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See detailEffect of antimicrobial drug on lung microbiota in healthy dogs.
Fastrès, Aline ULiege; Vangrinsven, Emilie ULiege; Tutunaru, Alexandru-Cosmin ULiege et al

in Journal of Veterinary Internal Medicine (2018, October 12)

In human and dogs, baseline differences in lung microbiota (LM) have been associated with important clinical features in chronic lung diseases (CLD) and there is growing evidence that an altered LM ... [more ▼]

In human and dogs, baseline differences in lung microbiota (LM) have been associated with important clinical features in chronic lung diseases (CLD) and there is growing evidence that an altered LM contributes to disease pathogenesis. The common use of antibiotic drugs throughout the management of CLD likely represents a major confounding factor in the study of the LM. However, the effect of antibiotic treatment on the LM in healthy individuals has not been specifically investigated yet. The aim of the present study was to assess the short and medium-term effect of an oral treatment with a large spectrum antimicrobial drug on the LM in healthy dogs. Six healthy experimental beagle dogs were included. Amoxycillin/clavulanic acid (AC) was administered at a dose of 20 mg/kg twice daily for 10 days. In each dog, bronchoalveolar lavage fluid (BALF) was collected at 3 different timepoints: before administration of AC (J0) and immediately (J10) as well as 16 days (J26) after interruption of AC. In each BALF, total and differentiated cell counts were obtained and metagenetic analyses were performed on the V1-V3 hypervariable region of 16S rDNA after total bacterial 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. Statistical comparisons between events for microbiota community and BALF cell counts were made using Friedman test and post-hoc t-tests with Bonferroni correction. Statistical differences in bacterial population relative abundance between timepoints were assessed using a mixed linear model with FDR correction for multiple comparisons. Administration of AC did not induce significant changes in BALF cellular counts and had no effect on the richness, evenness and alpha diversity. Bacteroidetes and Proteobacteria abundance increased from J0 to J10 (mean: 12.0 to 24.1, and 12.3 to 26.2%, respectively; p<0.001) and decreased at J26 (13.1 and 9.3%; p<0.001). Firmicutes abundance decreased from J0 to J10 (45.6 to 18.3%; p<0.001) and increased at J26 (28.8%; p<0.001). Actinobacteria abundance increased at J26, compared with J0 and J10 (43.7 compared with 23.2 and 23.5% respectively; p<0.001). We failed to show significant differences between timepoints at the level of genera and species. In conclusion, in healthy dog, oral administration of a commonly used large spectrum antimicrobial drug induces significant changes in microbial population abundance at the phyla level. Most but not all of these changes normalize within 2 weeks after discontinuation of the drug. [less ▲]

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See detailMonitoring of hygiene in institutional kitchens in Belgium
Duthoo, E; Krings, Simone ULiege; Daube, Georges ULiege et al

Poster (2018, October)

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See detailAnalysis of the lung microbiota in healthy dogs and in canine idiopathic pulmonary fibrosis, a possible spontaneous model for human IPF.
Fastrès, Aline ULiege; Roels, Elodie ULiege; Taminiau, Bernard ULiege et al

Poster (2018, September 16)

Canine idiopathic pulmonary fibrosis (CIPF) mimics human IPF and affects old dogs from one breed: the West Highland white terrier (WHWT). In human IPF, lung microbiota is suspected to be associated with ... [more ▼]

Canine idiopathic pulmonary fibrosis (CIPF) mimics human IPF and affects old dogs from one breed: the West Highland white terrier (WHWT). In human IPF, lung microbiota is suspected to be associated with disease pathogenesis and might serve as a therapeutic target. The aim of the study was to characterize microbial alteration associated with breed, environment and CIPF disease. Bronchoalveolar lavage fluid was sampled from 4 groups of dogs: client-owned WHWTs affected with CIPF from Belgium-BE (n=7, 11.6y), healthy client-owned BE-WHWTs (n=5, 11.2y), healthy client-owned WHWTs from Finland-FI (n=5, 11.0y) and healthy experimental BE-beagle dogs (n=6, 8.8y). Metagenetic analysis was performed on V1-V3 hypervariable region of 16S rDNA after total bacterial DNA extraction and sequencing on a MiSeq Illumina sequencer. Data were compared between healthy BE-WHWTs and BE-beagles, healthy BE-WHWTs and FI-WHWTs, and healthy BE-WHWTs and CIPF BE-WHWTs. Data analyses demonstrated that the same phyla (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) predominated in all groups. Significant differences (p<005) were mainly noted for species with relative abundance of less than 1%, except for Micrococcus luteus, higher in FI-WHWTs (7.4%) compared to BE-WHWTs (0%) and for Pseudomonas JF766687 and EU373411, Serratia and Hydrogenophilus spp, higher in BE-beagles (15.3, 8.1, 5.0 and 1.5%) compared with BE-WHWTs (0.3, 0.3, 0, 0.1%, respectively). Results indicate that further analysis of the impact of breed or environment and inclusion of larger amounts of dogs are needed before investigation of lung microbiota as a biomarker or therapeutic target in CIPF. [less ▲]

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See detailEar canal microbiota – a comparison between healthy dogs and atopic dogs without clinical signs of otitis externa.
ngo, jerome; Taminiau, Bernard ULiege; Daube, Georges ULiege et al

in Veterinary Dermatology (2018), 29

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See detailUse of propidium monoazide followed by 16S rDNA sequencing in order to discriminate live and dead cells in surface and food samples from collective kitchens
Krings, Simone ULiege; Duthoo, Evelyne; De Reu, Koen et al

Poster (2018, June 19)

Classical microbiological analyses have been the “Gold standard” in food microbiology in order to detect and quantify bacteria in food and surface samples. However, these methods bear the limitation of ... [more ▼]

Classical microbiological analyses have been the “Gold standard” in food microbiology in order to detect and quantify bacteria in food and surface samples. However, these methods bear the limitation of the ability of bacteria to grow on the used media. Advances in sequencing methods have allowed analysing the bacterial DNA in different types of samples, but these techniques do not evaluate if the bacteria are viable. In order to detect viable bacteria by sequencing methods, the interest in sample treatment with propidium monoazide (PMA) prior to DNA extraction is growing (Li et al., 2017; Nocker et al., 2010). This study addresses the possible benefit of this method for surface and food samples taken in collective kitchen. Samples have been collected aseptically, stored at 4°C and analysed by classical microbiological methods and 16S rDNA sequencing (with and without prior treatment by PMA). The bacterial counts in many samples were low or under the detection limit (< 1 CFU/mL or < 10 CFU/g) and did rarely exceed the 5 log CFU/surface (mostly for sinks). This fact implied problems for the 16S rDNA sequencing, as a minimal bacterial load of 100-1000 CFU/mL is required in order to achieve reliable results. In addition, PMA-treated samples did generally generate fewer sequences. However, variations of relative population abundances are rendered visible and allow performing correlations between the bacterial populations and their occurrence in untreated and treated samples. This could give hints about residual bacterial DNA and bacteria that colonise kitchen surfaces, which have not been described previously. References: • Li, R. et al. (2017). Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water. Scientific reports, 7(1), 5752. • Nocker, A., et al. (2010). Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing. Int Microbiol, 13(2), 59-65. [less ▲]

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See detailPotential resident bacterial microbiota in udder tissues of culled cows sampled in abattoir
Pirard, Barbara ULiege; Crevecoeur, Sébastien ULiege; Taminiau, Bernard ULiege et al

Poster (2018, June 19)

Aseptic milking samples and microbiological analyses are used in routine for bovine mastitis diagnosis. Few papers treated about a resident microbiota in the ruminant healthy mammary gland tissues (Spuria ... [more ▼]

Aseptic milking samples and microbiological analyses are used in routine for bovine mastitis diagnosis. Few papers treated about a resident microbiota in the ruminant healthy mammary gland tissues (Spuria et al., 2017), or about immunological consequences related with a such cohabitation (Rainard, 2017). In practice, it’s difficult to sample mammary gland tissues out of risks for cows health or milk production. We thus design a study based on samples taken at abattoir. It aimed at identify, quantify, compare the cow milk and mammary gland tissues microbiota of macroscopically healthy mammary glands, by classical microbiological analyses and by amplicon sequencing. We harvested thirteen couples of milk secretion and tissue samples, originated from the same quarter of reformed cows. Aseptic milking has been done just before culling and mammary gland tissues had been taken of the carcasses on slaughterline. Total and specific microbiological counting and metagenetic analysis were performed. Metagenetic analyses showed one main bacterial genus, Corynebacterium, generally found in the milk in higher proportions than in tissues. When it dominates clearly other populations in milk secretions, it can be found in the same quarter tissues. In case of identification of pathogenic bacteria in milk samples, the same pathogen were detected in tissues from the same quarters but in very different proportions: higher for Streptococcus uberis, lower for Staphylococcus spp or Enterococcus faecium. In tissues, Flavobacterium and Atopostipes genera were statistically more abundant than in milk. Data show also that species evenness and beta diversity are greater in mammary glands than in milk secretions. In opposition, species richness is higher in milk samples. These results show a potential resident microflora in mammary glands of culled cows in abattoirs. Metagenetic analysis of milk samples could be a good indicator of the udder microbiota and health in the future but our first results must be completed and confirmed on a larger number of samples. Hypothesis about nature of such a resident flora will have to be confirmed on producing cows before studying bacterial-host interactions. Rainard, P. 2017. Mammary microbiota of dairy ruminants: fact or fiction? Vet.Res., , 48 (25), 1-10. Spuria, L. et al. 2017. Microbial agents in macroscopically healthy mammary gland tissues of small ruminants. PeerJ, 5. [less ▲]

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See detailGUT MICROBIOTA AND FAECAL LEVELS OF SHORT CHAIN FATTY ACIDS DIFFER UPON BLOOD PRESSURE LEVELS IN MAN
HUART, Justine ULiege; Leenders, Justine ULiege; Taminiau, Bernard ULiege et al

in Nephrology Dialysis Transplantation (2018, May 18), 33(Issue suppl_1), 368369

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See detailImpact of microbial composition of Cambodian traditional dried starters (Dombea) on flavor compounds of rice wine: combining amplicon sequencing with HP-SPME-GCMS
Ly, Sokny ULiege; Mith, Hasika; Tarayre, Cédric ULiege et al

in Frontier in microbiology (2018)

Dombae is a traditional ferment starter which has been used for starchy based wine production in Cambodia. However, the production technology of rice wine in Cambodia is not optimized. The current study ... [more ▼]

Dombae is a traditional ferment starter which has been used for starchy based wine production in Cambodia. However, the production technology of rice wine in Cambodia is not optimized. The current study aimed to investigate the microbiota associated in five ferment starters and the effect of a traditional fermentation process using a metagenomics sequencing analysis and HS-SPME-GCMS for the characterization of the aromatic profiles at the end of fermentation. Most of bacteria identified in this study were lactic acid bacteria including Weissella cibaria, Pediococcus sp. MMZ60A, Lactobacillus fermentum and Lactobacillus plantarum. Saccharomyces cerevisiae and Saccharomycopsis fibuligera were found to be abundant yeasts while the only amylolytic filamentous fungus was Rhizopus oryzae. A total of 25 aromatic compounds were detected and identified as esters, alcohols, acids, ketones and aldehydes. The alcohol group was dominant in each rice wine. Significant changes were observed at the level of microbial communities during fermentation, suggesting microbial succession for the assimilation of starch and subsequently assimilation of fermentation by-products leading to the production of flavor compounds. At this level, the presence of Weissella, Pediococcus and Lactobacillus genus was strongly correlated with most of the flavor compounds detected. [less ▲]

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See detailPerinatal short-chain fructooligosaccharides program intestinal microbiota and improve enteroinsular axis function and inflammatory status in high-fat diet-fed adult pigs.
Le Bourgot, Cindy; Ferret-Bernard, Stephanie; Apper, Emmanuelle et al

in FASEB Journal (2018)

Perinatal nutrition programs physiologic and metabolic functions, with consequences on the susceptibility to develop metabolic diseases in adulthood. The microbiota represents a key factor of such ... [more ▼]

Perinatal nutrition programs physiologic and metabolic functions, with consequences on the susceptibility to develop metabolic diseases in adulthood. The microbiota represents a key factor of such programming. We investigated whether perinatal prebiotic [short-chain fructooligosaccharides (scFOS)] supplementation improved adult metabolic health in association with microbiota changes in pigs used as human model. Sows were supplemented with scFOS or not during the end of gestation and the entire lactation, and offspring received scFOS accordingly during 1 mo after weaning. Pigs were then fed a standard diet for 5 mo, followed by a high-fat diet for 3 mo once adults. Perinatal scFOS supplementation induced a persistent modulation of the composition of the fecal microbiota in adulthood, notably by increasing the Prevotella genus. Meanwhile, scFOS animals displayed improved capacity to secrete glucagon-like peptide-1 and improved pancreas sensitivity to glucose without any changes in peripheral insulin sensitivity. Perinatal scFOS supplementation also increased ileal secretory IgA secretion and alkaline phosphatase activity and decreased TNF-alpha expression in adipose tissue. In conclusion, perinatal scFOS supplementation induced long-lasting modulation of intestinal microbiota and had beneficial consequences on the host physiology in adulthood. Our results highlight the key role of perinatal nutrition on later microbiota and host metabolic adaptation to an unbalanced diet.-Le Bourgot, C., Ferret-Bernard, S., Apper, E., Taminiau, B., Cahu, A., Le Normand, L., Respondek, F., Le Huerou-Luron, I., Blat, S. Perinatal short-chain fructooligosaccharides program intestinal microbiota and improve enteroinsular axis function and inflammatory status in high-fat diet-fed adult pigs. [less ▲]

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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 ULiege et al

in Animal (2018), 12

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 detailThe use of predictive models in the context of food spoilage: the case of white pudding
Cauchie, Emilie ULiege; Korsak Koulagenko, Nicolas ULiege; Ellouze, Mariem et al

in Proceedings of the First international Conference on "Innovative Food Ingredients and Food safety" (2018)

Food spoilage is a major issue for the food industry and consumers as products become unacceptable for human consumption leading to significant food waste and economic losses. This study combines the use ... [more ▼]

Food spoilage is a major issue for the food industry and consumers as products become unacceptable for human consumption leading to significant food waste and economic losses. This study combines the use of predictive microbiology and metagenetics in order to predict bacterial evolution in Belgian white pudding. The ecology of the product was studied at several times during the storage at constant temperatures, and under three different packaging (food wrap, modified atmosphere 30% CO2 – 70% N2 and vacuum packaging), by association of classical microbiological plate counting and 16S rRNA metagenetic analysis on each sample. The bacterial evolution could thus be deduced for the two major spoilage populations in the product, Brochothrix thermosphacta and Pseudomonas spp. The growth parameters were estimated using the nlsMicrobio package from R and then used to simulate the microbial behavior in dynamic conditions with three different softwares: ComBase, Sym’Previus and baranyi growth function in R. These results are compared with validation curves, obtained from these durability studies. A relatively good agreement was obtained between the validation data set and the simulations, showing that the approach combining the metagenetics and the simulations based on an accurate database is promising. Performance factors (bias and accuracy factors) indicated no significant structural deviation of the maximal growth rates simulations between observed and predicted values with R and Sym’Previus. An overestimation was mainly observed with R, while an underestimation was generally observed with Sym’Previus and ComBase. None of predictive simulations give an identical microbial curve that the validation data set, but all models show relatively good statistical fittings. This work gives a proof of concept on the feasibility to combine predictive models and metagenetics in order to predict bacterial evolution using different predictive tools. In the future, predictive models needed to be more accurate by taking into account as many growth parameters as possible. [less ▲]

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

Scientific conference (2017, October 13)

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See detailComparison between swabbing devices in order to analyse the microbial flora found on surfaces by classical microbiology and 16S rDNA amplicon sequencing
Krings, Simone ULiege; Crevecoeur, Sébastien ULiege; Rodriguez Diaz, Cristina ULiege et al

Conference (2017, October 13)

The work in community kitchens involves several steps bearing the risk of transmitting pathogenic microorganisms from static or dynamic surfaces to the food. This study aimed at comparing the recovery ... [more ▼]

The work in community kitchens involves several steps bearing the risk of transmitting pathogenic microorganisms from static or dynamic surfaces to the food. This study aimed at comparing the recovery efficiency of different sampling devices in terms of culture methods and culture-independent 16S rDNA amplicon sequencing for characterising the microbial flora. In order to mimic surfaces found in community kitchens, sterile stainless steel and polypropylene surfaces were spiked with a two-fold raw chicken meat dilution and several concentrations of Escherichia coli, Salmonella Enteritidis and Bacillus cereus, respectively. The ideal swab for kitchen analyses should recover the highest number of viable bacteria with the highest population diversity. Classical culture method showed that recovery was possible with all swabs, but that Sponge-Sticks had the highest recovery rates. The 16S rDNA amplicon sequencing revealed a high relative abundance of Bacillus genus in cotton pad, gauze pad and Sponge-Stick samples and low proportions of Salmonella genus in all samples. Analysis of Escherichia genus results was problematic for molecular analyses as it appeared as a source of DNA contamination of the reagents used during library creation. Furthermore, it was possible to decipher bias populations in the sponge samples (from neutralising buffer) and in controls (due to low amounts of DNA), allowing the exclusion of those in the following analyses, if needed. In the end, these results attest the similarity of population diversity in cotton pad, gauze pad and Sponge-Stick samples, leaving the final choice to the operator. [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 detailComparison between swabbing devices in order to analyse the microbial flora found on surfaces of community kitchens by classical microbiology and amplicon sequencing
Krings, Simone ULiege; Crevecoeur, Sébastien ULiege; Rodriguez Diaz, Cristina ULiege et al

Poster (2017, September 22)

Introduction The work in community kitchens involves several manual steps bearing the risk of transmitting pathogenic microorganisms. This fact justifies accurate control of surfaces to prevent foodborne ... [more ▼]

Introduction The work in community kitchens involves several manual steps bearing the risk of transmitting pathogenic microorganisms. This fact justifies accurate control of surfaces to prevent foodborne illnesses. Different sampling methods have been proposed to isolate bacteria from surfaces, while the ensuing analysis of the surfaces is usually performed by classical microbiology and molecular biology methods [1,2,3]. Studies found in the literature do compare different swabbing devices for surfaces but not in terms of 16S rRNA gene profiling [4]. This study aimed at comparing the efficiency of recovery of different swabbing devices in terms of culture methods and culture-independent 16S rRNA amplicon sequencing, the latter in order to characterise the present microbial flora. Material and Methods To mimic surfaces which can be found in community kitchens, sterile polypropylene cutting boards and stainless steel surfaces were spiked with 1mL of 2-fold dilution of chicken and with 1mL of several concentrations of Escherichia coli, Salmonella enterica ssp. enterica serovar Enteriditis and Bacillus cereus, respectively. Collection of the bacteria was performed with sterile moistened swabs, including commercial swabs (sponges HS10NB2G, Sponge-Sticks SSL100; 3M) and cotton and gauze pads. Enumeration was carried out by aerobic plate count on Plate Count Agar (Bio-Rad), Rapid’E.coli 2 (Bio-Rad), Rapid’Salmonella (Bio-Rad) and MYP (Oxoid) which were incubated for 24h at 37°C. The V1-V3 16S rRNA amplicon libraries obtained from total bacterial DNA extraction were sequenced on a Illumina MiSeq sequencer and the resulting sequences were analysed with mothur software. Statistical analyses were performed by 2-way ANOVA and Tukey’s multiple comparisons by using Prism 7. Discussion The perfect swab for kitchen analyses should recover the highest number of viable bacteria with a high population diversity from the surfaces. Classical culture method showed best recovery numbers for the three inoculated bacteria with Sponge-Sticks (no significant difference between inoculation dosis and recovered number of bacteria, p > 0.1234). The 16S rRNA amplicon sequencing allows to conclude that sponge samples were loaded with DNA from Microbacterium genus (from Neutralizing buffer). Furthermore, a high relative abundance of Bacillus genus was found in cotton pad, gauze pad and Sponge-Stick samples. Salmonella genus was detected in only low proportions, whereas Escherichia genus are problematic as their DNA can be contaminants of reagents used during library creation. However, differences in recovery or enumeration with each method must be considered, as they can induce estimation bias on the initial concentration or recovered CFU/mL. Finally, low amount of DNA in controls leads to the emergence of free DNA contaminants like Elizabethkingia population, which can be considered as a bias. References 1. Ismaïl R. et al. (2013). Int. J. Environ. Res. Public Health, 10, 6169-6183. 2. Biranjia-Hurdoyal, S., & Latouche, M. C. (2016). Can J Infect Dis Med Microbiol., 2016. 3. Malorny B. et al. (2008). Appl Environ Microbiol, 74(5), 1299-1304. 4. Lahou, E., & Uyttendaele, M. (2014). Int. J. Environ. Res. Public Health, 11(1), 804-814. [less ▲]

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