Publications by authors named "Geert Huys"

101 Publications

Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients.

Int J Mol Sci 2020 Mar 14;21(6). Epub 2020 Mar 14.

Department of Diagnostic Sciences, Laboratory Bacteriology Research, Ghent University, 9000 Ghent, Belgium.

In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as -cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, spp. and spp., and higher and with impaired kidney function, confirming an altered gut microbial composition in CKD.
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http://dx.doi.org/10.3390/ijms21061986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139965PMC
March 2020

Synthetic ecology of the human gut microbiota.

Nat Rev Microbiol 2019 12 2;17(12):754-763. Epub 2019 Oct 2.

Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium.

Despite recent advances in sequencing and culturing, a deep knowledge of the wiring and functioning of the human gut ecosystem and its microbiota as a community is still missing. A holistic mechanistic understanding will require study of the gut microbiota as an interactive and spatially organized biological system, which is difficult to do in complex natural communities. Synthetic gut microbial ecosystems can function as model systems to further current understanding of the composition, stability and functional activities of the microbiota. In this Review, we provide an overview of the current synthetic ecology strategies that can be used towards a more comprehensive understanding of the human gut ecosystem. Such approaches that integrate in vitro experiments using cultured isolates with mathematical modelling will enable the ultimate goal: translating mechanistic and ecological knowledge into novel and effective therapies.
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http://dx.doi.org/10.1038/s41579-019-0264-8DOI Listing
December 2019

Design of synthetic microbial consortia for gut microbiota modulation.

Curr Opin Pharmacol 2019 12 17;49:52-59. Epub 2019 Aug 17.

Department of Microbiology and Immunology, Rega Institute, KU Leuven, University of Leuven, Leuven, Belgium; VIB, Center for Microbiology, Leuven, Belgium. Electronic address:

The use of and interest in probiotics to modulate the human intestinal microbiota have strongly increased in recent years. However, most of the current probiotic products have been limited to single-strain formulations of easily culturable food-grade microorganisms and often resulted in mixed results or limited effects on host health. Therefore, a revision of current probiotic strategies by using synthetic human-derived microbial multispecies consortia is necessary. In light of this ongoing evolution of the field, novel approaches are needed to design and assemble bacterial cocktails targeted to restore dysbiotic states in microbiota-associated diseases. This review discusses the steps in the process for identifying effective targets, predicting putative multistrain communities, assembling ecosystems in silico and in vitro and monitoring stability and outputs before in vivo trials.
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http://dx.doi.org/10.1016/j.coph.2019.07.005DOI Listing
December 2019

Go with the flow or solitary confinement: a look inside the single-cell toolbox for isolation of rare and uncultured microbes.

Curr Opin Microbiol 2018 08 13;44:1-8. Epub 2018 Jun 13.

Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium; VIB, Center for Microbiology, Leuven, Belgium. Electronic address:

With the vast majority of the microbial world still considered unculturable or undiscovered, microbiologists not only require more fundamental insights concerning microbial growth requirements but also need to implement miniaturized, versatile and high-throughput technologies to upscale current microbial isolation strategies. In this respect, single-cell-based approaches are increasingly finding their way to the microbiology lab. A number of recent studies have demonstrated that analysis and separation of free microbial cells by flow-based sorting as well as physical stochastic confinement of individual cells in microenvironment compartments can facilitate the isolation of previously uncultured species and the discovery of novel microbial taxa. Still, while most of these methods give immediate access to downstream whole genome sequencing, upscaling to higher cell densities as required for metabolic readouts and preservation purposes can remain challenging. Provided that these and other technological challenges are addressed in future innovation rounds, integration of single-cell tools in commercially available benchtop instruments and service platforms is expected to trigger more targeted explorations in the microbial dark matter at a depth comparable to metagenomics.
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http://dx.doi.org/10.1016/j.mib.2018.05.002DOI Listing
August 2018

Diversity and Antibiotic Susceptibility of Strains From Milk Powder Produced in Germany.

Front Microbiol 2018 27;9:536. Epub 2018 Mar 27.

Department of Microbiology and Biotechnology, Max Rubner-Institut, Kiel, Germany.

Forty-seven spp. isolates from milk powder obtained from a powdered milk producer in Germany were investigated for their antibiotic resistance susceptibilities, in order to assess whether strains from food harbor multiple antibiotic resistances and whether the food route is important for dissemination of resistance genes. The strains were identified by 16S rRNA and B gene sequencing, as well as by whole genome sequencing of selected isolates and their DNA-DNA hybridization (DDH). Furthermore, they were genotyped by rep-PCR together with reference strains of pan-European groups I, II, and III strains of . Of the 47 strains, 42 were identified as , 4 as , and 1 as based on 16S rRNA gene sequencing. DDH with the genome sequence data of selected strains and B gene sequencing data suggested that the five non- strains all belonged to , suggesting that the B gene is more reliable than the 16S rRNA gene for species level identification in this genus. Rep-PCR genotyping of the strains showed that these could be grouped into four groups, and that some strains clustered together with reference strains of pan-European clinical group II and III strains. All strains in this study were intrinsically resistant toward chloramphenicol and oxacillin, but susceptible toward tetracycline, tobramycin, erythromycin, and ciprofloxacin. For cefotaxime, 43 strains (91.5%) were intermediate and 3 strains (6.4%) resistant, while 3 (6.4%) and 21 (44.7%) strains exhibited resistance to cefepime and streptomycin, respectively. Forty-six (97.9%) strains were susceptible to amikacin and ampicillin-sulbactam. Therefore, the strains in this study were generally not resistant to the clinically relevant antibiotics, especially tobramycin, ciprofloxacin, cefepime, and meropenem, suggesting that the food route probably poses only a low risk for multidrug resistant strains or resistance genes.
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http://dx.doi.org/10.3389/fmicb.2018.00536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880893PMC
March 2018

Sphingobacterium cellulitidis sp. nov., isolated from clinical and environmental sources.

Int J Syst Evol Microbiol 2017 05 5;67(5):1415-1421. Epub 2017 Jun 5.

Department of Microbiology, Faculty of Medicine, Kuwait University, Jabriya, Kuwait.

The taxonomic position of two isolates belonging to the genus Sphingobacterium was determined. The first isolate, R-53603T, was obtained from purulent discharge from the toe of a cellulitis patient in Kuwait. Comparative 16S rRNA gene sequence analysis revealed 99.87 % similarity of R-53603T with environmental isolate P031 (=R-53745) originating from activated sludge in Singapore. The two isolates were phylogenetically positioned on the same sub-branch. Highest 16S rRNA gene sequence similarity was found with the type strains of Sphingobacterium mizutaii (98.23 %), Sphingobacterium lactis (97.78 %) and Sphingobacterium daejeonense (97.14 %). DNA-DNA hybridizations revealed <70 % relatedness between the two isolates and the type strains of the close phylogenetic neighbours S. mizutaii(18.0-24.5 %), S. lactis(20.3-25.9 %) and S. daejeonense(13.2-20.0 %). The high relative contribution of iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c) in the cellular fatty acid profiles of R-53603T and R-53745, the presence of sphingophospholipids, MK-7 as the dominant menaquinone and phosphatidylethanolamine as the major polar lipid in strain R-53603T are typical chemotaxonomic characteristics for members of the genus Sphingobacterium. Phenotypic features most useful for differentiation of the two novel strains from the most closely related species S. mizutaii include growth on MacConkey agar, and utilization of stachyose, guanidine HCl and lithium chloride in Biolog GEN III tests. Strains R-53603T and R-53745 thus represent a novel species, for which the name Sphingobacterium cellulitidis sp. nov. is proposed. The type strain is R-53603T (=LMG 28764T=DSM 102028T).
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http://dx.doi.org/10.1099/ijsem.0.001832DOI Listing
May 2017

Acinetobacter dijkshoorniae sp. nov., a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries.

Int J Syst Evol Microbiol 2016 Oct 15;66(10):4105-4111. Epub 2016 Jul 15.

Department of Clinical Microbiology and ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic-Universitat de Barcelona, Barcelona, Spain.

The recent advances in bacterial species identification methods have led to the rapid taxonomic diversification of the genus Acinetobacter. In the present study, phenotypic and molecular methods have been used to determine the taxonomic position of a group of 12 genotypically distinct strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, initially described by Gerner-Smidt and Tjernberg in 1993, that are closely related to Acinetobacter pittii. Strains characterized in this study originated mostly from human samples obtained in different countries over a period of 15 years. rpoB gene sequences and multilocus sequence typing were used for comparisons against 94 strains representing all species included in the ACB complex. Cluster analysis based on such sequences showed that all 12 strains grouped together in a distinct clade closest to Acinetobacter pittiithat was supported by bootstrap values of 99 %. Values of average nucleotide identity based on blast between the genome sequence of strain JVAP01T (NCBI accession no. LJPG00000000) and those of other species from the ACB complex were always <91.2 %, supporting the species status of the group. In addition, the metabolic characteristics of the group matched those of the ACB complex and the analysis of their protein signatures by matrix-assisted laser desorption ionization time-of-flight MS identified some specific peaks. Our results support the designation of these strains as representing a novel species, for which the name Acinetobacter dijkshoorniae sp. nov. is proposed. The type strain is JVAP01T (=CECT 9134T=LMG 29605T).
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http://dx.doi.org/10.1099/ijsem.0.001318DOI Listing
October 2016

Specific members of the predominant gut microbiota predict pouchitis following colectomy and IPAA in UC.

Gut 2017 01 30;66(1):79-88. Epub 2015 Sep 30.

Translational Research Center for Gastrointestinal Disorders (TARGID), University Hospital Leuven, KU Leuven, Leuven, Belgium.

Objective: Pouchitis is the most common complication after colectomy with ileal pouch-anal anastomosis (IPAA) for UC and the risk is the highest within the 1st year after surgery. The pathogenesis is not completely understood but clinical response to antibiotics suggests a role for gut microbiota. We hypothesised that the risk for pouchitis can be predicted based on the faecal microbial composition before colectomy.

Design: Faecal samples from 21 patients with UC undergoing IPAA were prospectively collected before colectomy and at predefined clinical visits at 1 month, 3 months, 6 months and 12 months after IPAA. The predominant microbiota was analysed using community profiling with denaturing gradient gel electrophoresis followed by quantitative real-time PCR validation.

Results: Cluster analysis before colectomy distinguished patients with pouchitis from those with normal pouch during the 1st year of follow-up. In patients developing pouchitis, an increase of Ruminococcus gnavus (p<0.001), Bacteroides vulgatus (p=0.043), Clostridium perfringens (p=0.011) and a reduction of two Lachnospiraceae genera (Blautia (p=0.04), Roseburia (p=0.008)) was observed. A score combining these five bacterial risk factors was calculated and presence of at least two risk factors showed a sensitivity and specificity of 100% and 63.6%, respectively.

Conclusions: Presence of R. gnavus, B. vulgatus and C. perfringens and absence of Blautia and Roseburia in faecal samples of patients with UC before surgery is associated with a higher risk of pouchitis after IPAA. Our findings suggest new predictive and therapeutic strategies in patients undergoing colectomy with IPAA.
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http://dx.doi.org/10.1136/gutjnl-2015-309398DOI Listing
January 2017

Integrated community profiling indicates long-term temporal stability of the predominant faecal microbiota in captive cheetahs.

PLoS One 2015 23;10(4):e0123933. Epub 2015 Apr 23.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium; BCCM/LMG Bacteria Collection, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium.

Understanding the symbiotic relationship between gut microbes and their animal host requires characterization of the core microbiota across populations and in time. Especially in captive populations of endangered wildlife species such as the cheetah (Acinonyx jubatus), this knowledge is a key element to enhance feeding strategies and reduce gastrointestinal disorders. In order to investigate the temporal stability of the intestinal microbiota in cheetahs under human care, we conducted a longitudinal study over a 3-year period with bimonthly faecal sampling of 5 cheetahs housed in two European zoos. For this purpose, an integrated 16S rRNA DGGE-clone library approach was used in combination with a series of real-time PCR assays. Our findings disclosed a stable faecal microbiota, beyond intestinal community variations that were detected between zoo sample sets or between animals. The core of this microbiota was dominated by members of Clostridium clusters I, XI and XIVa, with mean concentrations ranging from 7.5-9.2 log10 CFU/g faeces and with significant positive correlations between these clusters (P<0.05), and by Lactobacillaceae. Moving window analysis of DGGE profiles revealed 23.3-25.6% change between consecutive samples for four of the cheetahs. The fifth animal in the study suffered from intermediate episodes of vomiting and diarrhea during the monitoring period and exhibited remarkably more change (39.4%). This observation may reflect the temporary impact of perturbations such as the animal's compromised health, antibiotic administration or a combination thereof, which temporarily altered the relative proportions of Clostridium clusters I and XIVa. In conclusion, this first long-term monitoring study of the faecal microbiota in feline strict carnivores not only reveals a remarkable compositional stability of this ecosystem, but also shows a qualitative and quantitative similarity in a defined set of faecal bacterial lineages across the five animals under study that may typify the core phylogenetic microbiome of cheetahs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123933PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408007PMC
April 2016

Wheat bran extract alters colonic fermentation and microbial composition, but does not affect faecal water toxicity: a randomised controlled trial in healthy subjects.

Br J Nutr 2015 Jan 12;113(2):225-38. Epub 2014 Dec 12.

Translational Research Center for Gastrointestinal Disorders (TARGID),O&N 1, Box 701, Herestraat 49,3000Leuven,Belgium.

Wheat bran extract (WBE), containing arabinoxylan-oligosaccharides that are potential prebiotic substrates, has been shown to modify bacterial colonic fermentation in human subjects and to beneficially affect the development of colorectal cancer (CRC) in rats. However, it is unclear whether these changes in fermentation are able to reduce the risk of developing CRC in humans. The aim of the present study was to evaluate the effects of WBE on the markers of CRC risk in healthy volunteers, and to correlate these effects with colonic fermentation. A total of twenty healthy subjects were enrolled in a double-blind, cross-over, randomised, controlled trial in which the subjects ingested WBE (10 g/d) or placebo (maltodextrin, 10 g/d) for 3 weeks, separated by a 3-week washout period. At the end of each study period, colonic handling of NH3 was evaluated using the biomarker lactose[15N, 15N']ureide, colonic fermentation was characterised through a metabolomics approach, and the predominant microbial composition was analysed using denaturing gradient gel electrophoresis. As markers of CRC risk, faecal water genotoxicity was determined using the comet assay and faecal water cytotoxicity using a colorimetric cell viability assay. Intake of WBE induced a shift from urinary to faecal 15N excretion, indicating a stimulation of colonic bacterial activity and/or growth. Microbial analysis revealed a selective stimulation of Bifidobacterium adolescentis. In addition, WBE altered the colonic fermentation pattern and significantly reduced colonic protein fermentation compared with the run-in period. However, faecal water cytotoxicity and genotoxicity were not affected. Although intake of WBE clearly affected colonic fermentation and changed the composition of the microbiota, these changes were not associated with the changes in the markers of CRC risk.
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http://dx.doi.org/10.1017/S0007114514003523DOI Listing
January 2015

Probiotics: an update.

J Pediatr (Rio J) 2015 Jan-Feb;91(1):6-21. Epub 2014 Oct 23.

Faculté de Médecine Vétérinaire, Département des Sciences des Denrées Alimentaires, University of Liège, Liège, Belgium.

Objective: Triggered by the growing knowledge on the link between the intestinal microbiome and human health, the interest in probiotics is ever increasing. The authors aimed to review the recent literature on probiotics, from definitions to clinical benefits, with emphasis on children.

Sources: Relevant literature from searches of PubMed, CINAHL, and recent consensus statements were reviewed.

Summary Of The Findings: While a balanced microbiome is related to health, an imbalanced microbiome or dysbiosis is related to many health problems both within the gastro-intestinal tract, such as diarrhea and inflammatory bowel disease, and outside the gastro-intestinal tract such as obesity and allergy. In this context, a strict regulation of probiotics with health claims is urgent, because the vast majority of these products are commercialized as food (supplements), claiming health benefits that are often not substantiated with clinically relevant evidence. The major indications of probiotics are in the area of the prevention and treatment of gastro-intestinal related disorders, but more data has become available on extra-intestinal indications. At least two published randomized controlled trials with the commercialized probiotic product in the claimed indication are a minimal condition before a claim can be sustained. Today, Lactobacillus rhamnosus GG and Saccharomyces boulardii are the best-studied strains. Although adverse effects have sporadically been reported, these probiotics can be considered as safe.

Conclusions: Although regulation is improving, more stringent definitions are still required. Evidence of clinical benefit is accumulating, although still missing in many areas. Misuse and use of products that have not been validated constitute potential drawbacks.
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http://dx.doi.org/10.1016/j.jped.2014.08.005DOI Listing
July 2015

Psychrotrophic lactic acid bacteria associated with production batch recalls and sporadic cases of early spoilage in Belgium between 2010 and 2014.

Int J Food Microbiol 2014 Nov 19;191:157-63. Epub 2014 Sep 19.

LFMFP, Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Member of Food2Know, Coupure Links 653, B-9000 Gent, Belgium.

Between 2010 and 2014 several spoilage cases in Belgium occurring in retail foodstuffs prior to the end of shelf-life have been reported to our laboratory. Overall, seven cases involved strictly psychrotrophic lactic acid bacteria (LAB) contamination in packaged and chilled-stored food products. The products derived either from recalls of entire production batches or as specimens of sporadic spoilage manifestations. Some of these samples were returned to the manufacturing companies by consumers who observed the alterations after purchasing the products. The products covered a wide range of foodstuffs (i.e. meat, dairy, vegetable, egg products and composite food) and denoted different spoilage defects. However, the microbiota determined by means of 16S rRNA gene high-throughput sequencing analysis underpin few LAB genera (i.e. Leuconostoc, Lactobacillus, Weissella and Lactococcus), which are frequently encountered nowadays as specific spoilage organisms (SSO) albeit overlooked by mesophilic enumeration methods due to their strictly psychrotrophic character. The present study confirms the spreading of psychrotrophic LAB in Belgian food processing environments leading to unexpected spoilage, corroborating their spoilage dynamics and prevalence in all kinds of packaged and refrigerated foodstuffs in Northern Europe.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.013DOI Listing
November 2014

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod.

Syst Appl Microbiol 2014 Jul 27;37(5):329-35. Epub 2014 May 27.

Department of Biotechnology, Delft University of Biotechnology, Delft, The Netherlands.

Five isolates from marine fish (W3(T), WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3(T), WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)5-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA-DNA hybridizations revealed 89% relatedness between isolate W3(T) and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543(T), P. kishitanii LMG 23890(T), P. aquimaris LMG 26951(T) and P. phosphoreum LMG4233(T). The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3(T) (=NCCB 100098(T)=LMG 27681(T)) as the type strain.
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http://dx.doi.org/10.1016/j.syapm.2014.05.003DOI Listing
July 2014

Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment.

Int J Food Microbiol 2014 Aug 15;185:7-16. Epub 2014 May 15.

LFMFP, Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Member of Food2Know, Coupure Links 653, Gent B-9000, Belgium.

A study monitoring lactic acid bacteria contamination was conducted in a company producing fresh, minimally processed, packaged and ready-to-eat (RTE) vegetable salads (stored at 4°C) in order to investigate the reason for high psychrotrophic LAB levels in the products at the end of shelf-life. Initially, high microbial counts exceeding the established psychrotrophic thresholds (>10(7)-10(8)CFU/g) and spoilage manifestations before the end of the shelf-life (7days) occurred in products containing an assortment of sliced and diced vegetables, but within a one year period these spoilage defects became prevalent in the entire processing plant. Environmental sampling and microbiological analyses of the raw materials and final products throughout the manufacturing process highlighted the presence of high numbers of Leuconostoc spp. in halved and unseeded, fresh sweet bell peppers provided by the supplier. A combination of two DNA fingerprinting techniques facilitated the assessment of the species diversity of LAB present in the processing environment along with the critical point of their introduction in the production facility. Probably through air mediation and surface adhesion, mainly members of the strictly psychrotrophic species Leuconostoc gelidum subsp. gasicomitatum and L. gelidum subsp. gelidum were responsible for the cross-contamination of every vegetable handled within the plant.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2014.05.009DOI Listing
August 2014

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003.

PLoS One 2014 28;9(5):e98111. Epub 2014 May 28.

Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098111PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4037206PMC
June 2015

Phylogenetic analysis of faecal microbiota from captive cheetahs reveals underrepresentation of Bacteroidetes and Bifidobacteriaceae.

BMC Microbiol 2014 Feb 18;14:43. Epub 2014 Feb 18.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium.

Background: Imbalanced feeding regimes may initiate gastrointestinal and metabolic diseases in endangered felids kept in captivity such as cheetahs. Given the crucial role of the host's intestinal microbiota in feed fermentation and health maintenance, a better understanding of the cheetah's intestinal ecosystem is essential for improvement of current feeding strategies. We determined the phylogenetic diversity of the faecal microbiota of the only two cheetahs housed in an EAZA associated zoo in Flanders, Belgium, to gain first insights in the relative distribution, identity and potential role of the major community members.

Results: Taxonomic analysis of 16S rRNA gene clone libraries (702 clones) revealed a microbiota dominated by Firmicutes (94.7%), followed by a minority of Actinobacteria (4.3%), Proteobacteria (0.4%) and Fusobacteria (0.6%). In the Firmicutes, the majority of the phylotypes within the Clostridiales were assigned to Clostridium clusters XIVa (43%), XI (38%) and I (13%). Members of the Bacteroidetes phylum and Bifidobacteriaceae, two groups that can positively contribute in maintaining intestinal homeostasis, were absent in the clone libraries and detected in only marginal to low levels in real-time PCR analyses.

Conclusions: This marked underrepresentation is in contrast to data previously reported in domestic cats where Bacteroidetes and Bifidobacteriaceae are common residents of the faecal microbiota. Next to methodological differences, these findings may also reflect the apparent differences in dietary habits of both felid species. Thus, our results question the role of the domestic cat as the best available model for nutritional intervention studies in endangered exotic felids.
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http://dx.doi.org/10.1186/1471-2180-14-43DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936777PMC
February 2014

Psychrotrophic members of Leuconostoc gasicomitatum, Leuconostoc gelidum and Lactococcus piscium dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium.

Food Microbiol 2014 May 19;39:61-7. Epub 2013 Nov 19.

LFMFP, Laboratory of Food Microbiology and Food Preservation, Department of Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University, Member of Food2Know, Coupure Links 653, B-9000 Gent, Belgium.

Previously, a considerable underestimation (+0.5-3.2 log CFU/g) on the contamination levels of psychrotrophic lactic acid bacteria (LAB) was observed for 33 retail, packaged food products stored at chilling temperature when the mesophilic enumeration technique was implemented as reference shelf-life parameter. In the present study, the microbial diversity of the dominant psychrotrophic LAB recovered after incubation of plates at 22 °C for 5 days was determined using a polyphasic taxonomic approach. A total of 212 LAB isolates were identified using a combination of rep-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and pheS gene sequencing. Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc spp., Lactococcus piscium and Lactobacillus algidus proved to be the most competent and predominant species that may go undetected by the widely applied mesophilic enumeration protocols (ISO 4833:2003 and ISO 15214:1998). This study has assessed the interspecific variation among potential spoilage LAB, and highlights the significance of implementing a reference shelf-life parameter based on the enumeration of the total psychrotrophic bacterial load for industrial microbiological routine analyses.
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http://dx.doi.org/10.1016/j.fm.2013.11.005DOI Listing
May 2014

Gut microbiota affects sensitivity to acute DSS-induced colitis independently of host genotype.

Inflamm Bowel Dis 2013 Nov;19(12):2560-7

*Department for Molecular Biomedical Research, VIB, Ghent, Belgium; †Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium; ‡Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium; §Department of Structural Biology, VIB, Brussels, Belgium; ‖Department of Bioscience Engineering, Vrije Universiteit Brussel, Brussels, Belgium; and ¶BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, Ghent, Belgium.

Caspase-deficient mice and wild-type (WT) mice show significant differences in their gut microbiota composition. These differences coincide with the observation that caspase-3-deficient mice carrying a natural caspase-11 mutation (Casp3/11(-/-)) are less sensitive to acute dextran sodium sulfate-induced colitis than WT mice. For these reasons, we investigated the role of the microbiota in the development of colitis by cohousing WT and Casp3/11(-/-) mice. Microbial community fingerprinting by denaturing gradient gel electrophoresis analysis revealed that the similarities in gut microbial composition of WT and Casp3/11(-/-) mice increased after cohousing. In the acute dextran sodium sulfate-induced colitis model, Casp3/11(-/-) mice that were cohoused with WT mice showed increased weight loss and disease activity scores and increased neutrophil infiltration and inflammatory cytokine levels in their colon tissue compared with Casp3/11(-/-) mice that were not cohoused with WT mice. Also, we demonstrate that only the microbiota of the Casp3/11(-/-) mice cohoused with WT mice showed an important increase in Prevotella species. In conclusion, our cohousing experiments revealed that the colitogenic activity of the WT microbiota is transferable to Casp3/11(-/-) mice and that Prevotella species are likely to be involved. By contrast, the relative protection of Casp3/11(-/-) mice against dextran sodium sulfate damage is not transferred to WT mice after cohousing. These results underscore the need for in-depth studies of the bilateral interaction of host genes and microbiota to gain insight into the mechanisms of disease pathogenesis. Our findings also have important implications for the experimental design of disease studies in genetically modified mice and conclusions drawn from them.
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http://dx.doi.org/10.1097/MIB.0b013e3182a8759aDOI Listing
November 2013

The Italian Hafnia alvei strain LMG 27376 is Hafnia paralvei.

Vet Microbiol 2013 Dec 6;167(3-4):742-3. Epub 2013 Aug 6.

Clinical Microbiology and Virology, Spirito Santo Hospital, Pescara (PE), Italy. Electronic address:

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http://dx.doi.org/10.1016/j.vetmic.2013.07.026DOI Listing
December 2013

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains.

Food Microbiol 2013 Dec 5;36(2):327-34. Epub 2013 Jul 5.

Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA) (CONICET-Facultad de Ciencias Exactas, UNLP), La Plata 1900, Argentina.

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)5-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.
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http://dx.doi.org/10.1016/j.fm.2013.06.022DOI Listing
December 2013

Acinetobacter kookii sp. nov., isolated from soil.

Int J Syst Evol Microbiol 2013 Dec 15;63(Pt 12):4402-4406. Epub 2013 Aug 15.

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.

Two Gram-stain-negative, non-fermentative bacterial strains, designated 11-0202(T) and 11-0607, were isolated from soil in South Korea, and four others, LUH 13522, LUH 8638, LUH 10268 and LUH 10288, were isolated from a beet field in Germany, soil in the Netherlands, and sediment of integrated fish farms in Malaysia and Thailand, respectively. Based on 16S rRNA, rpoB and gyrB gene sequences, they are considered to represent a novel species of the genus Acinetobacter. Their 16S rRNA gene sequences showed greatest pairwise similarity to Acinetobacter beijerinckii NIPH 838(T) (97.9-98.4 %). They shared highest rpoB and gyrB gene sequence similarity with Acinetobacter johnsonii DSM 6963(T) and Acinetobacter bouvetii 4B02(T) (85.4-87.6 and 78.1-82.7 %, respectively). Strain 11-0202(T) displayed low DNA-DNA reassociation values (<40 %) with the most closely related species of the genus Acinetobacter. The six strains utilized azelate, 2,3-butanediol, ethanol and dl-lactate as sole carbon sources. Cellular fatty acid analyses showed similarities to profiles of related species of the genus Acinetobacter: summed feature 3 (C16 : 1ω7c, C16 : 1ω6c; 24.3-27.2 %), C18 : 1ω9c (19.9-22.1 %), C16 : 0 (15.2-22.0 %) and C12 : 0 (9.2-14.2 %). On the basis of the current findings, it is concluded that the six strains represent a novel species, for which the name Acinetobacter kookii sp. nov. is proposed. The type strain is 11-0202(T) ( = KCTC 32033(T) = JCM 18512(T)).
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http://dx.doi.org/10.1099/ijs.0.047969-0DOI Listing
December 2013

Amoxicillin-clavulanic acid resistance in fecal Enterobacteriaceae from patients with cystic fibrosis and healthy siblings.

J Cyst Fibros 2013 Dec 16;12(6):780-3. Epub 2013 Jul 16.

Laboratory of Microbiology, Faculty of Sciences, Ghent University, Belgium.

Background: The present study set out to detect and identify amoxicillin-clavulanic acid (AMC)-resistant Enterobacteriaceae in fecal samples of two patients with cystic fibrosis (CF) and their respective siblings.

Methods: Fecal Enterobacteriaceae were enumerated onto EMB agar containing amoxicillin (AMX). A total of 173 CF isolates and 41 sibling isolates were grouped into seven Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) clusters and identified through 16S rRNA and rpoB sequence analysis.

Results: The fecal microbiota of patients with CF revealed a higher prevalence of AMX resistant Enterobacteriaceae compared to that of their healthy siblings. Whereas all selected isolates of healthy siblings were assigned to Escherichia coli, isolates of patients with CF belonged to Klebsiella oxytoca (58.4%), E. coli (28.3%), Klebsiella variicola (7.5%) or Citrobacter sp. (5.8%). All tested CF isolates showed a high resistance rate to AMX, and a lower level of resistance to the combination with clavulanic acid. In contrast, all tested sibling isolates were susceptible for both AMX and AMC.

Conclusion: The higher abundance of AMX resistance in the investigated patients with CF suggests that frequent AMC administration may be one of the major contributing factors in the proliferation of Enterobacteriaceae and the development of resistant strains in the gastrointestinal tract.
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http://dx.doi.org/10.1016/j.jcf.2013.06.006DOI Listing
December 2013

Microbial characterization of probiotics--advisory report of the Working Group "8651 Probiotics" of the Belgian Superior Health Council (SHC).

Mol Nutr Food Res 2013 Aug 25;57(8):1479-504. Epub 2013 Jun 25.

Laboratory for Microbiology & BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, Ghent, Belgium.

When ingested in sufficient numbers, probiotics are expected to confer one or more proven health benefits on the consumer. Theoretically, the effectiveness of a probiotic food product is the sum of its microbial quality and its functional potential. Whereas the latter may vary much with the body (target) site, delivery mode, human target population, and health benefit envisaged microbial assessment of the probiotic product quality is more straightforward. The range of stakeholders that need to be informed on probiotic quality assessments is extremely broad, including academics, food and biotherapeutic industries, healthcare professionals, competent authorities, consumers, and professional press. In view of the rapidly expanding knowledge on this subject, the Belgian Superior Health Council installed Working Group "8651 Probiotics" to review the state of knowledge regarding the methodologies that make it possible to characterize strains and products with purported probiotic activity. This advisory report covers three main steps in the microbial quality assessment process, i.e. (i) correct species identification and strain-specific typing of bacterial and yeast strains used in probiotic applications, (ii) safety assessment of probiotic strains used for human consumption, and (iii) quality of the final probiotic product in terms of its microbial composition, concentration, stability, authenticity, and labeling.
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http://dx.doi.org/10.1002/mnfr.201300065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910143PMC
August 2013

Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota.

J Cyst Fibros 2013 May 11;12(3):206-15. Epub 2012 Nov 11.

Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.

Background: Recurrent antimicrobial interventions and disease-related intestinal dysfunction are suspected to contribute to the dysbiosis of the gastrointestinal microbial ecosystem in patients with cystic fibrosis (CF). The present study set out to detect and identify microbial discriminants in the gut microbiota composition that are associated with CF-related intestinal dysbiosis.

Methods: An in-depth description of CF-associated gut dysbiosis was obtained by screening denaturing gradient gel electrophoresis (DGGE) fingerprints for potentially discriminating bacterial species, and quantification by means of real-time PCR analyses using group-specific primers.

Results: A total of 8 DGGE band-classes assigned to the genus Bifidobacterium (n=3), and members of Clostridium clusters XIVa (n=3) and IV (n=2), were significantly (p<0.05) underrepresented in samples of patients with CF. Real-time PCR analyses confirmed a significantly lower abundance and temporal stability of bifidobacteria and Clostridium cluster XIVa in the fecal microbiota of patients with CF.

Conclusion: This study is the first to report specific microbial determinants of dysbiosis in patients with CF.
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http://dx.doi.org/10.1016/j.jcf.2012.10.003DOI Listing
May 2013

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning.

Forensic Sci Int 2012 Sep 11;221(1-3):11-5. Epub 2012 Apr 11.

BCCM/LMG Bacteria Collection & Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.
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http://dx.doi.org/10.1016/j.forsciint.2012.03.017DOI Listing
September 2012

Food fermentations: microorganisms with technological beneficial use.

Int J Food Microbiol 2012 Mar 31;154(3):87-97. Epub 2011 Dec 31.

Danone Research, RD128, 91 767 Palaiseau Cedex, France.

Microbial food cultures have directly or indirectly come under various regulatory frameworks in the course of the last decades. Several of those regulatory frameworks put emphasis on "the history of use", "traditional food", or "general recognition of safety". Authoritative lists of microorganisms with a documented use in food have therefore come into high demand. One such list was published in 2002 as a result of a joint project between the International Dairy Federation (IDF) and the European Food and Feed Cultures Association (EFFCA). The "2002 IDF inventory" has become a de facto reference for food cultures in practical use. However, as the focus mainly was on commercially available dairy cultures, there was an unmet need for a list with a wider scope. We present an updated inventory of microorganisms used in food fermentations covering a wide range of food matrices (dairy, meat, fish, vegetables, legumes, cereals, beverages, and vinegar). We have also reviewed and updated the taxonomy of the microorganisms used in food fermentations in order to bring the taxonomy in agreement with the current standing in nomenclature.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2011.12.030DOI Listing
March 2012

Cross-sectional and longitudinal comparisons of the predominant fecal microbiota compositions of a group of pediatric patients with cystic fibrosis and their healthy siblings.

Appl Environ Microbiol 2011 Nov 16;77(22):8015-24. Epub 2011 Sep 16.

Faculty of Sciences, Laboratory of Microbiology, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.

Although only poorly documented, it can be assumed that intensive antibiotic treatments of chronic lung infections in patients with cystic fibrosis (CF) also affect the diversity and metabolic functioning of the gastrointestinal microbiota and potentially lead to a state of dysbiosis. A better knowledge of the differences in gut microbiota composition and stability between patients with CF and healthy subjects could lead to optimization of current antibiotic therapies and/or development of add-on therapies. Using conventional culturing and population fingerprinting by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA amplicons, we compared the predominant fecal microbiota of 21 patients with CF and 24 healthy siblings in a cross-sectional study. General medium counts, as well as counts on media specific for lactic acid bacteria, clostridia, Bifidobacterium spp., Veillonella spp., and Bacteroides-Prevotella spp., were consistently higher in sibling samples than in CF samples, whereas the reverse was found for enterobacterial counts. DGGE fingerprinting uncovered large intersubject variations in both study groups. On the other hand, the cross-sectional data indicated that the predominant fecal microbiota of patients and siblings had comparable species richness. In addition, a longitudinal study was performed on 7 or 8 consecutive samples collected over a 2-year period from two patients and their respective siblings. For these samples, DGGE profiling indicated an overall trend toward lower temporal stability and lower species richness in the predominant fecal CF microbiota. The observed compositional and dynamic perturbations provide the first evidence of a general dysbiosis in children with CF compared to their siblings.
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http://dx.doi.org/10.1128/AEM.05933-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208981PMC
November 2011

Safety assessment of probiotics for human use.

Gut Microbes 2010 May-Jun;1(3):164-85. Epub 2010 Mar 4.

Dairy and Food Culture Technologies, Centennial, CO, USA.

The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicogenicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects must be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. The field of probiotic safety is characterized by the scarcity of studies specifically designed to assess safety contrasted with the long history of safe use of many of these microbes in foods.
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http://dx.doi.org/10.4161/gmic.1.3.12127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023597PMC
September 2011

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food.

Syst Appl Microbiol 2011 May 6;34(3):227-34. Epub 2011 Feb 6.

Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africa.

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0-6h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12-54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.
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http://dx.doi.org/10.1016/j.syapm.2010.11.016DOI Listing
May 2011