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See detailReducing agent can be omitted in the incubation medium of the batch in vitro fermentation model of the pig intestines
Poelaert, Christine; Nollevaux, Geraldine; Boudry, Christelle et al

in Animal (in press)

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by ... [more ▼]

Over the past decade, in vitro methods have been developed to study intestinal fermentation in pigs and its influence on the digestive physiology and health. In these methods, ingredients are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na2S or cysteine-HCl generates the required anaerobic environment by releasing metabolites similar to those produced when protein is fermented, possibly inducing a dysbiosis. An experiment was conducted to study the impact of two reducing agents on results yielded by such in vitro fermentation models. Protein (soybean proteins, casein) and carbohydrate (potato starch, cellulose) ingredients were fermented in vitro by bacteria isolated from fresh feces obtained from three sows in three carbonate-based incubation media differing in reducing agent: (i) Na2S, (ii) cysteine-HCl and (iii) control with a mere saturation with CO2 and devoid of reducing agent. The gas production during fermentation was recorded over 72 h. Short chain fatty acids (SCFA) production after 24 and 72 h and microbial composition of the fermentation broth after 24 h were compared between ingredients and between reducing agents. The fermentation residues after 24 h were also evaluated in terms of cytotoxicity using Caco-2 cell monolayers. Results showed that the effect of the ingredient induced higher differences than the reducing agent. Among the latter, cysteine-HCl induced the strongest differences compared with the control, whereas Na2S was similar to the control for most parameters. For all ingredients, final gas produced per g of substrate was similar ( P>0.10) for the three reducing agents whereas the maximum rate of gas production ( Rmax) was reduced ( P<0.05) when carbohydrate ingredients were fermented with cysteine-HCl in comparison to Na2S and the control. For all ingredients, total SCFA production was similar ( P>0.10) after 24 h of fermentation with Na2S and in the control without reducing agent. Molar ratios of branched chain-fatty acids were higher ( P<0.05) for protein (36.5% and 9.7% for casein and soybean proteins, respectively) than for carbohydrate (<4%) ingredients. Only fermentation residues of casein showed a possible cytotoxic effect regardless of the reducing agent ( P<0.05). Concerning the microbial composition of the fermentation broth, most significant differences in phyla and in genera ascribable to the reducing agent were found with potato starch and casein. In conclusion, saturating the incubation media with CO2 seems sufficient to generate a suitable anaerobic environment for intestinal microbes and the use of a reducing agent can be omitted. [less ▲]

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See detailMeat retail conditions within the establishments of Kigali city (Rwanda): bacteriological quality and risk factors for Salmonella occurrence
Niyonzima, Eugene ULiege; Ongol, Martin Patrick; Brostaux, Yves ULiege et al

in Tropical Animal Health and Production (in press)

Meat constitutes one of the major vehicles for human foodborne infections. This study aimed to assess the retail conditions and to determine the microbiological quality and safety of meat retailed within ... [more ▼]

Meat constitutes one of the major vehicles for human foodborne infections. This study aimed to assess the retail conditions and to determine the microbiological quality and safety of meat retailed within the establishments of Kigali (Rwanda). A questionnaire survey was carried out in 150 retail outlets to characterise meat retail conditions. Additionally, 270 retail meat samples were analysed for the enumeration of hygiene indicator bacteria (total mesophilic bacteria and Escherichia coli) and for the qualitative detection of Salmonella, using conventional culture methods. The results revealed that beef was the predominant meat sold within the retail premises of Kigali city, while meat from non-bovine animal species was mainly sold in large establishments. Salmonella was detected in 19.6% of all the retailed meat samples evaluated, whereas the mean loads for total mesophilic bacteria and E. coli were 7.3 and 3.5 log cfu/g, respectively. Three factors, namely the temperature conditions of the meat under retail, the cleanability of the used meat cutting boards, and the training of personnel in hygienic meat handling practices, were found to be significantly (p ≤ 0.05) associated with the risk of Salmonella occurrence in the retailed meat. The findings from this study highlight the need for improvements in hygienic meat handling practices, particularly, in small and medium meat retail establishments in Kigali. [less ▲]

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See detailWhat is the landscape of artisanal cheese production in Belgium?
Gerard, Amaury ULiege; Daube, Georges ULiege; Sindic, Marianne ULiege

Conference (2018, February 08)

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See detailWhat is the landscape of artisanal cheese production in Belgium?
Gerard, Amaury ULiege; Daube, Georges ULiege; Sindic, Marianne ULiege

Poster (2018, February 08)

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See detailComparison of faecal microbiota of horses suffering from atypical myopathy and healthy co-grazers
Cerri, Simona ULiege; Taminiau, Bernard ULiege; Votion, Dominique ULiege et al

Poster (2017, November 02)

Objectives: To characterize faecal microbiota of horses with atypical myopathy (AM) compared with healthy co-grazers (HcG). Methods: Fresh faecal samples were obtained from 6 horses (1 stallion, 3 ... [more ▼]

Objectives: To characterize faecal microbiota of horses with atypical myopathy (AM) compared with healthy co-grazers (HcG). Methods: Fresh faecal samples were obtained from 6 horses (1 stallion, 3 geldings and 2 females; mean age of 11.810 years) with confirmed AM and 6 HcG (4 geldings and 2 females; mean age of 13.68 years) during autumn-2016 and spring-2017 AM outbreaks in Belgium. Bacterial taxonomy profiling obtained by 16S amplicon sequencing of faeces was used to identify differentially distributed bacterial taxa between AM and HcG. Results were statistically compared using Welch's t-test with STAMP software. Results: A total of 90,407 sequences were analysed and clustered to 8,066 operational taxonomic units. Bacterial populations were distributed between 17 phylas, although 20% of sequences could not be attributed to an existing phylum. Horses with AM harboured a significantly higher relative abundance of Ruminococcaeae family with a significantly lower Lachnospiraceae when compared to HcG. Discussion: AM is caused by hypoglycin A intoxication, but only a part of horses pasturing in the same toxic environment develops the pathology, suggesting that there may be protective factors at the horse level. The results of this study show significant differences in faecal microbiota between AM cases and HcG, which could suggest that microbiota could play a role in the development or prevention of clinical disease. Conclusions: Results demonstrate that microbiota of AM affected horses is significantly different compared to HcG. Significance: Microbiome could influence the development of AM, but this role deserves further investigation. [less ▲]

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See detailPossible effect of environment on lung microbiota in the healthy West Highland white terrier.
Fastrès, Aline ULiege; Roels, Elodie ULiege; Taminiau, Bernard ULiege et al

Diverse speeche and writing (2017)

Over the past few years, the number of publications about lung microbiota has considerably increased due to the advancement of new molecular technologies. In dogs, only two studies in healthy experimental ... [more ▼]

Over the past few years, the number of publications about lung microbiota has considerably increased due to the advancement of new molecular technologies. In dogs, only two studies in healthy experimental beagles have been published. Interestingly, we recently reported major differences in the lung microbiota composition and diversity between experimental healthy beagles and healthy client-owned West Highland white terriers (WHWT) suggesting an impact of the breed or of the living environment on lung microbiota composition. Accordingly, to further assess the effect of living environment, the aim of the present study was to compare lung microbiota data obtained in healthy WHWT (n = 5) living in Finland with those previously obtained in healthy WHWT (n = 5) living in Belgium. For this purpose, bronchoalveolar lavage was retrospectively selected. Metagenetic analysis were performed after DNA extraction and sequencing on a MiSeq Illumina sequencer. Taxonomical assignation and microbiota community analysis were done with MOTHUR V1.35 with an OTU clustering distance of 0.03. Data analyses demonstrated that the same 4 major phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes predominated in both groups of dogs. Significant but subtle differences were observed between groups. Results of the present study do not demonstrate a clear impact of the living environment on lung microbiota composition. In conclusion, analysis of larger cohorts of healthy dogs with various living conditions appears necessary. [less ▲]

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See detailClostridium difficile in beef cattle farms, farmers and their environment: assessing the spread of the bacterium
Rodriguez Diaz, Cristina ULiege; Hakimi, Djalal-Eddine; Vanleyssem, Raphael et al

in Veterinary Microbiology (2017), 2010

n recent years, several studies have described the presence of Clostridium difficile in healthy and diarrhoeic farm and domestic animals. In pigs and cattle, the isolation of some PCR-ribotypes associated ... [more ▼]

n recent years, several studies have described the presence of Clostridium difficile in healthy and diarrhoeic farm and domestic animals. In pigs and cattle, the isolation of some PCR-ribotypes associated with human infection, especially PCR-ribotypes 014 and 078, has led us to hypothesize about the zoonotic transmission of C. difficile infections. If these animals are reservoirs of C. difficile, farmers in close contact with their animals are particularly at risk of acquiring and spreading the bacterium. This study investigates the presence of C. difficile in closely associated populations, beef cattle and farmers, as well as in the animal feed, manure and dust in five different farms in Belgium. C. difficile was isolated from calves and cattle with a prevalence varying between 5.5% and 11.3%. Furthermore, all of the isolates were toxigenic. An important age and breed effect was observed in the colonization of C. difficile. For age, there was a higher probability of colonization in calves of less than 6 months in age than in cattle over 11 months of age. For the type of breed a higher prevalence of the bacterium was detected in the Limousin breed than in the Belgian Bleu breed. By contrast, none of the human and animal feed samples tested positive for C. difficile. The results obtained indicate a persistent animal reservoir of C. difficile, but an indirect dissemination to humans, probably via the environment. [less ▲]

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See detailIn vitro evaluation of the competing effect of Carnobacterium maltaromaticum isolated from vacuum packed meat against food pathogens
Maia Danielski, Gabriela ULiege; Didimo Imazaki, Pedro Henrique ULiege; Daube, Georges ULiege et al

Conference (2017, August 15)

Foodborne disease outbreaks are one of the leading causes of infections, hospitalizations and deaths provoked by pathogenic bacteria including Escherichia coli O157:H7, Listeria monocytogenes and ... [more ▼]

Foodborne disease outbreaks are one of the leading causes of infections, hospitalizations and deaths provoked by pathogenic bacteria including Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp. Carnobacterium maltaromaticum is a lactic acid bacteria, which could prevent the growth of pathogens in refrigerated food. The aim of this study was to determine the bioprotective potential of three strains of C. maltaromaticum isolated from chilled vacuum packed beef. They were tested in vitro against the pathogens described above. The results indicate that the selected strains have an antilisterial activity, which is optimized at low temperatures. Moreover, when the strains were combined with EDTA it was observed a slight, but significant, inhibition of the gram-negative bacteria used in this study. [less ▲]

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See detailImpact of 3’-­‐sialyllactose and Bifidobacterium crudilactis on infant feces microbial composition and virulence modulation of Escherichia coli O157:H7, using the SHIME® gastrointes6nal model
Bondue, Pauline ULiege; Lebrun, Sarah ULiege; Crevecoeur, Sébastien ULiege et al

Poster (2017, June)

Bifidobacterium crudilactis, a bacterial species from bovine origin, growths on bovine milk oligosaccharides (BMO). Cell free spent media (CFSM) from B. crudilactis and 3’-sialyllactose (3’SL), a major ... [more ▼]

Bifidobacterium crudilactis, a bacterial species from bovine origin, growths on bovine milk oligosaccharides (BMO). Cell free spent media (CFSM) from B. crudilactis and 3’-sialyllactose (3’SL), a major BMO, modulated Escherichia coli O157:H7 virulence gene expressiona. In this study, the SHIME® gastrointestinal model was inoculated with 4 different treatments: 3’SL (ttm1), B. crudilactis (ttm2), 3’SL and B. crudilactis (ttm3) and CFSM from 3’SL and B. crudilactis culture (ttm4). In each section of the colon, samples were collected and analysed for short chain fatty acid (SCFA) concentration using HPLC, microbial populations using 16S rDNA metagenetic analysis and correlated with E. coli O157:H7 virulence gene expression. The results showed that SCFA levels were stable during the experiments. Metagenetic analysis showed a microbial diversity in transverse (TC) and descending colon (DC) close to feces, dominated by Bacteroides, Prevotella and Fusobacterium, while the ascending colon (AC) showed a different microbial diversity dominated by Veillonella. Ttm4 and ttm2 induced mainly a down-regulation of virulence genes: fliC in DC with ttm4, and luxS, stx1, qseA in AC, DC or TC with ttm2. Ttm1 also showed a down-regulation of fliC in DC, similar to the one observed with ttm4, but this was associated with an up-regulation of fliC and stx1 in AC or TC. Finally, ttm3 showed slight upregulation of ler, fliC and qseA in AC. These results show that ttm4 and ttm2 might have a positive effect against virulence expression of E. coli O157:H7. However, this trend has to be validated with the further replicates on the SHIME® system. [less ▲]

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See detailAssessment of bacterial superficial contamination in classical or ritually slaughtered cattle using metagenetics and microbiological analysis
Korsak Koulagenko, Nicolas ULiege; Taminiau, Bernard ULiege; Hupperts, Caroline et al

in International Journal of Food Microbiology (2017), 247

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 ... [more ▼]

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. [less ▲]

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See detailUse of a metagenetic approach to monitor the bacterial microbiota of “Tomme d’Orchies” cheese during the ripening process
Ceugniez, Alexandre; Taminiau, Bernard ULiege; Coucheney, Françoise et al

in International Journal of Food Microbiology (2017), 247

The study of microbial ecosystems in artisanal foodstuffs is important to complete in order to unveil its diversity. The number of studies performed on dairy products has increased during the last decade ... [more ▼]

The study of microbial ecosystems in artisanal foodstuffs is important to complete in order to unveil its diversity. The number of studies performed on dairy products has increased during the last decade, particularly those performed on milk and cheese derivative products. In this work, we investigated the bacterial content of "Tomme d'Orchies" cheese, an artisanal pressed and uncooked French cheese. To this end, a metagenetic analysis, using Illumina technology, was utilized on samples taken from the surface and core of the cheese at 0, 1, 3, 14 and 21 days of ripening process. In addition to the classical microbiota found in cheese, various strains likely from environmental origin were identified. A large difference between the surface and the core content was observed within samples withdrawn during the ripening process. The main species encountered in the core of the cheese were Lactococcus spp. and Streptococcus spp., with an inversion of this ratio during the ripening process. Less than 2.5% of the whole population was composed of strains issued from environmental origin, as Lactobacillales, Corynebacterium and Brevibacterium. In the core, about 85% of the microbiota was attributed to the starters used for the cheese making. In turn, the microbiota of the surface contained less than 30% of these starters and interestingly displayed more diversity. The predominant genus was Corynebacterium sp., likely originating from the environment. The less abundant microbiota of the surface was composed of Bifidobacteria, Brevibacterium and Micrococcales. To summarize, the “Tomme d’Orchies” cheese displayed a high diversity of bacterial species, especially on the surface, and this diversity is assumed to arise from the production environment and subsequent ripening process. [less ▲]

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See detailIdentification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC
Fakih, Ibrahim; Thiry, Damien ULiege; Duprez, Jean-Noël ULiege et al

in Veterinary Microbiology (2017), 202

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC ... [more ▼]

Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar [less ▲]

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See detailOptimization of DNA extraction from the Algerian traditional date’s product “Btana”
Abekhti, Abdel; Dgeghati, S.; Taminiau, Bernard ULiege et al

in Applied Biology in Saharan Areas (2017), 1(1), 23-32

Btana is traditional preservation method that can sustain date’s supply during many years in comparaison to the commercial storage methods. However no scientific informations are available about ... [more ▼]

Btana is traditional preservation method that can sustain date’s supply during many years in comparaison to the commercial storage methods. However no scientific informations are available about biological factors that contribute to the successful of this method. Bacterial communities are a major key in preservation of many foods. Culture independent techniques are the most powerful tools to enhance bacterial diversity studies, but their efficiencies start with DNA extraction step. Therefore we have studied 3 protocols of DNA extraction from 11 Btana samples to evaluate their yield in total microbial DNA recovery. Protocols were based on a commercial kit DNeasy (QIAGEN, Germany) and two modified CTAB extraction methods (combined CTAB-DNeasy protocol, modified CTAB protocol) using polyvinyl pyrrolidone (PVP) and treatment with high salt solution (5M NaCl). Protocols were compared for quantity of DNA extracted using NanoDrop® ND-1000 Spectrophotometer and quality of DNA by 260/280 nm absorption ratio. The total extracted DNA was cheeked by PCR amplification of the 16S rRNA and visualized by electrophoresis on agarose gel (0.8%). Results showed that CTAB modified method provide the best DNA yield; however purification with NucleoSpin® Kit (Clontech, UK) was mostly needed for amplifying the DNA template. DNeasy kit protocol gave an amplified high quality DNA, but poor yields were obtained from date samples [less ▲]

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See detailDetection, isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs
De Witte, C.; Flahou, B.; Ducatelle, R. et al

in Systematic & Applied Microbiology (2017), 40(1), 42-50

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6-8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2 hours exposure to air ... [more ▼]

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6-8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2 hours exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, α-galactosidase, β-galactosidase, β-galactosidase-6-phosphate or α-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16 : 0 and C18 : 1ω9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (= DSM 101753(T) = LMG 29236(T)). We also demonstrated that Clostridium rectum and Fusobacterium mortiferum represent the same species, with nomenclatural priority for the latter. [less ▲]

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