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Vesth T.,Technical University of Denmark | Ozen A.,Technical University of Denmark | Andersen S.C.,Technical University of Denmark | Sommer Kaas R.,Technical University of Denmark | And 4 more authors.
Standards in Genomic Sciences | Year: 2013

The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the (mainly) Gram positive phylum of the Firmicutes. Further studies are required to unveil the evolutionary history of the Veillonella and other Negativicutes. © retained by original authors.


Ugarte-Ruiz M.,Complutense University of Madrid | Wassenaar T.M.,Molecular Microbiology and Genomics Consultants | Gomez-Barrero S.,Complutense University of Madrid | Porrero M.C.,Complutense University of Madrid | And 2 more authors.
Letters in Applied Microbiology | Year: 2013

We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006-1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA-SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla-genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla-genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla-genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared. Significance and Impact of Study: The tracing of Campylobacter through the food chain remains important to control campylobacteriosis in humans. Our study points out that the isolation method used affects the genotypes obtained, and this should be considered as a variant when comparing the results of surveillance studies. Significance and Impact of Study: The tracing of Campylobacter through the food chain remains important to control campylobacteriosis in humans. Our study points out that the isolation method used affects the genotypes obtained, and this should be considered as a variant when comparing the results of surveillance studies. © 2013 The Society for Applied Microbiology.


Ugarte-Ruiz M.,Complutense University of Madrid | Gomez-Barrero S.,Complutense University of Madrid | Porrero M.C.,Complutense University of Madrid | Alvarez J.,Instituto Ramon Y Cajal Of Investigacion Sanitaria Irycis | And 4 more authors.
Journal of Applied Microbiology | Year: 2012

Aims: To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture-dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n=38), neck skin (n=38) and packed fresh meat (n=38). Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006-1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest number of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real-time qPCR yielded the highest number of positive samples. Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less-contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensitive and powerful evaluation tool. Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distributed, which suggests this is still a significant risk for consumers. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.


Jun S.-R.,Oak Ridge National Laboratory | Jun S.-R.,University of Tennessee at Knoxville | Leuze M.R.,Oak Ridge National Laboratory | Nookaew I.,Oak Ridge National Laboratory | And 16 more authors.
FEMS Microbiology Reviews | Year: 2015

The 2014 Ebola outbreak in West Africa is the largest documented for this virus. To examine the dynamics of this genome, we compare more than 100 currently available ebolavirus genomes to each other and to other viral genomes. Based on oligomer frequency analysis, the family Filoviridae forms a distinct group from all other sequenced viral genomes. All filovirus genomes sequenced to date encode proteins with similar functions and gene order, although there is considerable divergence in sequences between the three genera Ebolavirus, Cuevavirus and Marburgvirus within the family Filoviridae. Whereas all ebolavirus genomes are quite similar (multiple sequences of the same strain are often identical), variation is most common in the intergenic regions and within specific areas of the genes encoding the glycoprotein (GP), nucleoprotein (NP) and polymerase (L). We predict regions that could contain epitope-binding sites, which might be good vaccine targets. This information, combined with glycosylation sites and experimentally determined epitopes, can identify the most promising regions for the development of therapeutic strategies.This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). © FEMS 2015. All rights reserved.


Land M.,Oak Ridge National Laboratory | Hauser L.,Oak Ridge National Laboratory | Hauser L.,University of Tennessee at Knoxville | Jun S.-R.,Oak Ridge National Laboratory | And 12 more authors.
Functional and Integrative Genomics | Year: 2015

Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date, there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them. © 2015, The Author(s).


Wassenaar T.M.,Molecular Microbiology and Genomics Consultants | Beimfohr C.,Vermicon AG | Geske T.,Medical Services | Zimmermann K.,SymbioPharm GmbH
Beneficial Microbes | Year: 2014

The ability of probiotic Escherichia coli to colonise the human gut was determined in a volunteer study following national (German) regulations. Five persons voluntarily took a single, high dose of Symbioflor®2, which contains 6 different probiotic E. coli genotypes, to assess tolerance of the product, after which presence of E. coli in their faeces was tested for a follow-up period of 30 weeks. Intake of the product did not result in severe side effect in any of the individuals, though mild side effects were observed. Stool analysis showed that the probiotic E. coli had colonised all five persons for a period of 10 to 30 weeks (mean: 18.7 weeks, median: 25.7 weeks). In two individuals there was evidence of competition between host E. coli and probiotic E. coli, while in two others total E. coli levels increased persistently with at least a factor of 10 as a result of the received dose. In one individual, who had lacked detectable levels of faecal E. coli at the start of the post-authorisation safety study, long-term colonisation was established, first by probiotic E. coli exclusively, which were later replaced by host E. coli strains. In four out of five individuals, total E. coli faecal counts were higher on average than at the start of the experiment, while in none total levels exceeded 5×107 cfu/g. When the specific genotypes of the 6 probiotic E. coli were analysed, it was found that one and the same common genotype was responsible for prolonged colonisation in all five individuals. © 2014 Wageningen Academic Publishers.


PubMed | Institute of Hygiene and Environmental Medicine and Molecular Microbiology and Genomics Consultants
Type: | Journal: Antimicrobial resistance and infection control | Year: 2016

Guidelines for the control of hospital-acquired MRSA include decolonization measures to end MRSA carrier status in colonized and infected patients. Successful decolonization typically requires up to 22days of treatment, which is longer than the average hospital length of stay (LOS). Incomplete decolonization is therefore common, with long-term MRSA carriage as a consequence. To overcome this, we developed an integrated MRSA Management (IMM) by extending MRSA decolonization to the outpatient and domestic setting. The protocol makes use of polyhexanide-based products, in view of reported qac-mediated resistance to chlorhexidine in S. aureus and MRSA.This is a prospective, single centre, controlled, non-randomized, open-label study to evaluate the efficiency of the IMM concept. The outcome of guideline-approved decolonization during hospital stay only (control group; n=201) was compared to the outcome following IMM treatment whereby decolonization was continued after discharge in the domestic setting or in a long-term care facility (study group; n=99). As a secondary outcome, the effect of MRSA-status of skin alterations was assessed.The overall decolonization rate was 47% in the IMM patient group compared to 12% in the control group (p<0.01). The continued treatment after hospital discharge was as effective as treatment completed during hospitalization, with microbiologically-confirmed decolonization (patients with completed regimes only) obtained with 55% for the IMM group and 43% for the control group (p>0.05). For patients with skin alterations (e.g. wounds and entry sites), decolonization success was 50% if the skin alterations were MRSA-negative at baseline, compared to 22% success for patients entering the study with MRSA-positive skin alterations (p<0.01).The IMM strategy offers an MRSA decolonization protocol that is feasible in the domestic setting and is equally effective compared with inpatient decolonization treatment when hospital LOS is long enough to complete the treatment. Moreover, for patients with average LOS, decolonization rates obtained with IMM are significantly higher than for in-hospital treatment. IMM is a promising concept to improve decolonization rates of MRSA-carriers for patients who leave the hospital before decolonization is completed.


Wassenaar T.M.,Molecular Microbiology and Genomics Consultants | Gunzer F.,TU Dresden
Gut Pathogens | Year: 2015

Now that microbial whole genome sequencing is in reach of many researchers, it is common to infer virulent properties of a given bacterial isolate based on the presence of virulence genes. However, this may lead to inaccurate presumptions of virulence. Using the findings of a recent publication (Da Silva Santos et al. Gut Pathog 7:2, 2015) where virulence was inferred from a genome sequence and subsequently confirmed by in vitro analysis, we present an alternative view on the case described in that publication. Our alternative view point, which is further substantiated by whole genome sequencing of probiotic E. coli strains, may contribute to a more balanced vision on the interactions between pathogens and host. © 2015 Wassenaar and Gunzer.


Lukjancenko O.,Technical University of Denmark | Ussery D.W.,Technical University of Denmark | Wassenaar T.M.,Molecular Microbiology and Genomics Consultants
Microbial Ecology | Year: 2012

Six bacterial genera containing species commonly used as probiotics for human consumption or starter cultures for food fermentation were compared and contrasted, based on publicly available complete genome sequences. The analysis included 19 Bifidobacterium genomes, 21 Lactobacillus genomes, 4 Lactococcus and 3 Leuconostoc genomes, as well as a selection of Enterococcus (11) and Streptococcus (23) genomes. The latter two genera included genomes from probiotic or commensal as well as pathogenic organisms to investigate if their non-pathogenic members shared more genes with the other probiotic genomes than their pathogenic members. The pan and core genome of each genus was defined. Pairwise BLASTP genome comparison was performed within and between genera. It turned out that pathogenic Streptococcus and Enterococcus shared more gene families than did the non-pathogenic genomes. In silico multilocus sequence typing was carried out for all genomes per genus, and the variable gene content of genomes was compared within the genera. Informative BLAST Atlases were constructed to visualize genomic variation within genera. The clusters of orthologous groups (COG) classes of all genes in the pan and core genome of each genus were compared. In addition, it was investigated whether pathogenic genomes contain different COG classes compared to the probiotic or fermentative organisms, again comparing their pan and core genomes. The obtained results were compared with published data from the literature. This study illustrates how over 80 genomes can be broadly compared using simple bioinformatic tools, leading to both confirmation of known information as well as novel observations. © 2012 The Author(s).


Wassenaar T.M.,Molecular Microbiology and Genomics Consultants
Letters in Applied Microbiology | Year: 2011

It has been known for decades that poultry meat is the most common single source for campylobacteriosis, yet the problem has not been solved. This review identifies some of the reasons why our attempts to reduce the incidence of this pathogen have largely failed. Based on the literature, the events a virtual population of Campylobacter may encounter, from growing in the gut of a broiler to eventually infecting humans and causing disease, are reviewed. Most steps in the farm-to-fork process are well studied, though there are gaps in our knowledge about survival and spread of Campylobacter populations before they enter the farm. Key events in the farm-to-fork chain that are suitable targets for prevention and control, to reduce food-borne campylobacteriosis, are indicated. Novel insights into the pathogenic mechanism responsible for disease in humans are summarized, which hypothesize that an overactive immune response is the reason for the typical inflammatory diarrhoea. A role of genetic microheterogeneity within a clonal population in this chain of events is being proposed here. The human host is not necessary for the survival of the bacterial species, nor have these bacteria specifically evolved to cause disease in that host. More likely, the species evolved for a commensal life in birds, and human disease can be considered as collateral damage owing to an unfortunate host-microbe interaction. The indirect environmental burden that results from poultry production should not be ignored as it may pose a diffuse, but possibly significant risk factor for disease. © 2011 The Author. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

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