Burkholderia multivorans septicemia in a pediatric liver transplant patient

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/148247/1/ajt15065_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/148247/2/ajt15065.pd

Comparative genomics of Burkholderia singularis sp. nov., a low G+C content, free-living bacterium that defies taxonomic dissection of the genus Burkholderia

Four Burkholderia pseudomallei-like isolates of human clinical origin were examined by a polyphasic taxonomic approach that included comparative whole genome analyses. The results demonstrated that these isolates represent a rare and unusual, novel Burkholderia species for which we propose the name B. singularis. The type strain is LMG 28154(T) (=CCUG 65685(T)). Its genome sequence has an average mol% G+C content of 64.34%, which is considerably lower than that of other Burkholderia species. The reduced G+C content of strain LMG 28154(T) was characterized by a genome wide AT bias that was not due to reduced GC-biased gene conversion or reductive genome evolution, but might have been caused by an altered DNA base excision repair pathway. B. singularis can be differentiated from other Burkholderia species by multilocus sequence analysis, MALDI-TOF mass spectrometry and a distinctive biochemical profile that includes the absence of nitrate reduction, a mucoid appearance on Columbia sheep blood agar, and a slowly positive oxidase reaction. Comparisons with publicly available whole genome sequences demonstrated that strain TSV85, an Australian water isolate, also represents the same species and therefore, to date, B. singularis has been recovered from human or environmental samples on three continents

Whole-Genome Sequencing of Three Clonal Clinical Isolates of B. cenocepacia from a Patient with Cystic Fibrosis.

Burkholderia cepacia complex bacteria are amongst the most feared of pathogens in cystic fibrosis (CF). The BCC comprises at least 20 distinct species that can cause chronic and unpredictable lung infections in CF. Historically the species B. cenocepacia has been the most prevalent in CF infections and has been associated in some centers with high rates of mortality. Modeling chronic infection by B. cenocepacia in the laboratory is challenging and no models exist which effectively recapitulate CF disease caused by BCC bacteria. Therefore our understanding of factors that contribute towards the morbidity and mortality caused by this organism is limited. In this study we used whole-genome sequencing to examine the evolution of 3 clonal clinical isolates of B. cenocepacia from a patient with cystic fibrosis. The first isolate was from the beginning of infection, and the second two almost 10 years later during the final year of the patients' life. These isolates also demonstrated phenotypic heterogeneity, with the first isolate displaying the mucoid phenotype (conferred by the overproduction of exopolysaccharide), while one of the later two was nonmucoid. In addition we also sequenced a nonmucoid derivative of the initial mucoid isolate, acquired in the laboratory by antibiotic pressure. Examination of sequence data revealed that the two late stage isolates shared 20 variant nucleotides in common compared to the early isolate. However, despite their isolation within 10 months of one another, there was also considerable variation between the late stage isolates, including 42 single nucleotide variants and three deletions. Additionally, no sequence differences were identified between the initial mucoid isolate and its laboratory acquired nonmucoid derivative, however transcript analysis indicated at least partial down regulation of genes involved in exopolysaccharide production. Our study examines the progression of B. cenocepacia throughout chronic infection, including establishment of sub-populations likely evolved from the original isolate, suggestive of parallel evolution. Additionally, the lack of sequence differences between two of the isolates with differing mucoid phenotypes suggests that other factors, such as gene regulation, come into play in establishing the mucoid phenotype

Antibacterial Activity of a Natural Clay Mineral against <i>Burkholderia cepacia</i> Complex and Other Bacterial Pathogens Isolated from People with Cystic Fibrosis

There is an impending crisis in healthcare brought about by a new era of untreatable infections caused by bacteria resistant to all available antibiotics. Thus, there is an urgent need to identify novel antimicrobial agents to counter the continuing threat posed by formerly treatable infections. We previously reported that a natural mineral clay known as Kisameet clay (KC) is a potent inhibitor of the organisms responsible for acute infections. Chronic bacterial infections present another major challenge to treatment by antimicrobials, due to their prolonged nature, which results in repeated exposure to antibiotics and a constant selection for antimicrobial resistance. A prime example is bacteria belonging to the Burkholderia cepacia complex (Bcc), which particularly causes some of the most serious chronic lung infections in patients with cystic fibrosis (CF) associated with unpredictable clinical outcomes, poor prognosis, and high mortality rates. Eradication of these organisms from CF patients with limited effective antimicrobial options is a major challenge. Novel therapeutic approaches are urgently required. Here, we report the in vitro antibacterial activity of KC aqueous suspensions (1–10% w/v) and its aqueous extract (L100) against a collection of extensively and multi-drug resistant clinical isolates of Bcc, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia isolated from patients with CF. These findings present a potential novel therapy for further investigation in the clinic

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

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

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