produce BAs from supplied precursors and no BA-producing genes were identified in its genome. Bacterial community analysis showed that in non-inoculated saeu-jeot (shrimp sauce) fermentation, Tetragenococcus predominated after 82 days, while in strain MJ4-inoculated saeu-jeot, Tetragenococcus predominated during the entire fermentation. Strain MJ4 repressed the growth of T. muriaticus, a known BA producer, during fermentation, but metabolite analysis demonstrated that metabolite profiles, including amino acids, were similar regardless of MJ4 inoculation. The metabolite analysis also showed that strain MJ4 clearly repressed the formation of cadaverine during fermentation. This study suggests that the use of strain MJ4 as a starter culture in salted fish fermentation may be a good strategy for the reduction of BA formation.

Virulence of Melissococcus plutonius and secondary invaders associated with European foulbrood disease of the honey bee.

European foulbrood is a globally distributed brood disease affecting honey bees. It may lead to lethal infections of larvae and, in severe cases, even to colony collapse. Lately, a profound genetic and phenotypic diversity was documented for the causative agent Melissococcus plutonius. However, experimental work on the impact of diverse M. plutonius strains on hosts with different genetic background is completely lacking and the role of secondary invaders is poorly understood. Here, we address these issues and elucidate the impact and interaction of both host and pathogen on one another. Moreover, we try to unravel the role of secondary bacterial invasions in foulbrood-diseased larvae. We employed in vitro infections with honey bee larvae from queens with different genetic background and three different M. plutonius strains. Larvae infection experiments showed host-dependent survival dynamics although M. plutonius strain 49.3 consistently had the highest virulence. This pattern was also reflected in significantly reduced weights of 49.3 strain-infected larvae compared to the other treatments. No difference was found in groups additionally inoculated with a secondary invader (Enterococcus faecalis or Paenibacillus alvei) neither in terms of larval survival nor weight. These results suggest that host background contributes markedly to the course of the disease but virulence is mainly dependent on pathogen genotype. Secondary invaders following a M. plutonius infection do not increase disease lethality and therefore may just be a colonization of weakened and immunodeficient, or dead larvae.

Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis.

BACKGROUND: Broad-spectrum antibiotics [Abx], including combination therapy with ciprofloxacin and metronidazole, are often prescribed during the treatment of inflammatory bowel disease [IBD] to alleviate symptoms, but with varying success. In this pilot study, we studied the effects of Abx on the course of experimental colitis, with a particular focus on sex as a determinant of the microbial and inflammatory responses. METHODS: The effects of Abx were tested on colonic inflammation and microbiome in male and female Rag-/- mice, using adoptive transfer of naïve T cells to induce colitis in a short-term [2-week] and long-term [9-week] study. RESULTS: We observed disparities between the sexes in both the response to adoptive T cell transfer and the effects of Abx. At baseline without Abx, female mice displayed a trend toward a more severe colitis than males. In both the short- and the long-term experiments, gut microbiota of some female mice exposed to Abx showed weak, delayed, or negligible shifts. Caecum weight was significantly lower in Abx-treated females. Abx exposure favoured a quick and persistent rise in Enterococcaceae exclusively in females. Males had higher relative abundance of Lactobacillaceae following Abx exposure relative to females. Abx-treated females trended toward higher colitis scores than Abx-treated males, and towards higher levels of IL-17A, NOS2, and IL-22. CONCLUSIONS: Although preliminary, our results suggest a differential response to both inflammation and Abx between male and female mice, The findings may be relevant to current practice and also as the basis for further studies on the differential gender effects during long-term antibiotic exposure in IBD.

Food to some, poison to others - honeybee royal jelly and its growth inhibiting effect on European Foulbrood bacteria.

Honeybee colonies (Apis mellifera) serve as attractive hosts for a variety of pathogens providing optimal temperatures, humidity, and an abundance of food. Thus, honeybees have to deal with pathogens throughout their lives and, even as larvae they are affected by severe brood diseases like the European Foulbrood caused by Melissococcus plutonius. Accordingly, it is highly adaptive that larval food jelly contains antibiotic compounds. However, although food jelly is primarily consumed by bee larvae, studies investigating the antibiotic effects of this jelly have largely concentrated on bacterial human diseases. In this study, we show that royal jelly fed to queen larvae and added to the jelly of drone and worker larvae, inhibits not only the growth of European Foulbrood-associated bacteria but also its causative agent M. plutonius. This effect is shown to be caused by the main protein (major royal jelly protein 1) of royal jelly.

Spoilage potential of Vagococcus salmoninarum in preservative-free, MAP-stored brown shrimp and differentiation from Brochothrix thermosphacta on streptomycin thallous acetate actidione agar.

AIMS: During a previous study concerning brown shrimp (Crangon crangon), selective streptomycin thallous acetate actidione (STAA) agar was used to determine the growth of Brochothrix thermosphacta. However, the growth of Vagococcus salmoninarum on this medium was also noticed. This study explores the spoilage potential of this organism when inoculated on sterile shrimp. METHODS AND RESULTS: Isolates growing on STAA were identified using (GTG)5 clustering followed by partial 16S rRNA gene sequence analysis. Their biochemical spoilage potential was analysed for H2 S production and enzymatic activities were tested using an APIZYM test. Headspace solid phase micro-extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to analyse the volatile organic compounds (VOCs) produced during storage of inoculated shrimp. CONCLUSION: Fifty-five per cent of isolates taken from STAA could be identified as V. salmoninarum, while no apparent morphological difference with B. thermosphacta isolates was identified upon the prescribed incubation conditions. For isolates identified as V. salmoninarum, production of 2-heptanone, 2-nonanone, 2-undecanone was found, as was the possibility to form H2 S. SIGNIFICANCE AND IMPACT OF THE STUDY: When using the STAA medium for detecting B. thermosphacta, one should consider the possible abundant presence of V. salmoninarum as well. Based on this study, V. salmoninarum does not exhibit great spoilage potential, although it can produce H2 S and formed VOCs which are also found in other spoiled seafood products.

Water quality in turkey farms in Khemisset (Morocco) and potential risk factors. / Qualité des eaux en élevage de dinde à Khémisset (Maroc) et hypothèses sur les facteurs de risques.

The objectives of this study were to assess the microbiological and physical/chemical quality of water in broiler turkey farms in the province of Khemisset (north-western Morocco) and, based on a questionnaire, to ascertain potential risk factors for contamination of drinking water with faecal coliforms. A total of 80 samples were collected and analysed in 20 farms (four from each farm). At the main inlet to the water line at the entrance to each turkey house, 100% of the samples were of unacceptable quality in terms of faecal coliforms, Escherichia coli, faecal streptococci, sulphitereducing anaerobes and enterococci. A significant reduction in microbiological contamination of the water line (p < 0.05) was observed on Day 60. While more than 90% of the samples were of satisfactory quality in terms of pH, nitrites, conductivity, nitrates and iron, only 35% were satisfactory in terms of total hardness and only 20% met quality standards for ammonium content. The factors affecting levels of contamination with faecal coliforms were water chlorination (p = 0.065; odds ratio = 14; 90% confidence interval [CI] = 1.14-71), cleaning and disinfection (p = 0.028; odds ratio = 14; 95% CI = 1.25-156.6) and antibiotic treatment (p = 0.001; odds ratio = 6; 95% CI = 2.1-35.2). To improve water quality in poultry farms, farmers are advised to protect wells from contamination and to install water purification units (pre-oxidation, coagulation, flocculation, disinfection). In addition, turkey houses and rearing equipment should be rigorously cleaned and disinfected between each batch of birds.

Cette étude a pour objectifs d'évaluer la qualité microbiologique et physico-chimique de l'eau des élevages de dinde de chair dans la province de Khémisset (nord-ouest du Maroc) et de formuler, au moyen d'un questionnaire, certaines hypothèses sur les facteurs de risque potentiels associés à la contamination de l'eau d'abreuvement par les coliformes fécaux. Au total, 80 échantillons ont été prélevés et analysés dans 20 élevages (quatre de chaque élevage). Au sas de la ligne d'abreuvement, 100 % des échantillons étaient de qualité inacceptable en ce qui concerne les coliformes fécaux, Escherichia coli, les streptocoques fécaux, les anaérobies sulfito-réducteurs et les entérocoques. Une réduction significative de la contamination microbiologique a été observée en bout de la ligne d'abreuvement (p < 0,05) au jour 60. Plus de 90 % des échantillons étaient de qualité satisfaisante pour ce qui concerne le pH, les nitrites, la conductivité, les nitrates et le fer ; en revanche, seulement 35 % et 20 % d'échantillons étaient conformes pour ce qui concerne la dureté totale et la présence d'ammonium, respectivement. Les facteurs associés à la contamination par les coliformes fécaux étaient la chloration de l'eau (p = 0,065 ; rapport des cotes = 14 ; intervalle de confiance [IC] à 90 % = 1,14-71), le nettoyage et la désinfection (p = 0,028 ; rapport des cotes = 14 ; IC à 95 % = 1,25-156,6) et le traitement par antibiotiques (p = 0,001 ; rapport des cotes = 6 ; IC à 95 % = 2,1-35,2). Afin d'améliorer la qualité de l'eau dans les élevages avicoles, il est recommandé aux éleveurs de protéger les puits contre la contamination et d'installer des stations de potabilisation de l'eau (pré-oxydation, coagulation, floculation, désinfection). Il convient également de procéder à un nettoyage et une désinfection rigoureux des bâtiments et du matériel d'élevage à la fin de chaque bande.

Los autores describen un estudio destinado a evaluar la calidad microbiológica y fisicoquímica del agua de explotaciones productoras de pavos de engorde de la provincia de Khemisset (noroeste de Marruecos) y a formular, mediante un cuestionario, ciertas hipótesis sobre los posibles factores de riesgo asociados a la contaminación del agua de bebida por coliformes fecales. En total se tomaron y analizaron 80 muestras de 20 explotaciones. En la esclusa de entrada del agua en la línea de bebederos, el 100% de las muestras era de calidad inaceptable por lo que respecta a la presencia de coliformes fecales, Escherichia coli, estreptococos fecales, bacterias anaerobias sulfitorreductoras y enterococos. A la salida de la línea de bebederos se observaba una reducción significativa de la contaminación microbiológica (p < 0,05) en el día 60. Más del 90% de las muestras eran de calidad satisfactoria por lo que respecta a pH, nitritos, conductividad, nitratos y hierro. En cambio, solo un 35% y un 20% de las muestras satisfacían los criterios relativos a la dureza total y la presencia de amonio, respectivamente. Los factores asociados a la contaminación por coliformes fecales eran la cloración del agua (p = 0,065; razón de probabilidades = 14; intervalo de confianza [IC] al 90% = 1,14-71), la limpieza y desinfección (p = 0,028; razón de probabilidades = 14; IC al 95% = 1,25-156,6) y el tratamiento con antibióticos (p = 0,001; razón de probabilidades = 6; IC al 95% = 2,1-35,2). Con el fin de mejorar la calidad del agua en las explotaciones avícolas se recomienda a los productores que protejan los pozos de la contaminación e instalen potabilizadoras de agua (preoxidación, coagulación, floculación y desinfección). Asimismo, conviene proceder a una limpieza y una desinfección rigurosas de los locales y el material de cría al final de cada banda.

Tetragenococcus koreensis is part of the microbiota in a traditional Italian raw fermented sausage.

This study reports the isolation of Tetragenococcus koreensis, a bacterial species currently represented only by the type strain isolated from kimchi, from a raw fermented and ripened Italian sausage, Ventricina Vastese, all over the ripening period of five months. Rep-PCR genotyping showed that different T. koreensis strains, identified by sequencing of the 16S rRNA gene, were present in the same production batch. Tests on representative isolates showed intra-species physiological variability and the possession of phenotypic traits relevant for the production of fermented sausages, i.e. ability to grow at high salt concentrations, to induce some changes in the peptide profile of the culture medium and inability to produce histamine and tyramine, confirmed by the absence of the respective decarboxylase genes. Therefore the opportunity to further investigate the suitability of T. koreensis as a starter for fermented meat products was suggested.

Microbial quality of industrial liquid egg white: assumptions on spoiling issues in egg-based chilled desserts.

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French egg processing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However, Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This study emphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts.

Identification of mutations involved in the requirement of potassium for growth of typical Melissococcus plutonius strains.

Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH(2)PO(4) to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na(+)/H(+) antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.

Complete genome sequence of Melissococcus plutonius DAT561, a strain that shows an unusual growth profile and is representative of an endemic cluster in Japan.

We report the complete genome sequence of Melissococcus plutonius DAT561, which is a causative agent of European foulbrood. M. plutonius DAT561 is a representative of nonfastidious strains isolated in Japan. The addition of potassium phosphate was not required for normal growth, unlike for typical M. plutonius strain/isolates.

[Colonization of the teat skin and the teat canal of lactating dairy cattle by mastitis pathogens]. / Besiedlung von Zitzenhaut und Zitzenkanal laktierender Milchrinder durch euterpathogene Mikroorganismen.

OBJECTIVE: The teat canal of lactating dairy cattle is the first barrier against mastitis pathogens invading the teat cistern and the udder lumen. However, in several studies it could be shown that the teat skin and the teat canal epithelium are colonized by mainly staphylococci as well as by other pathogenic or facultative pathogenic micro-organisms. The aim of the study was to evaluate the pathogen pressure on the bovine mammary gland and to establish data on the prevalence of relevant micro-organisms. MATERIAL AND METHODS: In 32 randomly selected German dairy farms the colonization of the teat skin and the teat canal by S.aureus (Baird Parker agar), Sc.uberis (modified Rambach agar according to Watts et al. [1993]), enterococci (kanamycin esculin azide agar) and coliforms (ChromoCult Coliform agar) was investigated by the wet/dry swab technique DIN10113-1:1997-07. RESULTS: In total 84.5% of all 1358 investigated teat canals of clinically healthy cattle were colonized by at least one of the four pathogen groups. S.aureus was isolated from 72.2% of the teat canals. For S.aureus , Sc.uberis and E.coli maximum bacterial counts of >10(6) cfu/swab and for other coliform bacteria>10(7) cfu/swab were detected. Correlations could be found between the bacterial counts on the teat skin and in the teat canal within the pathogen groups S.aureus, Sc.uberis, E.coli and other coliforms (Spearman's rank correlation coefficient r≥0.30, p<0.001) as well as between the environmental udder pathogens Sc.uberis and coliforms (r≥0.25, p<0.01). CONCLUSION AND CLINICAL RELEVANCE: The variation of the bacterial counts on teat epithelia could be an indicator that the microflora of the teat canal epithelium can be affected by management practices in dairy farms. The results of this study point out that under field conditions the efficiency of hygienic practices (husbandry, milking) can be assessed by sampling of the bovine teat canal by the wet/dry swab technique.

Using temperature and time criteria to control the effectiveness of continuous thermal sanitation of piggery effluent in terms of set microbial indicators.

AIM: To determine the minimal conditions (temperature-time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. METHODS AND RESULTS: The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite-reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+-specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4-5 log(10), but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3.1 log(10) for SRC and of 1.4 log(10) for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. CONCLUSION: Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). SIGNIFICANCE AND IMPACT OF THE STUDY: Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low-cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist.

Effect of alginate and inulin on intestinal microbial ecology of weanling pigs reared under different husbandry conditions.

The effects of inulin and alginate on intestinal microbial ecophysiology were investigated in piglets fed a diet (C) with 0.1% alginate (C+A) or 1.5% inulin (C+I) from weaning at day 28. The experiment was performed at an experimental farm (EF) and a commercial farm (CF). Digesta was collected from the ileum, caecum and colon of four piglets from each group on days 29, 30, 33 and 39. The metabolite concentrations changed with age. Colonic and caecal metabolites were affected by prebiotic treatment. Changes in microbiota composition were assessed by cultivation and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. Enterococci increased in C+A at EF and decreased in C+I at both farms. Lactobacilli decreased in all segments in the experimental groups on days 30 and 33. Yeasts in C+I were five times lower at CF than at EF on day 39. The richness and diversity of DGGE profiles increased in the experimental groups. The evenness of colon digesta-derived DGGE profiles was higher in the experimental groups than in C and this situation was reversed in the distal small intestine. Multivariate redundancy analysis confirmed the recorded effects. In summary, both prebiotics affected the intestinal microbiota, and the changes were more pronounced at the CF.