Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates

Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients

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

Burkholderia pseudomultivorans sp. nov., a novel Burkholderia cepacia complex species from human respiratory samples and the rhizosphere

A

Allergic disease in the first year of life is associated with differences in subsequent neurodevelopment and behaviour

Background: Recent trials suggest a link between neuropsychological function, atopy and allergic disease particularly in early childhood; however the nature of this association remains unclear. Aims: To investigate the relationship between early allergic disease and sensitisation at 12 months of age and neurodevelopmental outcomes at 18 months. Study design: Linear or binary logistic regression analysis was used to determine whether allergic diseases or sensitization at 12 months of age was a significant predictor of neurodevelopmental test scores at the 18 months. Subjects: Infants with a maternal history of allergic disease (n=324). Outcome measures: Allergic outcomes at 12 months of age included allergen sensitisation, eczema, IgE-mediated and food allergy, and neurodevelopmental outcomes at 18 included the Bayley Scales of Infant Toddler Development III Edition, the Achenbach Child Behaviour Checklist and the Macarthur Scales of Infant Toddler Development. Results: Children with any diagnosed allergic disease at 12months had evidence of reduced motor scores (p=.016), and this was most apparent for a diagnosis of eczema (p=.007). Non-IgE mediated food allergy was significantly positively associated with problem Internalising Behaviours (p=.010), along with a trend for effects on the Social–Emotional composite score for IgE-Mediated food allergies (p=.052). Allergic sensitisation was not independently associated with any effects on neurodevelopmental outcomes. Conclusion: This study provides evidence that an allergic phenotype in infancy is associated with effects on neurodevelopment. Further research is required to investigate the nature of this relationship

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

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

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