Bifidobacterium pseudolongum are efficient indicators of animal fecal contamination in raw milk cheese industry

Background: The contamination of raw milk cheeses (St-Marcellin and Brie) from two plants in France was studied at several steps of production (raw milk, after addition of rennet - St-Marcellin - or after second maturation - Brie -, after removal from the mold and during ripening) using bifidobacteria as indicators of fecal contamination. Results: Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. B. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) at the different production steps) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2.40 log cfu ml-1) of Brie cheeses samples. Mean counts of B. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further characterization of these populations is currently under process. Forty-eight percents (St-Marcellin) and 70 % (Brie) of the samples were B. pseudolongum positive / E. coli negative while only 10 % (St-Marcellin) and 3 % (Brie) were B. pseudolongum negative / E. coli positive. Conclusions: The increase of total bifidobacteria during ripening in Marcellin’s process does not allow their use as fecal indicator. The presence of B. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. B. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. B. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses. Results: Bifidobacterium semi-quantitative counts were compared using PCR-RFLP and real-time PCR. Bif. pseudolongum were detected in 77% (PCR-RFLP; 1.75 to 2.29 log cfu ml-1) at the different production steps) and 68% (real-time PCR; 2.19 to 2.73 log cfu ml-1) of St-Marcellin samples and in 87% (PCR-RFLP; 1.17 to 2. 40 log cfu ml-1) of Brie cheeses samples. Mean counts of Bif. pseudolongum remained stable along both processes. Two other populations of bifidobacteria were detected during the ripening stage of St-Marcellin, respectively in 61% and 18% of the samples (PCR-RFLP). The presence of these populations explains the increase in total bifidobacteria observed during ripening. Further identification of these species is currently under process. Forty-eight percents (St-Marcellin) and 70 % (Brie) of the samples were Bif. pseudolongum positive / E. coli negative while only 10 % (St-Marcellin) and 3 % (Brie) were Bif. pseudolongum negative / E. coli positive. Conclusions: The increase of total bifidobacteria during ripening in Marcellin’s process does not allow their use as fecal indicator. The presence of Bif. pseudolongum along the processes defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. Bif. pseudolongum was more sensitive as an indicator than E. coli along the two different cheese processes. Bif. pseudolongum should be used as fecal indicator rather than E. coli to assess the quality of raw milk and raw milk cheeses.BIFI

Discrimination between Bifidobacterium species from human and animal origin by PCR-restriction fragment length polymorphism

peer reviewedBifidobacteria are normal intestinal flora in humans and animals. The genus Bifidobacterium includes 31 species of significant host specificity. Taking into account their properties, we proposed to use bifidobacteria as fecal contamination indicators. PCR-restriction fragment length polymorphism on the 16S rDNA gene was used to distinguish the different Bifidobacterium species. Sixty-four strains belonging to 13 different species were differentiated from animal or human origin using one or two restriction enzymes. Moreover, the primers used were specifics of the Bifidobacterium genus. Therefore, this method made it possible to determine both the presence of bifidobacteria in a sample and its origin of contamination

Assessment of bacterial superficial contamination in classical or ritually slaughtered cattle using metagenetics and microbiological analysis

The aim of this study was to investigate the influence of the slaughter technique (Halal vs. Classical slaughter) on the superficial contamination of cattle carcasses, by using traditional microbiological procedures and 16S rDNA metagenetics. The purpose was also to investigate the neck area to identify bacteria originating from the digestive or the respiratory tract. Twenty bovine carcasses (10 from each group) were swabbed at the slaughterhouse, where both slaughtering methods are practiced. Two swabbing areas were chosen: one “legal” zone of 1,600 cm2 (composed of zones from rump, flank, brisket and forelimb) and locally on the neck area (200 cm2). Samples were submitted to classical microbiology for aerobic Total Viable Counts (TVC) at 30°C and Enterobacteriaceae counts, while metagenetic analysis was performed on the same samples. The classical microbiological results revealed no significant differences between both slaughtering practices; with values between 3.95 and 4.87 log CFU/100 cm2 and 0.49 and 1.94 log CFU/100 cm2, for TVC and Enterobacteriaceae respectively. Analysis of pyrosequencing data showed that differences in the bacterial population abundance between slaughtering methods were mainly observed in the “legal” swabbing zone compared to the neck area. Bacterial genera belonging to the Actinobacteria phylum were more abundant in the “legal” swabbing zone in “Halal” samples, while Brevibacterium and Corynebacterium were encountered more in “Halal” samples, in all swabbing areas. This was also the case for Firmicutes bacterial populations (families of Aerococcaceae, Planococcaceae). Except for Planococcoceae, the analysis of Operational Taxonomic Unit (OTU) abundances of bacteria from the digestive or respiratory tract revealed no differences between groups. In conclusion, the slaughtering method does not influence the superficial microbiological pattern in terms of specific microbiological markers of the digestive or respiratory tract. However, precise analysis of taxonomy at the genus level taxonomy highlights differences between swabbing areas. Although not clearly proven in this study, differences in hygiene practices used during both slaughtering protocols could explain the differences in contamination between carcasses from both slaughtering groups

Prévalence, résistance aux antibiotiques et profile des gènes de virulence de Escherichia coli partagée entre les aliments et d'autres sources. une revue systématique

peer reviewedBackground and Aim: Foodborne diseases caused by Escherichia coli are prevalent globally. Treatment is challenging due to antibiotic resistance in bacteria, except for foodborne infections due to Shiga toxin-producing E. coli, for which treatment is symptomatic. Several studies have been conducted in Africa on antibiotic resistance of E. coli isolated from several sources. The prevalence and distribution of resistant pathogenic E. coli isolated from food, human, and animal sources and environmental samples and their virulence gene profiles were systematically reviewed. Materials and Methods: Bibliographic searches were performed using four databases. Research articles published between 2000 and 2022 on antibiotic susceptibility and virulence gene profile of E. coli isolated from food and other sources were selected. Results: In total, 64 articles were selected from 14 African countries: 45% of the studies were conducted on food, 34% on animal samples, 21% on human disease surveillance, and 13% on environmental samples. According to these studies, E. coli is resistant to ~50 antimicrobial agents, multidrug-resistant, and can transmit at least 37 types of virulence genes. Polymerase chain reaction was used to characterize E. coli and determine virulence genes. Conclusion: A significant variation in epidemiological data was noticed within countries, authors, and sources (settings). These results can be used as an updated database for monitoring E. coli resistance in Africa. More studies using state-of-theart equipment are needed to determine all resistance and virulence genes in pathogenic E. coli isolated in Africa

In vitro evaluation of the aryl hydrocarbon receptor transactivity induced by human intestinal metabolites

The gastrointestinal tract is directly exposed to the environment and constitutes one of the first lines of defense against harmful antigens in the diet or the microbiota. Highly expressed in intestinal cells, the aryl hydrocarbon receptor (AhR) plays a crucial role in gut mucosal integrity and in intestinal immune function. Substances derived from food and the microbiota in the intestinal lumen can induce AhR transcriptional activity and thus maintain intestinal barrier homeostasis. We suggested the combination of a simulator of the human gut microbial ecosystem (SHIME) with cell based AhR transactivity bioassays. Thereby, the effects of human gut microbiota metabolism on the AhR signaling pathways could be evaluated. Three AhR reporter gene cell lines (T47D, HepG2, HT29) from human mammary, hepatic, and intestinal tissues, respectively, were used to test mixtures of microbiota-derived metabolites that were generated in a 3-days experiment on a SHIME system. The SHIME samples induced an AhR agonist activation in the different cell lines, with slightly tissue-dependent effects. Microbiota produced metabolites, such as short-chain fatty acids (SCFA) and tryptophan catabolites were quantified chromatographically in the SHIME samples, and they might be causing the AhR agonist activity of the mixture. However, more detailed investigations are needed to identify the contribution of individual metabolites to AhR activation. This is an innovative suggestion to study in vitro the AhR modulation caused by the human intestinal metabolism.GutTransit Projec

Sucralose and maltodextrin affect differently the gut microbiota of healthy individuals and IBD patients

editorial reviewedIntestinal fibrosis is a long–term complication of inflammatory bowel diseases (IBD). Changes in microbial populations have been linked with the onset of fibrosis and some food additives are known to promote intestinal inflammation facilitating fibrosis induction. Most of these studies have been performed using murine models or healthy donors while the effects of food additives on intestinal microbiota of patients suffering from IBD is less understood. The aim of this work was therefore to determine how food additives affect intestinal microbiota of both healthy and “IBD” donors. Two food additives, sucralose (SUC) and maltodextrin (MDX), were tested in a short–term (72h) in vitro model of the human intestinal microbiota. Three groups of donors were investigated: healthy persons (H), patients in remission of IBD (R) and patients with an active period of IBD (A). Short-chain fatty acid production was assessed using SPME-GC/MS while the evolution of microbial populations positively or negatively correlated with health, inflammation and/or fibrosis was assessed using qPCR. MDX and SUC increased propionate and butyrate production in H and R donors. In addition, MDX increased butyrate in A donors and decreased the butyrate–producer genus Roseburia in H and R donors. Both, SUC and MDX decreased the beneficial bacteria F. prausnitzii in A donors. Moreover, in R and A donors, SUC and MDX decreased A. muciniphila. MDX promoted the growth of Enterococcus in H and R groups and Streptococcus in the three groups of donors, both genus being associated with intestinal inflammation. SUC induced increases of Escherichia/Shigella in H and Enterococcus in H and R groups. Ruminococcus, correlated with higher risk of fibrosis, was increased in the three groups of donors treated with MDX while SUC increased Ruminococcus only in IBD donors. Oscillospira, correlated with reduced risk of fibrosis, was decreased in the three groups of donors with MDX and in H and A donors with SUC. This study demonstrates how strongly the human microbiota can be affected by some food additives. In addition, to our knowledge, this is the first human in vitro study focusing on the impact of food additives on microbiota of IBD patients

Human Adult Microbiota in a Static Colon Model: AhR Transcriptional Activity at the Crossroads of Host–Microbe Interaction

peer reviewedFunctional symbiotic intestinal microbiota regulates immune defense and the metabolic processing of xenobiotics in the host. The aryl hydrocarbon receptor (AhR) is one of the transcription factors mediating host–microbe interaction. An in vitro static simulation of the human colon was used in this work to analyze the evolution of bacterial populations, the microbial metabolic output, and the potential induction of AhR transcriptional activity in healthy gut ecosystems. Fifteen target taxa were explored by qPCR, and the metabolic content was chromatographically profiled using SPME-GC-MS and UPLC-FLD to quantify short-chain fatty acids (SCFA) and biogenic amines, respectively. Over 72 h of fermentation, the microbiota and most produced metabolites remained stable. Fermentation supernatant induced AhR transcription in two of the three reporter gene cell lines (T47D, HepG2, HT29) evaluated. Mammary and intestinal cells were more sensitive to microbiota metabolic production, which showed greater AhR agonism than the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) used as a positive control. Some of the SCFA and biogenic amines identified could crucially contribute to the potent AhR induction of the fermentation products. As a fundamental pathway mediating human intestinal homeostasis and as a sensor for several microbial metabolites, AhR activation might be a useful endpoint to include in studies of the gut microbiota.GutTransit Project (Convention N  8225, Pôle–N  27), Wagralim, Wallonie, Belgiu

In Vitro and In Vivo Assessments of Two Newly Isolated Bacteriophages against an ST13 Urinary Tract Infection Klebsiella pneumoniae.

peer reviewedAntibiotic resistance represents a major public health concern requiring new alternatives including phage therapy. Klebsiella pneumoniae belongs to the ESKAPE bacteria and can cause urinary tract infections (UTIs). The aims of this study were to isolate and characterize new bacteriophages against a K. pneumoniae strain isolated from UTIs and to assess their efficacy in vitro and in vivo in a Galleria (G.) mellonella larvae model. For this purpose, two bacteriophages were newly isolated against an ST13 K. pneumoniae strain isolated from a UTI and identified as K3 capsular types by wzi gene PCR. Genomic analysis showed that these bacteriophages, named vB_KpnP_K3-ULINTkp1 and vB_KpnP_K3-ULINTkp2, belong to the Drulisvirus genus. Bacteriophage vB_KpnP_K3-ULINTkp1 had the narrowest host spectrum (targeting only K3), while vB_KpnP_K3-ULINTkp2 also infected other Klebsiella types. Short adsorption times and latent periods were observed for both bacteriophages. In vivo experiments showed their ability to replicate in G. mellonella larvae and to decrease host bacterial titers. Moreover, both bacteriophages improved the survival of the infected larvae. In conclusion, these two bacteriophages had different in vitro properties and showed in vivo efficacy in a G. mellonella model with a better efficiency for vB_KpnP_K3-ULINTkp2

Use of an in vitro gastrointestinal model to evaluate the potential impact of a vegetal extract on human intestinal health

editorial reviewedThere are numerous medicinal plants and fruits traditionally used to treat gastrointestinal disorders. However, the effects caused by these vegetal products on the intestinal microbial populations are poorly understood. METHODS: An in vitro simulator of human digestion (SHIME®) was used to analyze the intestinal effects of two weeks of treatment with three increasing doses of a vegetal extract. Gut microbiota community and metabolites were studied on ascending (AC), transverse (TC), and descending (DC) colons using qPCR and SPME-GC/MS methods, respectively. RESULTS: A significant increase of acetic acid in TC, and of butyrate in all colons were observed by the end of treatment, while propionate levels remained unchanged. On 11 targeted microbial populations, most decreased in DC during the treatment, and Bacteroidetes decreased in all colon compartments, while Firmicutes increased. Bifidobacterium increased in AC even two weeks after completing the treatment. Akkermansia muciniphila increased in TC and DC following treatment with the higher doses. During the two weeks after completing the treatment, Bacteroides-Prevotella populations significantly increased in TC and DC regions, probably as a residual effect induced by the vegetal extract. Overall, the studied vegetal extract increased health-promoting bacteria which could have a beneficial impact on gastrointestinal health and gut barrier.GutTansit Projec