The qacC Gene Has Recently Spread between Rolling Circle Plasmids of Staphylococcus, Indicative of a Novel Gene Transfer Mechanism

Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. These qac gene products belong to the Small Multidrug Resistant (SMR) protein family, and are often encoded by rolling-circle (RC) replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance). The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations

Comparative Genomics of Bifidobacterium, Lactobacillus and Related Probiotic Genera

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

Review and phylogenetic analysis of <i>qac </i>genes that reduce susceptibility to quaternary ammonium compounds in <i>Staphylococcus </i>species

The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera

Evidence of host-virus co-evolution in tetranucleotide usage patterns of bacteriophages and eukaryotic viruses

BACKGROUND: Virus taxonomy is based on morphologic characteristics, as there are no widely used non-phenotypic measures for comparison among virus families. We examined whether there is phylogenetic signal in virus nucleotide usage patterns that can be used to determine ancestral relationships. The well-studied model of tail morphology in bacteriophage classification was used for comparison with nucleotide usage patterns. Tetranucleotide usage deviation (TUD) patterns were chosen since they have previously been shown to contain phylogenetic signal similar to that of 16S rRNA. RESULTS: We found that bacteriophages have unique TUD patterns, representing genomic signatures that are relatively conserved among those with similar host range. Analysis of TUD-based phylogeny indicates that host influences are important in bacteriophage evolution, and phylogenies containing both phages and their hosts support their co-evolution. TUD-based phylogeny of eukaryotic viruses indicates that they cluster largely based on nucleic acid type and genome size. Similarities between eukaryotic virus phylogenies based on TUD and gene content substantiate the TUD methodology. CONCLUSION: Differences between phenotypic and TUD analysis may provide clues to virus ancestry not previously inferred. As such, TUD analysis provides a complementary approach to morphology-based systems in analysis of virus evolution

In silico Selection of Amplification Targets for Rapid Polymorphism Screening in Ebola Virus Outbreaks

To achieve maximum transmission chain tracking in the current Ebola outbreak, whole genome sequencing (WGS) has been proposed to provide optimal information. However, WGS remains a costly and time-intensive procedure that is poorly suited for the large numbers of samples being generated, especially under severe time and work-environment constraints as in the present DRC outbreak. To better prepare for future outbreaks, where an apparent single outbreak may actually represent overlapping outbreaks caused by independent variants, and where rapid identification of emerging new transmission chains will be essential, a more practical method would be to amplify and sequence genomic areas that reveal the highest information to differentiate EBOV variants. We have identified four highly informative polymorphism PCR sequencing targets, suitable for rapid tracing of transmission chains and identification of new sources of Ebola outbreaks, an approach which will be far more practical in the field than WGS

Insights from Comparative Genomics of the Genus Salmonella

Comparative genomics have become a standard approach to gain insights into the interrelationships of microorganisms. Here, we have applied variable bioinformatic techniques to compare over 200 Salmonella genomes. First, we present a tree of all sequenced different members of the Enterobacteriaceae family, based on comparison of average amino acid identities. This technique was also applied to zoom in on the genomes of the genus Salmonella. The pan and core genomes of this genus were established and compared to experimental data available on the literature that identified essential genes. Difficulties and shortcomings of both approaches are discussed. Metabolic pathways unique for Salmonella were identified. Finally, we present an analysis of genes coding for small RNAs, an important part of the genetic repertoire of bacteria that is often ignored. The findings reported here are discussed and compared with available literature

Genomic Characterization of Campylobacter jejuni Strain M1

Campylobacter jejuni strain M1 (laboratory designation 99/308) is a rarely documented case of direct transmission of C. jejuni from chicken to a person, resulting in enteritis. We have sequenced the genome of C. jejuni strain M1, and compared this to 12 other C. jejuni sequenced genomes currently publicly available. Compared to these, M1 is closest to strain 81116. Based on the 13 genome sequences, we have identified the C. jejuni pan-genome, as well as the core genome, the auxiliary genes, and genes unique between strains M1 and 81116. The pan-genome contains 2,427 gene families, whilst the core genome comprised 1,295 gene families, or about two-thirds of the gene content of the average of the sequenced C. jejuni genomes. Various comparison and visualization tools were applied to the 13 C. jejuni genome sequences, including a species pan- and core genome plot, a BLAST Matrix and a BLAST Atlas. Trees based on 16S rRNA sequences and on the total gene families in each genome are presented. The findings are discussed in the background of the proven virulence potential of M1

Identification of hyperinvasive Campylobacter jejuni strains isolated from poultry and human clinical sources

Campylobacter jejuni causes gastroenteritis with a variety of symptoms in humans. In the absence of a suitable animal model, in vitro models have been used to study virulence traits such as invasion and toxin production. In this study, 113 C. jejuni isolates from poultry and poultry-related (n=74) environments as well as isolates from human cases (n=39) of campylobacteriosis and bacteraemia were tested for invasiveness using INT 407 cells. The method was sufficiently reproducible to observe a spectrum of invasiveness amongst strains. As a result, strains were classified as low, high and hyper-invasive. The majority of strains (poultry and human) were low invaders (82 % and 88 %, respectively). High invasion was found for 5 % of human strains and 11 % of poultry-related isolates. However, only 1 % of poultry strains were classified as hyperinvasive compared to 13 % of human isolates (P=0.0182). Of those isolates derived from the blood of bacteraemic patients, 20 % were hyperinvasive, though this correlation was not statistically significant. An attempt was made to correlate invasiveness with the presence of seven genes previously reported to be associated with virulence. Most of these genes did not correlate with invasiveness, but gene cj0486 was weakly over-represented, and a negative correlation was observed for the gene ciaB. This trend was stronger when the two genes were analysed together, thus ciaB– cj0486+ was over-represented in high and hyperinvasive strains, with low invaders more commonly found to lack these genes (P=0.0064)

Ebolavirus comparative genomics

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).Fil: Jun, Se Ran. Oak Ridge National Laboratory; Estados Unidos. University of Tennessee; Estados UnidosFil: Leuze, Michael R.. Oak Ridge National Laboratory; Estados UnidosFil: Nookaew, Intawat. Oak Ridge National Laboratory; Estados UnidosFil: Uberbacher, Edward C.. Oak Ridge National Laboratory; Estados UnidosFil: Land, Miriam. Oak Ridge National Laboratory; Estados UnidosFil: Zhang, Qian. Oak Ridge National Laboratory; Estados Unidos. University of Tennessee; Estados UnidosFil: Wanchai, Visanu. Oak Ridge National Laboratory; Estados UnidosFil: Chai, Juanjuan. Oak Ridge National Laboratory; Estados UnidosFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Trolle, Thomas. Technical University of Denmark; DinamarcaFil: Lund, Ole. Technical University of Denmark; DinamarcaFil: Buzard, Gregory S.. Booze Allen Hamilton; Estados UnidosFil: Pedersen, Thomas D.. Technical University of Denmark; Dinamarca. Assays; DinamarcaFil: Wassenaar, Trudy M.. Molecular Microbiology and Genomics Consultants; AlemaniaFil: Ussery, David W.. Oak Ridge National Laboratory; Estados Unidos. University of Tennessee; Estados Unidos. Technical University of Denmark; Dinamarc