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Anorexigenic substances released during infection may hinder the therapeutic efficacy of infeed antibiotics. Paracetamol (acetaminophen; PARA) inhibits the anorexia of infection and seems to improve the clinical efficacy of doxycycline (DOX) against bacterim respiratory disease in swine herds. In order to verify whether PARA selectively stimulates intake of DOX-medicated feed in diseased pigs, we documented the pharmacokinetics (PK) of DOX when coadministered with PARA and examined the effect of in-feed PARA on the interindividual variability in plasma concentrations after systemic exposure to in-feed DOX in swine herds with respiratory disease. Systemic exposure to DOX was measured with the area under the curve (AUC) of its plasma concentrations over time. First, a rich-sampling PK study of in-feed and i.v. DOX (10 mg/kg of BW) and PARA (30 and 10 mg/kg of BW, respectively) was performed on 5 pigs. The PK profiles of in-feed DOX were used in mathematical simulations to determine 5 optimal sampling times for the farm-based population PK study. A randomized, blind, parallel PK study was performed in 2 herds with bacterial respiratory disease, where liquid feed was fortified with DOX alone (5 mg * kg of [BW.sup.-1] * [meal.sup.-1]) or combined with PARA (15 mg * kg of [BW.sup.-1] * [meal.sup.-1]). Medicated meals were given twice, 12 h apart, to group-housed growing pigs (n > 50 pigs * [treatment.sup.-1] * [herd.sup.-1], totaling 215 pigs). Plasma concentrations of DOX and PARA were measured with HPLC. At variance with our expectations, PARA decreased (P = 0.069) mean AUC of in-feed DOX and did not decrease its variability (P > 0.34). Mean AUC of DOX increased with feed intake and with initial exposure to DOX, and was greater in sick animals. Therefore, symptomatic PARA-induced improvement in bacterial respiratory disease control with DOX is more likely caused by its analgesic/antipyretic effects than by its orexigenic effect. Interindividual variation in the AUC of DOX was large in pigs given group medication, even when sufficient feeding space was allowed and the amount of feed offered was greater than their requirements. Therefore, future studies to improve the efficacy of group antibiotic therapy should focus on feeding behavior characteristics as well as biopharmaceutical properties of medicated feeds. Key words: acetaminophen, doxycycline, feed supplement, pharmacokinetics, pig, respiratory disease

The kinetics of cortisol disposition and production were investigated in six healthy horses given [3H]cortisol. Exercise following cortisol administration resulted in two- to three-fold increases in plasma cortisol. Exercise also improved the clearance and distribution of cortisol. However, plasma cortisol concentration immediately returned to pre-exercise levels after exercise. These results suggest that exercise induce increases in cortisol by stimulating adrenal gland secretion during exercise.

Anorexigenic substances released during infection may hinder the therapeutic efficacy of in-feed antibiotics. Paracetamol (acetaminophen; PARA) inhibits the anorexia of infection and seems to improve the clinical efficacy of doxycycline (DOX) against bacterial respiratory disease in swine herds. In order to verify whether PARA selectively stimulates intake of DOX-medicated feed in diseased pigs, we documented the pharmacokinetics (PK) of DOX when coadministered with PARA and examined the effect of in-feed PARA on the interindividual variability in plasma concentrations after systemic exposure to in-feed DOX in swine herds with respiratory disease. Systemic exposure to DOX was measured with the area under the curve (AUC) of its plasma concentrations over time. First, a rich-sampling PK study of in-feed and i.v. DOX (10 mg/kg of BW) and PARA (30 and 10 mg/kg of BW, respectively) was performed on 5 pigs. The PK profiles of in-feed DOX were used in mathematical simulations to determine 5 optimal sampling times for the farm-based population PK study. A randomized, blind, parallel PK study was performed in 2 herds with bacterial respiratory disease, where liquid feed was fortified with DOX alone (5 mg·kg of BW-1·meal-1) or combined with PARA (15 mg·kg of BW-1·meal-1). Medicated meals were given twice, 12 h apart, to group-housed growing pigs (n > 50 pigs·treatment-1·herd-1, totaling 215 pigs). Plasma concentrations of DOX and PARA were measured with HPLC. At variance with our expectations, PABA decreased (P = 0.069) mean AUC of in-feed DOX and did not decrease its variability (P > 0.34). Mean AUC of DOX increased with feed intake and with initial exposure to DOX, and was greater in sick animals. Therefore, symptomatic PARA-induced improvement in bacterial respiratory disease control with DOX is more likely caused by its analgesic/antipyretic effects than by its orexigenic effect. Interindividual variation in the AUC of DOX was large in pigs given group medication, even when sufficient feeding space was allowed and the amount of feed offered was greater than their requirements. Therefore, future studies to improve the efficacy of group antibiotic therapy should focus on feeding behavior characteristics as well as biopharmaceutical properties of medicated feeds. [ABSTRACT FROM AUTHOR]

Anorexigenic substances released during infection may hinder the therapeutic efficacy of in-feed antibiotics. Paracetamol (acetaminophen; PARA) inhibits the anorexia of infection and seems to improve the clinical efficacy of doxycycline (DOX) against bacterial respiratory disease in swine herds. In order to verify whether PARA selectively stimulates intake of DOX-medicated feed in diseased pigs, we documented the pharmacokinetics (PK) of DOX when coadministered with PARA and examined the effect of in-feed PARA on the interindividual variability in plasma concentrations after systemic exposure to in-feed DOX in swine herds with respiratory disease. Systemic exposure to DOX was measured with the area under the curve (AUC) of its plasma concentrations over time. First, a rich-sampling PK study of in-feed and i.v. DOX (10 mg/kg of BW) and PARA (30 and 10 mg/kg of BW, respectively) was performed on 5 pigs. The PK profiles of in-feed DOX were used in mathematical simulations to determine 5 optimal sampling times for the farm-based population PK study. A randomized, blind, parallel PK study was performed in 2 herds with bacterial respiratory disease, where liquid feed was fortified with DOX alone (5 mg·kg of BW-1·meal-1) or combined with PARA (15 mg·kg of BW-1·meal-1). Medicated meals were given twice, 12 h apart, to group-housed growing pigs (n > 50 pigs·treatment-1·herd-1, totaling 215 pigs). Plasma concentrations of DOX and PARA were measured with HPLC. At variance with our expectations, PABA decreased (P = 0.069) mean AUC of in-feed DOX and did not decrease its variability (P > 0.34). Mean AUC of DOX increased with feed intake and with initial exposure to DOX, and was greater in sick animals. Therefore, symptomatic PARA-induced improvement in bacterial respiratory disease control with DOX is more likely caused by its analgesic/antipyretic effects than by its orexigenic effect. Interindividual variation in the AUC of DOX was large in pigs given group medication, even when sufficient feeding space was allowed and the amount of feed offered was greater than their requirements. Therefore, future studies to improve the efficacy of group antibiotic therapy should focus on feeding behavior characteristics as well as biopharmaceutical properties of medicated feeds. [ABSTRACT FROM AUTHOR]

1. The loss of metabolic capacities during culture time constitutes a major limitation for the use of hepatocyte primary cultures in in vitro metabolism measurements. A new strategy is presented that permits one to calculate the Michaelis-Menten parameters V[sub max] and K[sub m] from extended experiments, by modelling V[sub max] as a variable dependent on time using exponential or sigmoidal equations. 2. This method was tested with cortisol depletion in cultured rat hepatocytes. V[sub max] and K[sub m] were used to calculate intrinsic clearance, and comparisons were made with methods already described in the literature. Intrinsic clearances given by our method were scaled to in vivo hepatic clearances that were close to those reported in the literature. 3. Our method could quantify the V[sub max] decrease with culture time from estimates of time parameters, t[sub 1/2] or t[sub 50]. In our system, this V[sub max] decrease was in agreement with P450 cytochrome inactivation rates published for the rat liver. 4. In conclusion, we propose a convenient, simple and useful general method for both Michaelis-Menten parameter estimation and modelling of variations in the metabolic capacities observed in in vitro systems. Such an approach should improve the usefulness of hepatocytes in primary cultures for long-term metabolism experiments. [ABSTRACT FROM AUTHOR]

Summary The aim of this study was to document the 24 h urinary Cortisol concentrations during a rest day and a day that included prolonged exercise. The influence of exercise on disposition (plasma, urine) of a dose of Cortisol (1 mg/kg) was also assessed. Six adult saddle horses were used. In one experiment, urinary Cortisol elimination rates were measured during rest and prolonged exercise (56 km, 12 km/h) corresponding to about 50% of the V̇O [ABSTRACT FROM AUTHOR]

Traditional cheesemaking processes often involve backslopping practice. However, over successive inoculations, acidification deficiencies may arise. In such cases, adding a starter is recommended to restore the ecosystem stability. This study examines the impact of an autochthonous starter composed of three Lactococcus lactis strains on a raw goat milk microbial community during their evolution. Bacterial composition and technological features (acidification and aroma) were analyzed during communities' evolution over 800 generations. 16S rRNA gene metabarcoding showed that Lactococcus lactis strains predominated. The raw goat milk community acidification capacities varied early in the evolution and then remained stable. Adding the L. lactis starter to this community stabilized the ecosystem from the beginning of the evolution. The acetoin production was associated with the starter presence, consistent with the establishment of the diacetylatis biovar strain from the starter in the raw goat milk community throughout the evolution. Increased or decreased production of some volatile organic compounds when the starter was added revealed a specific aroma footprint due to interactions between the two communities. This study showed that adding a starter could help to achieve the maximum acidification rate from the early inoculation cycles and could significantly modify the aroma profile during long-term backslopping., Competing Interests: Declaration of competing interest The authors of the manuscript “Interaction between a Lactococcus lactis autochthonous starter and a raw goat milk microbial community during long-term backslopping” submitted at Food Microbiol. have no conflict of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by its main symptom, visceral hypersensitivity (VH), which is aggravated by stress. Gut-brain interactions and gut bacteria may alleviate IBS symptoms, including VH. γ-amino butyric acid (GABA), produced notably by lactic acid bacteria (LAB), shows promising result in IBS symptoms treatment. In bacteria, GABA is generated through glutamate decarboxylase (GAD) metabolism of L-glutamic acid, maintaining intracellular pH. In mammals, GABA acts as an inhibitory neurotransmitter, modulating pain, stress, and anxiety. Therefore, utilizing GABA-producing LAB as a therapeutic approach might be beneficial. Our previous work showed that a GABA-producing Lactococcus lactis strain, NCDO2118, reduced VH induced by acute stress in rats after a 10-day oral treatment. Here, we identified the strain CNCM I-5388, with a four-fold higher GABA production rate under the same conditions as NCDO2118. Both strains shared 99.1% identical GAD amino acid sequences and in vitro analyses revealed the same optimal pH for GAD activity; however, CNCM I-5388 exhibited 17 times higher intracellular GAD activity and increased resistance to acidic pH. Additionally, in vivo experiments have demonstrated that CNCM I-5388 has faster anti-VH properties in rats compared with NCDO2118, starting from the fifth day of treatment. Finally, CNCM I-5388 anti-VH effects partially persisted after 5-day treatment interruption and after a single oral treatment. These findings highlight CNCM I-5388 as a potential therapeutic agent for managing VH in IBS patients., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M)., Results: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the P gadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the P gadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production., Conclusions: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Twenty-four strains of Lactococcus lactis isolated from raw goat milk collected in the Rocamadour PDO area were analysed by MLST typing and phenotypic characterisation. The strains were combined to design an indigenous starter for the production of Rocamadour PDO cheese. The strains were divided into three classes based on their technological properties: acidifying and proteolytic strains in class I (12/24 strains), slightly acidifying and non-proteolytic strains in class II (2/24 strains), and non-acidifying and non-proteolytic strains in class III (10/24 strains). Interestingly, all but three strains (21/24) produced diacetyl/acetoin despite not having citrate metabolism genes, as would classically be expected for the production of these aroma compounds. Three strains (EIP07A, EIP13D, and EIP20B) were selected for the indigenous starter based on the following inclusion/exclusion criteria: (i) no negative interactions between included strains, (ii) ability to metabolize lactose and at least one strain with the prtP gene and/or capable of producing diacetyl/acetoin, and (iii) selected strains derived from different farms to maximise genetic and phenotypic diversity. Despite consisting exclusively of L. lactis strains, the designed indigenous starter allowed reproducible cheese production with performances similar to those obtained with an industrial starter and with the sensory qualities expected of Rocamadour PDO cheese., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Gut disorders associated to irritable bowel syndrome (IBS) are combined with anxiety and depression. Evidence suggests that microbially produced neuroactive molecules, like γ-aminobutyric acid (GABA), can modulate the gut-brain axis. Two natural strains of Lactococcus lactis and one mutant were characterized in vitro for their GABA production and tested in vivo in rat by oral gavage for their antinociceptive properties. L. lactis NCDO2118 significantly reduced visceral hypersensitivity induced by stress due to its glutamate decarboxylase (GAD) activity. L. lactis NCDO2727 with similar genes for GABA metabolism but no detectable GAD activity had no in vivo effect, as well as the NCDO2118 ΔgadB mutant. The antinociceptive effect observed for the NCDO2118 strain was mediated by the production of GABA in the gastro-intestinal tract and blocked by GABA B receptor antagonist. Only minor changes in the faecal microbiota composition were observed after the L. lactis NCDO2118 treatment. These findings reveal the crucial role of the microbial GAD activity of L. lactis NCDO2118 to deliver GABA into the gastro-intestinal tract for exerting antinociceptive properties in vivo and open avenues for this GRAS (Generally Recognized As safe) bacterium in the management of visceral pain and anxious profile of IBS patients., Competing Interests: VL, CB, PG, SN, VT, MD, VT, HE, MM, MC No competing interests declared, (© 2022, Laroute, Beaufrand et al.)

GABA (γ-aminobutyric acid) production has been widely described as an adaptive response to abiotic stress, allowing bacteria to survive in harsh environments. This work aimed to clarify and understand the relationship between GABA production and bacterial growth conditions, with particular reference to osmolarity. For this purpose, Lactococcus lactis NCDO 2118, a GABA-producing strain, was grown in glucose-supplemented chemically defined medium containing 34 mM L-glutamic acid, and different concentrations of salts (chloride, sulfate or phosphate ions) or polyols (sorbitol, glycerol). Unexpectedly, our data demonstrated that GABA production was not directly related to osmolarity. Chloride ions were the most significant factor influencing GABA yield in response to acidic stress while sulfate ions did not enhance GABA production. We demonstrated that the addition of chloride ions increased the glutamic acid decarboxylase (GAD) synthesis and the expression of the gad BC genes. Finally, under fed-batch conditions in a complex medium supplemented with 0.3 M NaCl and after a pH shift to 4.6, L. lactis NCDO 2118 was able to produce up to 413 mM GABA from 441 mM L-glutamic acid after only 56 h of culture, revealing the potential of L. lactis strains for intensive production of this bioactive molecule.

Lactic acid bacteria (LAB) potential in the food industry and in the biotechnological sector is a well-established interest. LAB potential in counteracting especially food-borne infections has received growing attention, but despite being a road full of promises is yet poorly explored. Furthermore, the ability of LAB to produce antimicrobial compounds, both by ribosomal synthesis and by decrypting them from proteins, is of high value when considering the growing impact of multidrug resistant strains. The antimicrobial potential of 14 food-derived lactic acid bacteria strains has been investigated in this study. Among them, four strains were able to counteract Listeria monocytogenes growth: Lactococcus lactis SN12 and L. lactis SN17 by high lactic acid production, whereas L. lactis 41FLL3 and Lactobacillus sakei I151 by Nisin Z and Sakacin P production, respectively. Strains Lactococcus lactis MG1363 , Lactobacillus rhamnosus 17D10 and Lactobacillus helveticus 4D5 were tested and selected for their potential attitude to hydrolyze caseins. All the strains were able to release bioactive peptides with already known antimicrobial, antihypertensive and opioid activities. These features render these strains or their bioactive molecules suitable for use in food as biocontrol agents, or as nutraceutical supplements to treat mild disorders such as moderate hypertension and children insomnia. These results highlight once again that LAB potential in ensuring food safety, food nutraceutical value and ultimately in favoring human health is still underexplored and underexploited.

Designing bacterial co-cultures adapted to ferment mixes of vegetal and animal resources for food diversification and sustainability is becoming a challenge. Among bacteria used in food fermentation, lactic acid bacteria (LAB) are good candidates, as they are used as starter or adjunct in numerous fermented foods, where they allow preservation, enhanced digestibility, and improved flavor. We developed here a strategy to design LAB co-cultures able to ferment a new food made of bovine milk and lupin flour, consisting in: (i) in silico preselection of LAB species for targeted carbohydrate degradation; (ii) in vitro screening of 97 strains of the selected species for their ability to ferment carbohydrates and hydrolyze proteins from milk and lupin and clustering strains that displayed similar phenotypes; and (iii) assembling strains randomly sampled from clusters that showed complementary phenotypes. The designed co-cultures successfully expressed the targeted traits i.e., hydrolyzed proteins and degraded raffinose family oligosaccharides of lupin and lactose of milk in a large range of concentrations. They also reduced an off-flavor-generating volatile, hexanal, and produced various desirable flavor compounds. Most of the strains in co-cultures achieved higher cell counts than in monoculture, suggesting positive interactions. This work opens new avenues for the development of innovative fermented food products based on functionally complementary strains in the world-wide context of diet diversification., (Copyright © 2020 Canon, Mariadassou, Maillard, Falentin, Parayre, Madec, Valence, Henry, Laroute, Daveran-Mingot, Cocaign-Bousquet, Thierry and Gagnaire.)

Staphylococcus xylosus is found in the microbiota of traditional cheeses, particularly in the rind of soft smeared cheeses. Despite its frequency, the molecular mechanisms allowing the growth and adaptation of S. xylosus in dairy products are still poorly understood. A transcriptomic approach was used to determine how the gene expression profile is modified during the fermentation step in a solid dairy matrix. S. xylosus developed an aerobic metabolism perfectly suited to the cheese rind. It overexpressed genes involved in the aerobic catabolism of two carbon sources in the dairy matrix, lactose and citrate. Interestingly, S. xylosus must cope with nutritional shortage such as amino acids, peptides, and nucleotides, consequently, an extensive up-regulation of genes involved in their biosynthesis was observed. As expected, the gene sigB was overexpressed in relation with general stress and entry into the stationary phase and several genes under its regulation, such as those involved in transport of anions, cations and in pigmentation were up-regulated. Up-regulation of genes encoding antioxidant enzymes and glycine betaine transport and synthesis systems showed that S. xylosus has to cope with oxidative and osmotic stresses. S. xylosus expressed an original system potentially involved in iron acquisition from lactoferrin.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis . The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilus via transit as a sub-dominant bacterium (10 3 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract.

Motivation: Technological advances in metabolomics have made it possible to monitor the concentration of extracellular metabolites over time. From these data, it is possible to compute the rates of uptake and excretion of the metabolites by a growing cell population, providing precious information on the functioning of intracellular metabolism. The computation of the rate of these exchange reactions, however, is difficult to achieve in practice for a number of reasons, notably noisy measurements, correlations between the concentration profiles of the different extracellular metabolites, and discontinuties in the profiles due to sudden changes in metabolic regime., Results: We present a method for precisely estimating time-varying uptake and excretion rates from time-series measurements of extracellular metabolite concentrations, specifically addressing all of the above issues. The estimation problem is formulated in a regularized Bayesian framework and solved by a combination of extended Kalman filtering and smoothing. The method is shown to improve upon methods based on spline smoothing of the data. Moreover, when applied to two actual datasets, the method recovers known features of overflow metabolism in Escherichia coli and Lactococcus lactis , and provides evidence for acetate uptake by L. lactis after glucose exhaustion. The results raise interesting perspectives for further work on rate estimation from measurements of intracellular metabolites., Availability and Implementation: The Matlab code for the estimation method is available for download at https://team.inria.fr/ibis/rate-estimation-software/ , together with the datasets., Contact: eugenio.cinquemani@inria.fr., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Lactococcus lactis is one of the most extensively used lactic acid bacteria for the manufacture of dairy products. Exploring the biodiversity of L. lactis is extremely promising both to acquire new knowledge and for food and health-driven applications. L. lactis is divided into four subspecies: lactis , cremoris , hordniae and tructae , but only subsp. lactis and subsp. cremoris are of industrial interest. Due to its various biotopes, Lactococcus subsp. lactis is considered the most diverse. The diversity of L. lactis subsp. lactis has been assessed at genetic, genomic and phenotypic levels. Multi-Locus Sequence Type (MLST) analysis of strains from different origins revealed that the subsp. lactis can be classified in two groups: "domesticated" strains with low genetic diversity, and "environmental" strains that are the main contributors of the genetic diversity of the subsp. lactis . As expected, the phenotype investigation of L. lactis strains reported here revealed highly diverse carbohydrate metabolism, especially in plant- and gut-derived carbohydrates, diacetyl production and stress survival. The integration of genotypic and phenotypic studies could improve the relevance of screening culture collections for the selection of strains dedicated to specific functions and applications., Competing Interests: The authors declare no conflict of interest.

Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes.

Staphylococcus xylosus is commonly used as starter culture for meat fermentation. Its technological properties are mainly characterized in vitro, but the molecular mechanisms for its adaptation to meat remain unknown. A global transcriptomic approach was used to determine these mechanisms. S. xylosus modulated the expression of about 40-50% of the total genes during its growth and survival in the meat model. The expression of many genes involved in DNA machinery and cell division, but also in cell lysis, was up-regulated. Considering that the S. xylosus population remained almost stable between 24 and 72 h of incubation, our results suggest a balance between cell division and cell lysis in the meat model. The expression of many genes encoding enzymes involved in glucose and lactate catabolism was up-regulated and revealed that glucose and lactate were used simultaneously. S. xylosus seemed to adapt to anaerobic conditions as revealed by the overexpression of two regulatory systems and several genes encoding cofactors required for respiration. In parallel, genes encoding transport of peptides and peptidases that could furnish amino acids were up-regulated and thus concomitantly a lot of genes involved in amino acid synthesis were down-regulated. Several genes involved in glutamate homeostasis were up-regulated. Finally, S. xylosus responded to the osmotic stress generated by salt added to the meat model by overexpressing genes involved in transport and synthesis of osmoprotectants, and Na(+) and H(+) extrusion.

The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters.

Objective: To measure florfenicol concentrations in ovine tear fluid after IM and SC administration and determine minimum inhibitory concentrations (MICs) of florfenicol against field isolates of Mycoplasma organisms potentially involved in infectious keratoconjunctivitis., Animals: 9 healthy adult Lacaune ewes., Procedures: Animals received an IM and SC administration of florfenicol (20 mg/kg) in a 2-way crossover design. Samples of blood and tear fluid were collected before and for 24 hours after administration. Concentrations of florfenicol in plasma and tear fluid were measured via high-performance liquid chromatography. The MIC of florfenicol for various Mycoplasma strains cultured from sheep and goats was determined via an agar dilution method., Results: Mean florfenicol concentration in tear fluid for the 24-hour period was significantly higher after IM administration (0.70 μg/mL) than after SC administration (0.22 μg/mL) and was maintained for a longer duration. The lacrimal fluid-to-plasma concentration ratio was not different between the 2 routes of administration, with mean values of 40.2% and 32.5% after IM and SC administration, respectively. The MIC for Mycoplasma agalactiae, Mycoplasma conjunctivae, and Mycoplasma mycoides isolates ranged from 0.5 to 8 μg of florfenicol/mL. Two strains of M agalactiae could be considered resistant to florfenicol., Conclusions and Clinical Relevance: Florfenicol readily penetrated the preocular tear fluid of sheep after IM and SC administration. For both routes of administration, doses > 20 mg/kg would be necessary to achieve tear fluid concentrations of florfenicol greater than the MICs for most strains of Mycoplasma organisms.

Lactococcus lactis subsp. lactis biovar diacetylactis strains are used in the dairy industry for generating acetoin and notably diacetyl which imparts a high level of buttery flavor notes. A collection of domesticated and environmental strains was screened for the production of diacetyl or acetoin (D/A), and citrate fermentation. Unexpectedly, both domesticated and environmental strains produced D/A. Domesticated strains belonging to the currently named "biovar diacetylactis" metabolized citrate and produced large amounts of D/A during early growth. They harbored the citP plasmid gene encoding citrate permease and a chromosomal region citM-citI-citCDEFXG involved in citrate metabolism. In these strains, citrate consumption was identified as the major determinant of aroma production. Environmental strains, specifically UCMA5716 and A12, produced as much D/A as the CitP(+) strains, though at slightly lower rates. UCMA5716 was found to contain the citM-citI-citCDEFXG cluster but not the citP gene. A12 had neither. In these strains, production rate of D/A was linearly correlated with pyruvate synthesis rate. However, the correlation factor was strain-dependent, suggesting different modes of regulation for pyruvate rerouting towards fermentation end-products and flavors. This work highlights the genetic and metabolic differences between environmental and domesticated strains. The introduction of environmental strains into industrial processes could considerably increase the diversity of starters, enhancing the delivery of new technological properties., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

GABA is a molecule of increasing nutraceutical interest due to its modulatory activity on the central nervous system and smooth muscle relaxation. Potentially probiotic bacteria can produce it by glutamate decarboxylation, but nothing is known about the physiological modifications occurring at the microbial level during GABA production. In the present investigation, a GABA-producing Lactococcus lactis strain grown in a medium supplemented with or without glutamate was studied using a combined transcriptome/proteome analysis. A tenfold increase in GABA production in the glutamate medium was observed only during the stationary phase and at low pH. About 30 genes and/or proteins were shown to be differentially expressed in glutamate-stimulated conditions as compared to control conditions, and the modulation exerted by glutamate on entire metabolic pathways was highlighted by the complementary nature of transcriptomics and proteomics. Most glutamate-induced responses consisted in under-expression of metabolic pathways, with the exception of glycolysis where either over- or under-expression of specific genes was observed. The energy-producing arginine deiminase pathway, the ATPase, and also some stress proteins were down-regulated, suggesting that glutamate is not only an alternative means to get energy, but also a protective agent against stress for the strain studied.

Neonatal maternal deprivation (NMD) increases gut paracellular permeability (GPP) through mast cells and nerve growth factor (NGF), and modifies corticotrophin-releasing factor (CRF) and corticosterone levels. CRF, corticosterone and mast cells are involved in stress-induced mucosal barrier impairment. Consequently, this study aimed to specify whether corticosteronaemia and colonic expression of both preproCRF and CRF are modified by NMD, and to determine if altered expression may participate in the elevated GPP in connection with NGF and mast cells. Male Wistar rat pups were either separated from postnatal days 2-14, or left undisturbed with their dam. At 12 weeks of age, adult rats were treated with mifepristone (an antagonist of corticoid receptors), alpha-helical CRF((9-41)) (a non-specific CRF receptor antagonist), or SSR-125543 (CRF-R(1) receptor antagonist). We also determined corticosteronaemia and both colonic preproCRF and CRF expression. Then, control rats were treated by CRF, doxantrazole (mast cell stabilizer), BRX-537A (a mast cell activator) and anti-NGF antibody. NMD did not modify colonic CRF level but increased colonic preproCRF expression and corticosteronaemia. Peripheral CRF, via CRF-R(1) receptor, but not corticosterone, was involved in the elevated GPP observed in these rats, through a mast-cell-mediated mechanism, since the increase of GPP induced by exogenous CRF was abolished by doxantrazole. Anti-NGF antibody treatment also reduced the elevated GPP induced by CRF or BRX-537A. CRF acts through CRF-R(1) receptors to stimulate NGF release from mast cells, which participates in the elevated GPP observed in NMD adult rats. This suggests that early traumatic experience induced neuro-endocrine dysfunction, involved in alterations of gut mucosal barrier.

The objectives of this study were to compare assay methods for plasma creatinine (Pl-creat) in cats and to describe the disposition of creatinine and iohexol in 12 healthy and moderately azotaemic cats. Exogenous creatinine and iohexol were injected simultaneously by intravenous bolus, and repeated blood samples were taken to determine the pharmacokinetic parameters of each marker. Pl-creat was assayed by high-performance liquid chromatography (HPLC), Jaffé and enzymatic methods. The enzymatic method was shown to be more reliable than the Jaffé method. Two stereoisomers, exo- and endo-iohexol were identified. The plasma clearance of creatinine (2.3+/-0.66 ml/min/kg) was significantly higher (P<0.001) than that of exo-iohexol (1.7+/-0.40 ml/min/kg). The volume of distribution (447+/-97 ml/kg) and elimination half-life (181+/-77 min) of creatinine were also higher (P<0.001) than those of exo- and endo-iohexol. The estimated daily endogenous production of creatinine was 65+/-23 mg/kg. None of the pharmacokinetic parameters was changed by the azotaemic status of the animals.