• Keywords
• Burkholderia, FT-IR, cystic fibrosis, epidemiology,
• MeSH
• Burkholderia cepacia complex * MeSH
• Burkholderia * MeSH
• Cystic Fibrosis * complications   MeSH
• Burkholderia Infections * MeSH
• Humans MeSH
• Spectroscopy, Fourier Transform Infrared methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.

• MeSH
• Burkholderia cenocepacia genetics   MeSH
• Chronic Disease MeSH
• Cystic Fibrosis complications   microbiology   MeSH
• Burkholderia Infections genetics   MeSH
• Respiratory Tract Infections microbiology   MeSH
• Humans MeSH
• Comparative Genomic Hybridization MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cystic fibrosis (CF) patients in the Czech Republic suffered in the late 1990s from an epidemic with ST32 strain of Burkholderia cepacia complex (Bcc). Cohort segregation of Bcc and of ST32 positive patients was introduced in 1999 and 2002, respectively. We performed a study to evaluate the molecular epidemiology of Bcc infection after implementation of these infection control measures. Patients attending the Prague CF adult Centre from 2000 to 2015 were included in the present study. Demographic data and microbial statuses were collected from patient records. All Bcc isolates were analyzed using multilocus sequence typing (MLST). The prevalences of epidemic strain ST32 and of other Bcc strains were calculated. Ninety out of 227 CF patients were infected with Bcc during the study period. The prevalence of ST32 cases significantly decreased from 46.5% in 2000-2001 to 10.4% in 2014-2015 (P < 0.001) due to occurrence of only one new case in 2003, as well as to the death of 72% of ST32-infected patients. Conversely, there was a significant increase in prevalence of other Bcc strains, which rose from 0 to 14.9% (P = 0.015) and of transient infections. A micro-epidemic of infection with ST630 strain was observed in 2014 in lung transplant patients hospitalized in intensive care unit. The prevalence of epidemic strain ST32 decreased, whereas that of non-clonal strains of Bcc increased. Routine use of MLST allowed early detection of new and potentially epidemic strains.

• Keywords
• Cystic Fibrosis, Cystic Fibrosis Patient, Molecular Genetic Method, Multilocus Sequence Typing, recA Group,
• MeSH
• Burkholderia cepacia complex classification   genetics   isolation & purification   MeSH
• Cystic Fibrosis epidemiology   microbiology   MeSH
• Burkholderia Infections epidemiology   microbiology   MeSH
• Humans MeSH
• Molecular Epidemiology MeSH
• Multilocus Sequence Typing MeSH
• Prevalence MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic epidemiology   MeSH

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.

• MeSH
• Bacteremia microbiology   MeSH
• Organelle Biogenesis * MeSH
• Burkholderia cenocepacia genetics   physiology   MeSH
• Cystic Fibrosis complications   microbiology   MeSH
• Adult MeSH
• Flagella genetics   physiology   MeSH
• Burkholderia Infections microbiology   MeSH
• Blood microbiology   MeSH
• Humans MeSH
• Locomotion MeSH
• Microarray Analysis MeSH
• Prognosis MeSH
• Retrospective Studies MeSH
• Sputum microbiology   MeSH
• Gene Expression Profiling * MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain. The clonality of the isolates was evident by multilocus sequence typing; however, fingerprinting profiles obtained by pulsed-field gel electrophoresis (PFGE) showed substantial band variability. We investigated whether the PFGE pattern diversity resulted from genomic rearrangements mediated by insertion sequences (IS); in addition, we determined whether stressful growth conditions altered the transposition activity of these IS. DNA probes for IS commonly found in B. cenocepacia were designed using the B. cenocepacia J2315 genome. Southern hybridization analysis of ST-32 isolates demonstrated diversity in both the copy number and the insertion site for a homologue of ISBcen20. Movement of the ISBcen20 homologue was detected when the ST-32 isolate CZ1238 was exposed to oxidative stress (growth in the presence of H(2)O(2)). PFGE analysis of CZ1238 derivatives exposed to oxidative stress demonstrated genomic rearrangements. Interestingly, when the closely related B. cenocepacia strain J2315 was exposed to oxidative stress, no movement of ISBcen20 was detected. Since frameshift mutations are present within the transposases of all copies of this IS in J2315, our data suggest that the transposase is inactive. In summary, we have demonstrated for the first time that IS movement can be mediated by oxidative stress and can lead to genomic rearrangements in the CF pathogen B. cenocepacia. These IS movements may alter the PFGE fingerprints of isolates that are clonal by other typing methods.

• MeSH
• Burkholderia classification   genetics   isolation & purification   MeSH
• Cystic Fibrosis complications   MeSH
• DNA, Bacterial genetics   MeSH
• DNA Fingerprinting methods   MeSH
• Disease Outbreaks MeSH
• Genetic Variation * MeSH
• Genotype MeSH
• Burkholderia Infections epidemiology   microbiology   MeSH
• Humans MeSH
• Molecular Epidemiology methods   MeSH
• Oxidative Stress * MeSH
• Electrophoresis, Gel, Pulsed-Field methods   MeSH
• Recombination, Genetic * MeSH
• Cluster Analysis MeSH
• Bacterial Typing Techniques methods   MeSH
• DNA Transposable Elements MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA Transposable Elements MeSH