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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). Source


Ugarte-Ruiz M.,Complutense University of Madrid | Florez-Cuadrado D.,Complutense University of Madrid | Wassenaar T.M.,Molecular Microbiology and Genomics Consultants | Porrero M.C.,Complutense University of Madrid | Dominguez L.,Complutense University of Madrid
International Journal of Environmental Research and Public Health | Year: 2015

Seeking a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species in untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment steps, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB), there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing better than mCCDA or CFA ones. Inclusion of an enrichment step increased the ratio of C. coli vs. C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer instances of resistance were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments. © 2015 by the authors; licensee MDPI, Basel, Switzerland. Source


Porrero M.C.,Complutense University of Madrid | Wassenaar T.M.,Molecular Microbiology and Genomics Consultants | Gomez-Barrero S.,Complutense University of Madrid | Garcia M.,Complutense University of Madrid | And 8 more authors.
Letters in Applied Microbiology | Year: 2012

Aims: Iberian pigs are bred in Spain for the production of high-value dry-cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin-resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. Methods and Results: From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. Conclusions: Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. Significance and Impact of the Study: To our knowledge, this is the first detection of MRSA in food animals raised in free-range conditions. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology. Source


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. Source


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. Source

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