Phylogenetic analysis of the concatenated MLST alleles for isolates of <i>B</i>. <i>cenocepacia recA</i> subgroup A representing common epidemic clones or isolates commonly used in laboratory studies of this species.

<p>A neighbor-joining tree was constructed using the Jukes-Cantor method for computing evolutionary distances. The branch lengths, indicated by the scale bar, are measured as the number of substitutions per site. <i>B</i>. <i>multivorans</i> type strain, LMG13010, was used as an outlier. Bootstrap values (from 1000 replicates) are shown next to the branches. * = isolates belonging to the ET-12 lineage. The tree was constructed using MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143472#pone.0143472.ref038" target="_blank">38</a>].</p

Quantitative RT-PCR of transcripts encoded by selected genes from the cepacian biosynthesis cluster show upregulation of <i>bce</i> transcripts in mucoid C3921 relative to its corresponding nonmucoid derivative C3921-CTZ32G.

<p>RNA extracted from triplicate stationary phase cultures of bacteria (16 hour) grown in yeast extract media was assayed by quantitative RT-PCR in triplicate, using transcripts from the <i>gyrB</i> gene to normalize expression values between experiments. Results are expressed relative to the nonmucoid isolate, C3921-CTZ32G, and error bars represent standard error of the mean.</p

Summary of SNVs.

<p>* = position relates to the full concatenated genome of all three chromosomes of the sequenced J2315 <i>B</i>. <i>cenocepacia</i> genome.</p><p>** = Predicted functional effect and score calculated by Provean (<a href="http://provean.jcvi.org/index.php" target="_blank">http://provean.jcvi.org/index.php</a>). A value of -2.5 or lower was used to determine if a mutation was deleterious or neutral.</p><p>⁺ = Previous transcriptome and proteome data comparing C8963 and C9343 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143472#pone.0143472.ref021" target="_blank">21</a>]. Values are relative overexpression in the mucoid C9343 vs the nonmucoid C8963.</p><p>N = insufficient evidence to make a confident base call at this position.</p><p>Summary of SNVs.</p

Genome sequences of isolates of <i>Burkholderia cenocepacia</i> C3921 (green), C8963 (blue) and C9343 (gold) compared to the reference genome of <i>B</i>. <i>cenocepacia</i> J2315 (purple), created using BRIG.

<p>Chromosomes 1 and 2 are drawn to scale. Black circles depict the relative sizes of chromosome 3 and the plasmid. To avoid implying absence where there was a lack of significance in the sequence, all low-confidence query sequences were called as their corresponding base pair in the J2315 sequence. Sequence coverage depth drawn with a maximum value cutoff of 150. The outer rings show the location of genomic islands described in J2315 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143472#pone.0143472.ref026" target="_blank">26</a>]. PS = putative or known polysaccharide synthesis genes. LPS = lipopolysaccharide biosynthesis genes. BCESM = <i>Burkholderia cepacia</i> epidemic strain marker.</p

Isolates examined in this study.

<p>Four isolates were sequenced in this study: i) C3921 –the first isolate of <i>B</i>. <i>cenocepacia</i> from this patient, this isolate displays the mucoid phenotype; ii) C8963 –isolated 9 years and 3 months after the initial isolate, this isolate displays the nonmucoid phenotype; iii) C9343 –isolated 10 years after C3921 and 3 months prior to the death of this patient, this isolate was mucoid and iv) C3921-CTZ32G a nonmucoid variant of C3921 isolated in the laboratory following exposure to higher than MIC levels of the antibiotic ceftazidime [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143472#pone.0143472.ref017" target="_blank">17</a>]. Scale is time in years from first isolate.</p

Carriage of Methicillin-Resistant <i>Staphylococcus aureus</i> by Wild Urban Norway Rats (<i>Rattus norvegicus</i>)

<div><p>Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is an important cause of multi-drug-resistant infections in people, particularly indigent populations. MRSA can be transmitted between people and domestic animals, but the potential for transmission between people and commensal pests, particularly rodents, had not been investigated. The objective of this study was to identify the presence and characterize the ecology of MRSA in rats (<i>Rattus</i> spp.) from in an impoverished, inner-city neighborhood. Oropharyngeal swabs were collected from rats trapped in 33 city blocks and one location within the adjacent port. Bacterial culture was performed and MRSA isolates were characterized using a variety of methods, including whole-genome sequencing (WGS). The ecology of MRSA in rats was described using phylogenetic analysis, geospatial analysis, and generalized linear mixed models. MRSA was identified 22 of 637 (3.5%) rats tested, although prevalence varied from 0 – 50% among blocks. Isolates belonged to 4 clusters according to WGS, with the largest cluster (n = 10) containing isolates that were genetically indistinguishable from community-acquired USA300 MRSA strains isolated from people within the study area. MRSA strains demonstrated both geographic clustering and dispersion. The odds of an individual rat carrying MRSA increased with increased body fat (OR = 2.53, 95% CI = 1.33 – 4.82), and in the winter (OR = 5.29, 95% CI = 1.04 – 26.85) and spring (OR = 5.50, 95% CI = 1.10 – 27.58) compared to the fall. The results show that urban rats carried the same MRSA lineages occurring in local human and/or animal populations, supporting recent transmission from external sources. MRSA carriage was influenced by season, most likely as a result of temporal variation in rat behavior and rat-human interactions.</p></div

Prevalence of MRSA-positive and -negative rats in each city block.

<p>Prevalence of MRSA-positive and -negative rats in each city block.</p

Relationship between MRSA-status, season, and morphometric characteristics among a population of wild Norway rats.

a<p>Frequencies and percentages may not add to 100% because of exclusion of rats with missing data for the variable in question.</p>b<p>Reference category.</p>c<p>Insufficient power for accurate estimation.</p

Number of deamination variants ranges widely amongst samples when fixation time is uncontrolled.

<p>Orange crosses represent number of deamination variants (y-axis; C->T/G->A). Blue dots represent all other possible variants. GR UNG = GeneRead uracil-N-glycosylase, QA = QiaAmp FFPE kit.</p

Targeted regions of amplification for the NGS assay.

<p>Targeted regions of amplification for the NGS assay.</p