Rationale: Infections by Burkholderia species bacteria in cystic fibrosis (CF) may be transmissible, necessitating infection control measures, and remain a serious cause of morbidity and mortality. The last major study of Burkholderia epidemiology in Canada included cases up until July 2000 and was marked by the dominance of a limited number of epidemic clones of Burkholderia cenocepacia.Objectives: Describe the nationwide epidemiology of Burkholderia species infections in people with cystic fibrosis in Canada over the 17-year period since 2000.Methods: Isolates were collected from across Canada between August 2000 and July 2017 and identified to the species and, for isolates between 2015 and 2017, strain level.Results: We analyzed 1,362 Burkholderia isolates from at least 396 people with CF. Forty-nine percent (n = 666) of all isolates and 47% (n = 179) of new incident infections were identified as B. multivorans. The incidence of Burkholderia infection in the Canadian CF population did not change between 2000 and 2017 at 6 cases per 1,000 annually. Multilocus sequence typing analysis suggested minimal sharing of clones in Canada.Conclusions: The epidemiology of Burkholderia in CF in Canada has shifted from limited numbers of epidemic strains of B. cenocepacia to largely nonclonal isolates of B. multivorans, B. cenocepacia, and other species. Despite widespread infection control, however, Burkholderia species bacteria continue to be acquired by people with CF at an unchanged rate, posing a continued hazard.

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 28154T (=CCUG 65685T). 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 28154T 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.

Rationale: We have been collecting Burkholderia species bacteria from patients with cystic fibrosis (CF) for the last 30 years. During this time, our understanding of their multispecies taxonomy and infection control has evolved substantially.Objectives: To evaluate the long-term (30 year) epidemiology and clinical outcome of Burkholderia infection in CF, and fully define the risks associated with infection by each species.Methods: Isolates from Burkholderia-positive patients (n=107) were speciated and typed annually for each infected patient. Microbiological and clinical data were evaluated by thorough review of patient charts, and statistical analyses performed to define significant epidemiological factors.Measurements and Main Results: Before 1995, the majority of new Burkholderia infections were caused by epidemic clones of Burkholderia cenocepacia. After implementation of new infection control measures in 1995, Burkholderia multivorans became the most prevalent species. Survival analysis showed that patients with CF infected with B. cenocepacia had a significantly worse outcome than those with B. multivorans, and a novel finding was that, after Burkholderia infection, the prognosis for females was significantly worse than for males.Conclusions: B. multivorans and B. cenocepacia have been the predominant Burkholderia species infecting people with CF in Vancouver. The implementation of infection control measures were successful in preventing new acquisition of epidemic strains of B. cenocepacia, leaving nonclonal B. multivorans as the most prevalent species. Historically, survival after infection with B. cenocepacia has been significantly worse than B. multivorans infection, and, of new significance, we show that females tend toward worse clinical outcomes.

ABSTRACT Burkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that can cause severe disease in cystic fibrosis (CF) patients and other immunocompromised individuals and are typically multidrug resistant. Here we observed that unlike other BCC species, most environmental and clinical Burkholderia vietnamiensis isolates were intrinsically susceptible to aminoglycosides but not to cationic antimicrobial peptides or polymyxin B. Furthermore, strains acquired aminoglycoside resistance during chronic CF infection, a phenomenon that could be induced under tobramycin or azithromycin pressure in vitro. In comparing susceptible and resistant B. vietnamiensis isolates, no gross differences in lipopolysaccharide structure were observed, all had lipid A-associated 4-amino-4-deoxy-l-arabinose residues, and all were resistant to the permeabilizing effects of aminoglycosides, a measure of drug entry via self-promoted uptake. However, susceptible isolates accumulated 5 to 6 times more gentamicin than a resistant isolate, and aminoglycoside susceptibility increased in the presence of an efflux pump inhibitor. B. vietnamiensis is therefore unusual among BCC bacteria in its susceptibility to aminoglycosides and capacity to acquire resistance. Aminoglycoside resistance appears to be due to decreased cellular accumulation as a result of active efflux.

The aim of the present study was to re-examine the taxonomic position and structure of taxon K (also known as group K) within the Burkholderia cepacia complex (Bcc). For this purpose, a representative set of strains was examined by a traditional polyphasic taxonomic approach, by multilocus sequence typing (MLST) analysis and by analysis of available whole-genome sequences. Analysis of the recA gene sequence revealed three different lineages, designated recA-I, recA-II and recA-III. DNA-DNA hybridization experiments demonstrated that recA-I and recA-II isolates each represented a single novel species. However, DNA-DNA hybridization values of recA-II strains towards recA-III strains and among recA-III strains were at the threshold level for species delineation. By MLST, recA-I isolates were clearly distinguished from the others and represented a distinct lineage referred to as MLST-I, whereas recA-II and recA-III isolates formed a second MLST lineage referred to as MLST-II. A divergence value of 3.5 % was obtained when MLST-I was compared with MLST-II. The internal level of concatenated sequence divergence within MLST-I and MLST-II was 1.4 and 2.7 %, respectively; by comparison with the level of concatenated sequence divergence in established Bcc species, these data demonstrate that the MLST-I and MLST-II lineages represent two distinct species within the Bcc. The latter conclusion was supported by comparison of the whole-genome average nucleotide identity (ANI) level of MLST-I and MLST-II strains with strains of established Bcc species and by a whole-genome-based phylogenetic analysis. We formally propose to classify taxon K bacteria from the MLST-I and MLST-II lineages as Burkholderia contaminans sp. nov. (with strain J2956T =LMG 23361T =CCUG 55526T as the type strain) and Burkholderia lata sp. nov. (with strain 383T =ATCC 17760T =LMG 22485T =CCUG 55525T as the type strain), respectively. The MLST approach was confirmed as a valuable instrument in polyphasic taxonomic studies; more importantly, the cumulative data for about 1000 Bcc isolates analysed demonstrate that the 3 % concatenated sequence divergence level correlates with the 70 % DNA-DNA hybridization or 95 % whole-genome ANI threshold levels for species delineation.

ABSTRACT Burkholderia multivorans is a prominent B. cepacia complex (BCC) species causing infection in people with cystic fibrosis. Despite infection control measures being introduced to reduce the spread of BCC there is a continued emergence of infections by B. multivorans . Our objective was to analyze a global collection of B. multivorans isolates, comparing those from environmental and clinical sources with those from reported outbreaks. Multilocus sequence typing (MLST) was performed on 107 B. multivorans isolates to provide a detailed analysis of the global population biology of this species. MLST resolved 64 B. multivorans sequence types. Twelve of these were globally distributed and associated with human infection; two of these (ST-21 and ST-375) were also composed of environmental isolates. These global lineages included strains previously linked to large outbreaks (e.g., French epidemic clone ST-16). Though few environmental isolates of B. multivorans were available for analysis, of six strains identified, three were identical to strains recovered from cystic fibrosis (CF) infection. Although the ability of B. multivorans to cause CF outbreaks is known, our report here concerning the existence of globally distributed B. multivorans CF strains is a new observation for this emerging B. cepacia complex pathogen and suggests that certain strain types may be better adapted to human infection than others. Common transmission-associated risk factors were not obviously linked to the globally distributed strains; however, the overlap in strains recovered from water environments, industrial products, and human infection suggests that environmental sources may be an important reservoir for infection with B. multivorans .

ABSTRACT We demonstrate that all nine species of the Burkholderia cepacia complex can express the mucoid phenotype. A survey of clinical isolates showed that strains of B. cenocepacia , the most virulent species of the complex, are most frequently nonmucoid. Additionally, isolates from patients with chronic infections can convert from mucoid to nonmucoid.

The taxonomic position of five recA gene clusters of Burkholderia cepacia complex (Bcc) isolates was determined using a polyphasic taxonomic approach. The levels of 16S rRNA and recA gene sequence similarity, multilocus sequence typing (MLST) data and the intermediate DNA-DNA binding values demonstrated that these five clusters represented five novel species within the Bcc. Biochemical identification of these species is difficult, as is the case for most Bcc species. However, identification of these novel species can be accomplished through recA gene sequence analysis, MLST and BOX-PCR profiling and by recA RFLP analysis. For diagnostic laboratories, recA gene sequence analysis offers the best combination of accuracy and simplicity. Based on these results, we propose five novel Bcc species, Burkholderia latens sp. nov. (type strain FIRENZE 3(T) =LMG 24064(T) =CCUG 54555(T)), Burkholderia diffusa sp. nov. (type strain AU1075(T) =LMG 24065(T) =CCUG 54558(T)), Burkholderia arboris sp. nov. (type strain ES0263A(T) =LMG 24066(T) =CCUG 54561(T)), Burkholderia seminalis sp. nov. (type strain AU0475(T) =LMG 24067(T) =CCUG 54564(T)) and Burkholderia metallica sp. nov. (type strain AU0553(T) =LMG 24068(T) =CCUG 54567(T)). In the present study, we also demonstrate that Burkholderia ubonensis should be considered a member of the Bcc.

Members of the Burkholderia cepacia complex (Bcc), found in many environments, are associated with clinical infections. Examining diverse species and strains from different environments with multilocus sequence typing, we identified >20% of 381 clinical isolates as indistinguishable from those in the environment. This finding links the natural environment with the emergence of many Bcc infections.

Introduction: Infection with the Burkholderia cepacia complex is an important cause of morbidity and mortality in cystic fibrosis (CF). We investigated the molecular clock speed of the seven genes used in the multilocus sequence typing (MLST) scheme for these bacteria.Methods: At least two isolates, separated by months to years, from each of 20 patients were typed using MLST. In total 41 isolates, providing 128 isolate-years, were analyzed. Mutation and recombination rates were estimated assuming a Poisson distribution.Results: Out of 20 patients, 15 had no change in sequence type over time (mean 7.07 years, range 1.09 to 14.24). One patient had strain replacement. Three patients had evidence of recombination involving one of the seven housekeeping genes, and one patient had evidence of recombination of two genes. The mutation rate was estimated as 2.36 x 10(-6) per nucleotide per year (50% confidence limit) and 1.02 x 10(-5) per nucleotide per year (upper 95% confidence limit). The rate of nucleotide changes due to recombination events was estimated as 0.676 to 0.839 per year (95% confidence limits).Conclusions: B. cepacia complex housekeeping genes have a slow molecular clock speed and MLST provides a robust and reliable typing technique for isolates from this complex. A low rate of point mutation was found, with a higher rate of recombination events, in keeping with previous cross-sectional epidemiological data. The study also demonstrated, for the first time, recombination in a longitudinal in vivo study. (C) 2006 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

OBJECTIVE:To determine whether contaminated ultrasound gel is the source of intermittent outbreaks of nosocomial infection due to Burkholderia cepacia complex in patients without cystic fibrosis since 1992.DESIGN:A prospective clinical and in vitro study of all in-use bottles of ultrasound gel, as well as a retrospective analysis of archived bacterial strains, were performed. Handling of gel for clinical purposes throughout the hospital was evaluated. Gel and archived clinical isolates of B. cepacia complex were speciated to genomovar level and characterized by pulsed-field gel electrophoresis, and the pulsed-field gel electrophoresis patterns were compared.SETTING:The Hospital for Sick Children, a 300-bed, tertiary care, pediatric academic health sciences center in Toronto, Canada.PATIENTS:All patients without cystic fibrosis from whom B. cepacia complex was recovered at the Hospital for Sick Children since 1992.RESULTS:No standardized protocol for storage or handling of ultrasound gel was found. Gel from 39% of bottles grew either B. cepacia (genomovar I) or Burkholderia stabilis (genomovar IV). These isolates had pulsed-field gel electrophoresis patterns identical to 2 of the 7 clinical pulsed-field gel electrophoresis types that are responsible for 88% of clinical isolates.CONCLUSIONS:Contaminated ultrasound gel contributed to nosocomial infection due to B. cepacia complex in this institution over the course of 10 years. Suggested guidelines for the handling of ultrasound gel are provided.

BACKGROUND:Colonisation with Burkholderia cepacia complex in patients with cystic fibrosis (CF) has been associated with adverse outcomes. The aim of the present study was to determine the actuarial survival of CF patients colonized with B. cepacia and to evaluate the efficacy of the Royal Prince Alfred Hospital segregation policy. A secondary aim was to characterize the specific genomovars and strains of B. cepacia isolated in an Australian clinic. METHODS:Retrospective review of spirometric and microbiological data on all patients colonized with B. cepacia. Each B. cepacia-colonized subject was matched with three case-control subjects. Phenotype and genomovar typing, random amplified polymorphic DNA strain type and B. cepacia epidemic strain marker analyses were performed. The effect of B. cepacia colonization on transplant-free survival was estimated by Cox's proportional hazards regression using the entire clinic population. RESULTS:Fifteen patients were colonized with B. cepacia, of whom six (40%) had died from CF-related disease by August 1998, compared with 30 of 173 (17.3%) of the entire clinic population. Cepacia status had a significant adverse effect on survival, with a hazard ratio of 2.16 (95% confidence interval 1.0-4.69; P = 0.05). The outcome was variable in subgroups of B. cepacia. DISCUSSION:Colonization with B. cepacia had a significant adverse effect on survival within the study population. Genomovar and strain typing contributed usefully in accessing the effectiveness of the hospital's segregation policy in preventing cross-colonization.

Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar (Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia-like bacteria.

Pseudomonas aeruginosa is the most common respiratory pathogen in patients with cystic fibrosis (CF), but the predominant mechanism by which it is acquired is controversial. To determine the frequency of patient-to-patient spread, we evaluated P. aeruginosa isolates from 174 patients treated at the CF clinics in Vancouver, BC, Canada, since 1981. Multiple isolates were obtained from each patient and genetically typed by random amplified polymorphic DNA and pulsed field gel electrophoresis analyses. A total of 157 genetic types of P. aeruginosa was identified, 123 of which were unique to individual patients. A total of 34 types was shared by more than one patient; epidemiologic evidence linked these individuals only in the cases of 10 sibships and 1 pair of unrelated patients. We conclude that there is an extremely low risk in Vancouver for patients with CF to acquire P. aeruginosa from other patients. It appears that prolonged close contact, such as occurs between siblings, is necessary for patient-to-patient spread. The major source of acquisition of P. aeruginosa in CF appears to be from the environment. Considering these observations, we do not recommend segregation of patients with CF on the basis of their colonization status with P. aeruginosa.