Understanding the Pathogenicity of Burkholderia contaminans, an Emerging Pathogen in Cystic Fibrosis.

Several bacterial species from the Burkholderia cepacia complex (Bcc) are feared opportunistic pathogens that lead to debilitating lung infections with a high risk of developing fatal septicemia in cystic fibrosis (CF) patients. However, the pathogenic potential of other Bcc species is yet unknown. To elucidate clinical relevance of Burkholderia contaminans, a species frequently isolated from CF respiratory samples in Ibero-American countries, we aimed to identify its key virulence factors possibly linked with an unfavorable clinical outcome. We performed a genome-wide comparative analysis of two isolates of B. contaminans ST872 from sputum and blood culture of a female CF patient in Argentina. RNA-seq data showed significant changes in expression for quorum sensing-regulated virulence factors and motility and chemotaxis. Furthermore, we detected expression changes in a recently described low-oxygen-activated (lxa) locus which encodes stress-related proteins, and for two clusters responsible for the biosynthesis of antifungal and hemolytic compounds pyrrolnitrin and occidiofungin. Based on phenotypic assays that confirmed changes in motility and in proteolytic, hemolytic and antifungal activities, we were able to distinguish two phenotypes of B. contaminans that coexisted in the host and entered her bloodstream. Whole genome sequencing revealed that the sputum and bloodstream isolates (each representing a distinct phenotype) differed by over 1,400 mutations as a result of a mismatch repair-deficient hypermutable state of the sputum isolate. The inferred lack of purifying selection against nonsynonymous mutations and the high rate of pseudogenization in the derived isolate indicated limited evolutionary pressure during evolution in the nutrient-rich, stable CF sputum environment. The present study is the first to examine the genomic and transcriptomic differences between longitudinal isolates of B. contaminans. Detected activity of a number of putative virulence factors implies a genuine pathogenic nature of this novel Bcc species.

Gene expression profiling of Burkholderia cenocepacia at the time of cepacia syndrome: loss of motility as a marker of poor prognosis?

Cepacia syndrome (CS) is a fatal septic condition that develops in approximately 20% of cystic fibrosis (CF) patients chronically infected with the Burkholderia cepacia complex (Bcc). The most common causative agent is Burkholderia cenocepacia, a clinically dominant Bcc species that contains the globally distributed epidemic strain sequence type 32 (ST32). Using microarrays, we compared the transcriptomes of ST32 isolates from the bloodstream at the time of CS with their sputum counterparts recovered 1 to 2 months prior to the development of CS. Global gene expression profiles of blood isolates revealed greater activities of the virulence genes involved in the type III secretion system, the bacterial exopolysaccharide cepacian, and quorum sensing, while reduced expression was demonstrated for flagellar genes. Furthermore, a nonmotile phenotype (as evaluated by a swimming motility assay) was identified in blood isolates from 6 out of 8 patients with CS; this phenotype was traceable to 24 months prior to the onset of CS. Loss of motility was not observed in any of the 89 ST32 isolates recovered over the course of chronic infection from 17 patients without CS. In conclusion, the gene expression of Bcc bacteria disseminated during CS has been elucidated for the first time. This study demonstrated marked differences at the transcriptome level between isogenic ST32 isolates that are attributable to the stage and site of infection. The finding of a nonmotile B. cenocepacia isolate may serve as a warning sign for the development of CS in the near future.

Novel diagnostic PCR assay for Burkholderia cenocepacia epidemic strain ST32 and its utility in monitoring infection in cystic fibrosis patients.

BACKGROUND: A highly transmissible Burkholderia cenocepacia sequence type (ST) 32 strain caused a major outbreak at the Prague Cystic Fibrosis (CF) Centre in the late 1990s and early 2000s. Because a large number of CF patients were affected by ST32, a rapid and easy-to-use diagnostic tool for ST32 infection was urgently needed for the detection of new cases as well as for long-term surveillance. The present study sought to identify unique DNA sequences within the ST32 genome to develop an ST32 strain-specific PCR assay. METHODS: Genomic subtractive hybridisation between B. cenocepacia ST32 and the closely related genome-sequenced strain B. cenocepacia ST28 identified a 325 bp long region that was absent in all but one Burkholderia strain, as demonstrated by our newly designed PCR. RESULTS: Out of 57 strains, only B. cenocepacia ST33 cross-reacted with ST32, resulting in a PCR specificity of 98.2%. This specificity was further tested by various genotyping methods, which revealed the practical indistinguishibility of ST32 and ST33. The PCR sensitivity, checked on a panel of 50 ST32 clinical isolates, was 100%. A closer examination of the ST32-specific sequence revealed no significant homology apart from a fragment of the ISBmu3 transposase. CONCLUSIONS: This novel ST32-specific PCR assay allows the rapid and reliable detection of a globally distributed B. cenocepacia epidemic strain. Its routine use is especially well suited to infection surveillance programs for CF populations with a high rate of ST32 infection. This PCR method can also be used to detect ST33, a clonal variant of ST32.

PCR detection of Burkholderia cepacia complex as one of key factors to handle a long-term outbreak.

BACKGROUND: Once the outbreak with Burkholderia cenocepacia ST32 was identified in the Prague cystic fibrosis (CF) centre, molecular tools were implemented into diagnostic routine in order to complement infection control measures with as accurate as possible microbiological service. In addition, genotyping techniques were applied as part of an infection surveillance program to assign species and strain status in samples positive for Burkholderia cepacia complex (Bcc). We sought to evaluate a usefulness of Bcc-specific PCR in infection control and to map evolution of the outbreak. METHODS: Since 2001, 6109 respiratory samples from 299 CF patients were examined not only by conventional culture, but also by PCR, detecting Bcc directly in sputum. RESULTS: Diagnosis of Bcc infection was established by culture in 54 patients already prior to 2001. As 39 more patients were diagnosed by culture and/or PCR during 2001-2010, this represented annual prevalence of 18.5%-28.9%. Twelve of 39 patients were culture negative at the time of their first PCR positivity. Although 2/3 of them became subsequently culture positive, the time delay in diagnostics of the infection by culture ranged from 1 to 22 months. New cases of Bcc infection were detected every year, however a dramatic drop was observed for the epidemic strain ST32. CONCLUSION: A likely factor contributing to the end of ST32 epidemic was segregation of Bcc infected patients that included also patients with no culture, but PCR positivity. The diagnostic PCR led to timely identification of cases with 'culture-invisible' infection.