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Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of nosocomial infections and a major public health concern worldwide. During the last decade, MRSA of CC398 have emerged as important colonizers of livestock. These strains also represent an increasing cause of human infections. A recent study reporting a new dominant spa type among MRSA from Finish fattening pigs (CC398/t2741) identified a strain lacking both the global virulence regulator gene locus agr and the adhesion gene fnbB. The aim of this study was to characterize this agr/fnbB-negative livestock-associated MRSA strain in terms of growth, hemolysis and adhesive capacity, and to provide data on its genomic background. To this end, growth curves and hemolysis patterns were generated and adhesion assays on human keratinocyte and porcine nasal mucosa cell lines were performed. Whole genome sequencing was used to determine the nature and extent of the relevant deletions in the livestock strains. For comparison, an agr-positive, fnbB-negative CC398/t2741 strain from the same pig herd, an agr/fnbB- positive CC398/t034 strain from another pig herd and one human MRSA strain and its isogenic Δagr knockout mutant were used. The agr-negative strains adhered significantly better to human and porcine host cells than the agr-positive control strains. For the agr-positive porcine MRSA strains, cytotoxic effects on porcine mucosal cells were observed. The strong adhesive capacity of the naturally agr-negative livestock-associated MRSA, in combination with diminished cytotoxic effects, is likely favorable for inducing persistent colonization in pigs. Independently of the host cell type, similar adhesive capacities of the naturally agr-negative livestock-associated MRSA and the human MRSA strain were shown. Our results indicate that loss of agr in the livestock-associated MRSA strain investigated in this study may have increased its potential to be transmitted to and amongst humans., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The use of whole-genome sequencing as a tool for the study of infectious bacteria is of growing clinical interest. Chlamydia trachomatis is responsible for sexually transmitted infections and the blinding disease trachoma, which affect hundreds of millions of people worldwide. Recombination is widespread within the genome of C. trachomatis, thus whole-genome sequencing is necessary to understand the evolution, diversity, and epidemiology of this pathogen. Culture of C. trachomatis has, until now, been a prerequisite to obtain DNA for whole-genome sequencing; however, as C. trachomatis is an obligate intracellular pathogen, this procedure is technically demanding and time consuming. Discarded clinical samples represent a large resource for sequencing the genomes of pathogens, yet clinical swabs frequently contain very low levels of C. trachomatis DNA and large amounts of contaminating microbial and human DNA. To determine whether it is possible to obtain whole-genome sequences from bacteria without the need for culture, we have devised an approach that combines immunomagnetic separation (IMS) for targeted bacterial enrichment with multiple displacement amplification (MDA) for whole-genome amplification. Using IMS-MDA in conjunction with high-throughput multiplexed Illumina sequencing, we have produced the first whole bacterial genome sequences direct from clinical samples. We also show that this method can be used to generate genome data from nonviable archived samples. This method will prove a useful tool in answering questions relating to the biology of many difficult-to-culture or fastidious bacteria of clinical concern.

Purpose of Review: Increasingly, genomics is being used to answer detailed clinical questions. Although genome analysis of bacterial sexually transmitted infections (STIs) lags far behind that of many other bacterial pathogens, genomics can reveal previously inaccessible aspects of pathogen biology., Recent Findings: Comparative genomic studies on the most common bacterial STI, chlamydia, have revolutionized our understanding of this intracellular bacterium, demonstrating that it undergoes extensive recombination and that the traditional typing schemes can be misleading. Genome projects can also help us to understand the recently observed phenomenon of 'diagnostic escape' seen in both Chlamydia trachomatis and Neisseria gonorrhoeae., Summary: The routine use of genomics in clinical settings is becoming a reality. For STIs, a primary requirement is an understanding of the diversity of circulating strains and how they change over time. This can help to inform future studies and allow us to address real clinical issues such as outbreak identification, global spread of successful clones and antimicrobial resistance monitoring.

XplA is a cytochrome P450 that mediates the microbial metabolism of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It has an unusual structural organisation comprising a heme domain that is fused to its flavodoxin redox partner. XplA along with its partnering reductase XplB are plasmid encoded and the gene xplA has now been found in divergent genera across the globe with near sequence identity. Importantly, it has only been detected at explosives contaminated sites suggesting rapid dissemination of this novel catabolic activity, possibly within the 50-year period since the introduction of RDX into the environment. The X-ray structure of XplA-heme has been solved, providing fundamental information on the heme binding site. Interestingly, oxygen is not required for the degradation of RDX, but its presence determines the final degradation products, demonstrating that the degradation chemistry is flexible with both anaerobic and aerobic pathways resulting in the release of nitrite from the substrate. Transgenic plants expressing xplA are able to remove saturating levels of RDX from soil leachate and may provide a low cost sustainable remediation strategy for contaminated military sites., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Clostridium difficile has rapidly emerged as the leading cause of antibiotic-associated diarrheal disease, with the transcontinental spread of various PCR ribotypes, including 001, 017, 027 and 078. However, the genetic basis for the emergence of C. difficile as a human pathogen is unclear. Whole genome sequencing was used to analyze genetic variation and virulence of a diverse collection of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1-85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages.