New in vitro colonic fermentation model for Salmonella infection in the child gut

The definitive version is available at ww3.interscience.wiley.comInternational audienceIn this study, a new in vitro continuous colonic fermentation model of Salmonella infection with immobilized child fecal microbiota and Salmonella serovar Typhimurium was developed for the proximal colon. This model was then used to test the effects of two amoxicillin concentrations (90 and 180 mg day(-1)) on the microbial composition and metabolism of the gut microbiota and on Salmonella serovar Typhimurium during a 43-day fermentation. Addition of gel beads (2%, v/v) colonized with Salmonella serovar Typhimurium in the reactor resulted in a high and stable Salmonella concentration (log 7.5 cell number mL(-1)) in effluent samples, and a concomitant increase of Enterobacteriaeceae, Clostridium coccoides-Eubacterium rectale and Atopobium populations and a decrease of bifidobacteria. During amoxicillin treatments, Salmonella concentrations decreased while microbial balance and activity were modified in agreement with in vivo data, with a marked decrease in C. coccoides-E. rectale and an increase in Enterobacteriaceae. After interruption of antibiotic addition, Salmonella concentration again increased to reach values comparable to that measured before antibiotic treatments, showing that our model can be used to simulate Salmonella shedding in children as observed in vivo. This in vitro model could be a useful tool for developing and testing new antimicrobials against enteropathogens

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

The definitive version is available at ww3.interscience.wiley.comInternational audienceAims: To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods. Methods and Results: A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose- 5-phosphate ⁄ fructose-6-phosphate phosphoketolase gene (xfp) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2.5 x 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods. Conclusions: The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces. Significance and Impact of the Study: This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods

Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria

<p>Abstract</p> <p>Background</p> <p>Reuterin produced from glycerol by <it>Lactobacillus reuteri</it>, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of <it>Lb. reuteri</it>. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin.</p> <p>The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by <it>Lb</it>. <it>reuteri </it>SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed.</p> <p>Results</p> <p>Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator <it>Escherichia coli </it>(7.5–15 mM). Lactobacilli and <it>Clostridium clostridioforme </it>were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found.</p> <p>Conclusion</p> <p>Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide.</p

Safety assessment of dairy microorganisms: Propionibacterium and Bifidobacterium.

International audienceThe genera Propionibacterium and Bifidobacterium are clustered in the class Actinobacteria and form the anaerobic branch of coryneform bacteria. The dairy propionibacteria comprising four species P. freudenreichii, P. acidipropionici, P. jensenii and P. thoenii are industrially important as starter cultures in hard-cheese ripening and recently also as protective bio-preservatives and probiotics. These four species are considered as safe whereas cutaneous Propionibacterium species (also named "acnes group") are pathogens. In contrast, bifidobacteria in fermented dairy products and milk powder are exclusively used as probiotics; selected strains of several species (out of more than thirty) contribute to this task. It has been only rarely found that commensal bifidobacteria have been connected with certain dental and other infections. Consequently, only one single species, Bifidobacterium dentium, is recognized as pathogenic. Genome sequence analysis of Bifidobacterium longum and molecular biological analysis of other probiotic strains confirmed so far the absence of virulence and pathogenecity factors. However, tetracycline resistance genes tet(W), although probably not easy transferable, were found in Bifidobacterium strains, also in Bifidobacterium animalis subsp. lactis, the worldwide most used industrial strain. Conclusively, strains from the Propionibacterium and Bifidobacterium species in dairy food generally represent so far no health hazards

Screening of Lactobacillus spp. for the prevention of Pseudomonas aeruginosa pulmonary infections

Background: Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host's health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. Results: Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. Conclusion: Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further studies are needed in order to test their innocuity and their capacity to behave such as an oropharyngeal barrier against Pseudomonas aeruginosa colonisation in vivo

Comparative effects of exopolysaccharides from lactic acid bacteria and fructo-oligosaccharides on infant gut microbiota tested in an in vitro colonic model with immobilized cells

ISSN:0168-6496ISSN:1574-694

In vitro inhibition activity of nisin A, nisin Z, pediocin PA-1 and antibiotics against common intestinal bacteria

The definitive version is available at ww3.interscience.wiley.comInternational audienceAims: To evaluate the sensitivity of 21 common intestinal bacteria to six antibiotics and three broad-spectrum bacteriocins (nisins Z and A and pediocin PA-1). Methods and Results: Neutralized cell-free culture supernatants containing active bacteriocins, and antibiotics were tested with the agar diffusion test and the disc-diffusion method, respectively. The tested intestinal strains showed high sensitivity to most antibiotics except for streptomycin and oxacillin. Nisins A and Z (8 lg per well) had similar activity spectra and inhibited all Gram-positive intestinal bacteria at different levels (except Streptococcus salivarius), with bifidobacteria (except Bifidobacterium breve and Bif. catenulatum), Collinsella aerofaciens and Eubacterium biforme being the most sensitive strains, but they were not active against Gram-negative bacteria. Surprisingly, none of the tested strains were inhibited by pediocin PA-1 (16 lg per well). Conclusion: Pediocin PA-1 which is very active against Listeria spp. and other food pathogens did not inhibit major intestinal species in the human intestine in contrast to both nisins A and Z. Significance and Impact of the Study: Our data suggest that pediocin PA-1 has potential to inhibit Listeria within the intestinal microbiota without altering commensal bacteria

Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces

The definitive version is available at ww3.interscience.wiley.comInternational audienceLactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This strain has been shown to prevent intestinal infections in vivo; however, its mechanisms of action, and more specifically whether reuterin production occurs within the intestinal tract, are not known. In this study, the effects of L. reuteri ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors were inoculated with adult immobilized fecal microbiota and the effects of daily addition of L. reuteri into one of the reactors (c.108 CFUmL1) without or with glycerol were tested on major bacterial populations and compared with addition of glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus–Enterococcus group and decreased Escherichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli concentration during the addition of glycerol (in presence or absence of L. reuteri) could be due to in situ reuterin production because 1,3-propanediol, a typical product of glycerol fermentation, was detected during the addition of glycerol

Cultivable epiphytic bacteria of the Chlorophyta Ulva sp.: diversity, antibacterial, and biofilm-modulating activities

International audienceAbstract Aims Macroalgae harbor a rich epiphytic microbiota that plays a crucial role in algal morphogenesis and defense mechanisms. This study aims to isolate epiphytic cultivable microbiota from Ulva sp. surfaces. Various culture media were employed to evaluate a wide range of cultivable microbiota. Our objective was to assess the antibacterial and biofilm-modulating activities of supernatants from isolated bacteria. Methods and results Sixty-nine bacterial isolates from Ulva sp. were identified based on 16S rRNA gene sequencing. Their antibacterial activity and biofilm modulation potential were screened against three target marine bacteria: 45%, mostly affiliated with Gammaproteobacteria and mainly grown on diluted R2A medium (R2Ad), showed strong antibacterial activity, while 18% had a significant impact on biofilm modulation. Molecular network analysis was carried out on four bioactive bacterial supernatants, revealing new molecules potentially responsible for their activities. Conclusion R2Ad offered the greatest diversity and proportion of active isolates. The molecular network approach holds promise for both identifying bacterial isolates based on their molecular production and characterizing antibacterial and biofilm-modulating activities