Carnobacterium maltaromaticum as bioprotective culture against spoilage bacteria in ground meat and cooked ham.

This study assessed the bioprotective effect of Carnobacterium maltaromaticum (CM) against Pseudomonas fluorescens (PF) and Brochothrix thermosphacta (BT) in ground beef and sliced cooked ham stored in high- and low-oxygen-modified atmospheres (66/4/30% O2/N2/CO2 and 70/30% N2/CO2, respectively). Both meat products were inoculated with CM, PF, and BT individually or in combination and stored for 7 days (3 days at 4 °C + 4 days at 8 °C) for ground beef and 28 days (10 days at 4 °C + 18 days at 8 °C) for sliced cooked ham. Each food matrix was assigned to 6 treatments: NC (no bacterial inoculation, representing the indigenous bacteria of meat), CM, BT, PF, CM + BT, and CM + PF. Bacterial growth, pH, instrumental color, and headspace gas composition were assessed during storage. CM counts remained stable from inoculation and throughout the shelf-life. CM reduced the population of inoculated and indigenous spoilage bacteria, including BT, PF, and enterobacteria, and showed a negligible impact on the physicochemical quality parameters of the products. Furthermore, upon simulating the shelf-life of ground beef and cooked ham, a remarkable extension could be observed with CM. Therefore, CM could be exploited as a biopreservative in meat products to enhance quality and shelf-life.

Phage Targeting Neonatal Meningitis E. coli K1 In Vitro in the Intestinal Microbiota of Pregnant Donors and Impact on Bacterial Populations.

Escherichia coli K1 is a leading cause of neonatal meningitis. The asymptomatic carriage of these strains in the maternal intestinal microbiota constitutes a risk of vertical transmission to the infant at birth. The aim of this work was to evaluate the efficacy of phage therapy against E. coli K1 in an intestinal environment and its impact on the intestinal microbiota. For this purpose, three independent experiments were conducted on the SHIME® system, the first one with only the phage vB_EcoP_K1_ULINTec4, the second experiment with only E. coli K1 and the last experiment with both E. coli K1 and the phage. Microbiota monitoring was performed using metagenetics, qPCR, SCFA analysis and the induction of AhR. The results showed that phage vB_EcoP_K1_ULINTec4, inoculated alone, was progressively cleared by the system and replicates in the presence of its host. E. coli K1 persisted in the microbiota but decreased in the presence of the phage. The impact on the microbiota was revealed to be donor dependent, and the bacterial populations were not dramatically affected by vB_K1_ULINTec4, either alone or with its host. In conclusion, these experiments showed that the phage was able to infect the E. coli K1 in the system but did not completely eliminate the bacterial load.

Impact Assessment of vB_KpnP_K1-ULIP33 Bacteriophage on the Human Gut Microbiota Using a Dynamic In Vitro Model.

New control methods are needed to counter antimicrobial resistances and the use of bacteriophages as an alternative treatment seems promising. To that end, the effect of the phage vB_KpnP_K1-ULIP33, whose host is the hypervirulent Klebsiella pneumoniae SA12 (ST23 and capsular type K1), was assessed on intestinal microbiota, using an in vitro model: the SHIME® system (Simulator of the Human Intestinal Microbial Ecosystem). After stabilization of the system, the phage was inoculated for 7 days and its persistence in the different colons was studied until its disappearance from the system. The concentration of short chain fatty acids in the colons showed good colonization of the bioreactors by the microbiota and no significant effect related to the phage treatment. Diversity (α and ß), the relative abundance of bacteria, and qPCR analysis targeting different genera of interest showed no significant variation following phage administration. Even if further in vitro studies are needed to assess the efficacy of this phage against its bacterial host within the human intestinal ecosystem, the phage ULIP33 exerted no significant change on the global colonic microbiota.

Impact of environmental conditions and gut microbiota on the in vitro germination and growth of Clostridioides difficile.

Clostridioides difficile is a spore-forming anaerobic Gram-positive bacterium responsible for a broad spectrum of intestinal symptoms and healthcare-associated diarrhoea. The hypothesis of this work was that different in vitro conditions, notably pH and human faecal microbiota composition, impact the germination and/or the growth of C. difficile. This study aimed to correlate growth kinetics of the bacterium with these two physiochemical parameters by using a static in vitro model. To better understand the initial gut colonisation, several growth curve assays were carried out to monitor the behaviour of the spores and vegetative forms of C. difficile strain 078 under different conditions mimicking the gut environment. When the faeces were added, no spore germination or growth was observed, but C. difficile spores germinated in vitro when the pH was maintained between 6.6 and 6.9 for four different faeces donors. The evolution of microbiota studied by 16S rDNA profiling showed high proportions of Enterobacteriaceae and E. coli/Shigella when C. difficile grew, regardless of the inoculated faeces. This model helped us to understand that the germination and growth of C. difficile are strongly pH dependent, and further research is needed to evaluate the potential impact of the gut microbiota composition on C. difficile.

The efficacy and effect on gut microbiota of an aflatoxin binder and a fumonisin esterase using an in vitro simulator of the human intestinal microbial ecosystem (SHIME®).

Mycotoxin intoxication is in general an acknowledged and tackled issue in animals. However, in several parts of the world, mycotoxicoses in humans still remain a relevant issue. The efficacy of two mycotoxin detoxifying animal feed additives, an aflatoxin bentonite clay binder and a fumonisin esterase, was investigated in a human child gut model, i.e. the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). Additionally, the effect of the detoxifiers on gut microbiota was examined in the SHIME. After an initial two weeks of system stabilisation, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) were added to the SHIME diet during one week. Next, the two detoxifiers and mycotoxins were added to the system for an additional week. The AFB1, FB1, hydrolysed FB1 (HFB1), partially hydrolysed FB1a and FB1b concentrations were determined in SHIME samples using a validated ultra-performance liquid chromatography-tandem mass spectrometry method. The short-chain fatty acid (SCFA) concentrations were determined by a validated gas chromatography-mass spectrometry method. Colonic bacterial communities were analysed using metabarcoding, targeting the hypervariable V1-V3 regions of the 16S rRNA genes. The AFB1 and FB1 concentrations significantly decreased after the addition of the detoxifiers. Likewise, the concentration of HFB1 significantly increased. Concentrations of SCFAs remained generally stable throughout the experiment. No major changes in bacterial composition occurred during the experiment. The results demonstrate the promising effect of these detoxifiers in reducing AFB1 and FB1 concentrations in the human intestinal environment, without compromising the gastrointestinal microbiota.

Effect of Bifidobacterium crudilactis and 3'-sialyllactose on the toddler microbiota using the SHIME® model.

The important changes in diet during the first years of life strongly modulate the intestinal microbiota of young children. Among in vitro digestive models, the simulator of human intestinal microbial ecosystem (SHIME®) model, seems particularly adapted to study the effects of prebiotics and/or probiotics on the dynamic microbiota of toddlers. The main purpose of this study was to investigate different formulations with prebiotic (3'-sialyllactose: 3'SL) and probiotic (Bifidobacterium crudilactis FR/62/b/3) effects on young child microbiota using the SHIME® model. The ascending (AC), transverse (TC) and descending (DC) colons of the SHIME® model were inoculated with feces from 3 donors aged between 1 and 2 years, in three separate vessels. After two weeks of microbiota stabilization, four treatments of one week (prebiotic, probiotic, synbiotic and cell-free spent media from the synbiotic) were administered. In all the colon vessels, the short chain fatty acid analyses, determined using high-performance liquid chromatography highlighted a ratio acetate/propionate/butyrate proportion of 5:19:6, situated between infant and adult normal values. As already observed in other validated studies focusing on the SHIME® model, the 16S rDNA sequencing highlighted a low richness and diversity in the AC, while the microbial communities in the TC and the DC remained similar to each other. Although some bacteria involved in biofilm development have been identified (Stenotrophomonas, Megasphera and Enterobacter), specific bacterial populations, proper to each colon were developed. Some bacteria associated to the upper intestinal tract, such as Lactobacillus and Veillonella genera, seemed to grow easily in the AC. The quantitative polymerase chain reaction (qPCR) targeting the hsp60 gene confirmed the ability of bifidobacteria to survive in this toddler model. In addition, the synbiotic treatment tended to a bifidogenic effect (P < 0.1). On the other hand, the feces of the donors and the content of the three colon vessels were filtered and placed in contact with Escherichia coli O157:H7 ATCC 43890 to evaluate the modulation of virulence gene expression using reverse transcription PCR. Finally, filtered supernatants from donor feces significantly up-regulated the expression of the luxS gene of E. coli O157:H7 (P = 0.013). In conclusion, despite the presence of biofilms, the toddler SHIME® model used in his study shared characteristics found both in adults and infants. Although additional investigations should be performed, combining 3'SL and B. crudilactis FR/62/b/3 could lead to a beneficial effect on infant microbiota by favoring bifidobacterial presence. Finally, the filtrated supernatant from young child feces could be able to modulate the quorum sensing mechanism for E. coli O157:H7.

A toddler SHIME® model to study microbiota of young children.

The 'first 1000 days of life' determine the gut microbiota composition and can have long-term health consequences. In this study, the simulator of the human intestinal microbial ecosystem (SHIME®) model, which represents the main functional sections of the digestive tract, was chosen to study the microbiota of young children. The aim of this study was to reproduce the digestive process of toddlers and their specific colonic environment. The ascending, transverse and descending colons of SHIME® model were inoculated with feces from three donors aged between 1 and 2 years-old, in three separate runs. For each run, samples from colon vessels were collected at days 14, 21 and 28 after microbiota stabilization period. Short chain fatty acid concentrations determined by HPLC showed that microbiota obtained in SHIME® model shared characteristics between adults and infants. In addition, microbial diversity and bacterial populations determined by 16S rRNA amplicon sequencing were specific to each colon vessel. In conclusion, the SHIME® model developed in this study seemed well adapted to evaluate prebiotic and probiotic impact on the specific microbiota of toddlers, or medicine and endocrine disruptor metabolism. Moreover, this study is the first to highlight some biofilm development in in vitro gastrointestinal modelling systems.

Baby-SPIME: A dynamic in vitro piglet model mimicking gut microbiota during the weaning process.

The study aimed to adapt the SHIME® model, developed to simulate human digestion and fermentation, to a baby-SPIME (baby Simulator of Pig Intestinal Microbial Ecosystem). What constitutes a unique feature of this model is its twofold objective of introducing an ileal microbial community and mimicking a dietary weaning transition. This model should then be ideally suited to test the dietary weaning strategies of piglets in vitro. Regarding the microbiota, the main phyla making up the model were Firmicutes, Bacteroidetes and Proteobacteria although Bacteroidetes decreased after inoculation (p = 0.043 in ileum, p = 0.021 in colon) and Delta-Proteobacteria were favoured (p = 0.083 in ileum, p = 0.043 in colon) compared to Gamma-Proteobacteria. The designed model led to a low representation of Bacilli - especially Lactobacillus sp. in the ileum and a weak representation of Bacteroidia in the proximal colon. However, Mitsuokella and Prevotella were part of the major genera of the model along with Bifidobacterium, Fusobacterium, Megasphaera and Bacteroides. As a result of weaning, two major changes - normally occurring in vivo - were detected in the system: firstly, Firmicutes diminished when Bacteroidetes increased particularly in the proximal colon; secondly, Bacteroides decreased and Prevotella increased (mean value for four runs). In terms of metabolite production, while a ratio acetate: propionate: butyrate of 60:26:14 was obtained in post-weaned colon, the expected inversion of the ratio propionate: butyrate in the post-weaned ileum was unfortunately not observed. To conclude, the so-called baby-SPIME model meets expectations regarding the resident microbiota of the proximal colon bioreactor and the metabolites produced thereof. In terms of the evolution of major groups of bacteria, the in vitro weaning process appeared to be successful. However, higher concentration of butyric acid would have been expected in ileum part of newly weaned piglets, as observed in vivo. The microbiota in the ileum bioreactor seemed in fact to act like a pre-colon. This suggests that microbial profile in ileum bioreactor had to be improved.

Development of an analytical method to detect short-chain fatty acids by SPME-GC-MS in samples coming from an in vitro gastrointestinal model.

Short chain fatty acids (SCFA) are end-products of intestinal bacterial fermentation. The concentrations of fermentation metabolites are closely related to the microbial activity that occurs in various digestive compartments. The fermentation products may vary qualitatively and quantitatively, especially within the colon. The Simulator of the Human Intestinal Microbial Ecosystem (SHIME), an in vitro dynamic and multicompartment model of the human intestinal tract, can be adapted to mimic the piglet gastrointestinal tract. In this context, a quantitative method, based on solid phase microextraction gas chromatography coupled to mass spectrometry (SPME-GC-MS), was developed for the determination of seven short chain fatty acids, i.e. acetic, propionic, butyric, isobutyric, valeric, isovaleric and hexanoic acids, in samples coming from this experimental in vitro gastrointestinal model. The advantage of the SPME-GC-MS technique is that the seven compounds could be determined in a single run, after a simple and rapid sample treatment, without any other extraction than the automatic SPME. The developed method was validated in accordance to the European and US FDA guidelines and showed good specificity/selectivity. In addition, limits of detection and quantification ranged from 8 to 72 mg L-1 and from 16 to 144 mg L-1, respectively. Two internal quality control samples spiked at different concentrations were analyzed to assess the trueness of the developed method, which ranged between 97.7 and 122.4% of the expected value, for the seven compounds analyzed. The method was successfully applied to twenty samples coming from a gastrointestinal model, with different inocula. The developed method might be used as a general method for measuring SCFA in biological samples.

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

[Value of infrared spectrophotometry morpho-constitutional analysis of double J stent encrustations for indirect determination of urinary stone composition]. / Intérêt de l'analyse morpho-constitutionnelle par spectrophotométrie infra-rouge des incrustations des sondes double J pour la détermination indirecte de la composition des calculs urinaires.

INTRODUCTION: A single stone analysis is necessary during the patient's clinical history in order to institute specific drug treatment and health and dietary measures to prevent stone recurrence. In practice, only one in every two stones is recovered for morpho-constitutional analysis. The objective of this study was to determine the place of double J stent encrustation analysis for indirect determination of stone composition. MATERIAL AND METHODS: Double J stents and stones from all patients treated in the same centre over 24 months were consecutively analysed by infrared spectrophotometry. The correlation coefficient 1, evaluating the concordance between the composition of stones and double J stent encrustation was estimated statistically by SPSS 12.0 software (0<1<1; 1=0: no concordance; 1=1: perfect concordance). RESULTS: 45 males and 27 females with a mean age of 45.3 years (range: 29-70) were included Double J stents were placed for: febrile obstruction (N=52; 72%), acute renal colic (N=15; 21%) and impaired renal function (N=5; 7%). Calculated values for 1 were: 0.78 for the concordance between the predominant constituent of the stone and the encrustation (N=72; p < 0.0005); 0.91 for the concordance between the nature of the encrustation of the upper loop and that of the lower loop of the stent (N=30, p < 0.0005). CONCLUSION: The composition of mineral encrustation of double J stents is a good marker of stone formation. This constitutes an alternative method that can be used by urologists when no stone is available for spectrophotometric analysis.