Deborah A. Henry

Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov.

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.

Epidemiology of Burkholderia cepacia complex in patients with cystic fibrosis, Canada

The Burkholderia cepacia complex is an important group of pathogens in patients with cystic fibrosis (CF). Although evidence for patient-to-patient spread is clear, microbial factors facilitating transmission are poorly understood. To identify microbial clones with enhanced transmissibility, we evaluated B. cepacia complex isolates from patients with CF from throughout Canada. A total of 905 isolates from the B. cepacia complex were recovered from 447 patients in 8 of the 10 provinces; 369 (83%) of these patients had genomovar III and 43 (9.6%) had B. multivorans (genomovar II). Infection prevalence differed substantially by region (22% of patients in Ontario vs. 5% in Quebec). Results of typing by random amplified polymorphic DNA analysis or pulsed-field gel electrophoresis indicated that strains of B. cepacia complex from genomovar III are the most potentially transmissible and that the B. cepacia epidemic strain marker is a robust marker for transmissibility.

Infection with Burkholderia cepacia Complex Genomovars in Patients with Cystic Fibrosis: Virulent Transmissible Strains of Genomovar III Can Replace Burkholderia multivorans

Infection with Burkholderia cepacia complex in patients with cystic fibrosis (CF) results in highly variable clinical outcomes. The purpose of this study was to determine if there are genomovar-specific disparities in transmission and disease severity. B. cepacia complex was recovered from 62 patients with CF on > or =1 occasions (genomovar III, 46 patients; genomovar II [B. multivorans], 19 patients; genomovar IV [B. stabilis], 1 patient; genomovar V [B. vietnamiensis], 1 patient; and an unclassified B. cepacia complex strain, 1 patient). Patient-to-patient spread was observed with B. cepacia genomovar III, but not with B. multivorans. Genomovar III strains replaced B. multivorans in 6 patients. Genomovar III strains were also associated with a poor clinical course and high mortality. Infection control practices should be designed with knowledge about B. cepacia complex genomovar status; patients infected with transmissible genomovar III strains should not be cohorted with patients infected with B. multivorans and other B. cepacia genomovars.

Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates.

A polyphasic taxonomic study, including amplified fragment length polymorphism (AFLP) fingerprinting, DNA-DNA hybridizations, DNA base-ratio determinations, phylogenetic analysis, whole-cell fatty acid analyses and an extensive biochemical characterization, was performed on 19 Burkholderia cepacia-like isolates from the environment and cystic fibrosis (CF) patients. Several of the environmental isolates have attracted considerable interest due to their biocontrol properties. The polyphasic taxonomic data showed that the strains represent a new member of the B. cepacia complex, for which the name Burkholderia ambifaria sp. nov. is proposed. The type strain is strain LMG 19182T. B. ambifaria can be differentiated from the other members of the B. cepacia complex by means of AFLP fingerprinting, whole-cell fatty acid analysis, biochemical tests (including ornithine and lysine decarboxylase activity, acidification of sucrose and beta-haemolysis) and a newly developed recA gene-based PCR assay. 16S rDNA-based RFLP analysis and PCR tests allowed differentiation of B. ambifaria from Burkholderia multivorans, Burkholderia vietnamiensis and B. cepacia genomovar VI, but not from B. cepacia genomovars I and III and Burkholderia stabilis. The finding that this new taxon includes both strains isolated from CF patients and potentially useful biocontrol strains supports the general consensus that the large-scale use of biocontrol strains belonging to the B. cepacia complex would be ill-advised until more is known about their potential pathogenic mechanisms.

Burkholderia latens sp nov., Burkholderia diffusa sp nov., Burkholderia arboris sp nov., Burkholderia seminalis sp nov and Burkholderia metallica sp nov., novel species within the Burkholderia cepacia complex

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.

Burkholderia anthina sp. nov. and Burkholderia pyrrocinia, two additional Burkholderia cepacia complex bacteria, may confound results of new molecular diagnostic tools

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.

Phenotypic Methods for Determining Genomovar Status of the Burkholderia cepacia Complex

ABSTRACT Recent taxonomic advances have demonstrated that Burkholderia cepacia is a cluster of at least seven closely related genomic species (or genomovars) collectively referred to as the B. cepacia complex, all of which may cause infections among cystic fibrosis patients and other vulnerable individuals. Thus, it is important for clinical microbiologists to be able to differentiate genomovars. Prior to this study, 361 B. cepacia complex isolates and 51 isolates easily confused with B. cepaciacomplex previously had been identified using a polyphasic approach, and in this study, a comparison of phenotypic and biochemical tests was carried out. It was determined that Burkholderia multivorans and Burkholderia stabilis could reliably be separated from other members of the B. cepacia complex by phenotypic methods. A combination of phenotypic and molecular tests such as recA PCR and 16S rRNA RFLP are recommended for differentiation among the genomovars of the B. cepaciacomplex. A biochemical reaction scheme for the identification ofB. gladioli, Pandoraea species, and Ralstonia pickettii and the differentiation of these species from theB. cepacia complex is also presented.

Environmental Burkholderia cepacia complex isolates in human infections

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.

Differential Mucoid Exopolysaccharide Production by Members of the Burkholderia cepacia Complex

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.

Persistent Bacteremia and Severe Thrombocytopenia Caused by Coagulase-Negative Staphylococcus in a Neonatal Intensive Care Unit

OBJECTIVE. Coagulase-negative Staphylococcus (CoNS) is the most frequent cause of late-onset sepsis in NICUs, but mortality is rare and morbidity is unusual. We report a new syndrome of CoNS sepsis characterized by significant morbidity and persistent bacteremia despite aggressive antibiotic therapy and no identified focus of infection. METHODS. We conducted a retrospective review of infants in the NICU with CoNS bacteremia between 2000 and 2002. Statistical analysis included an initial exploratory analysis followed by logistic regression. Microbiological identification of all isolates and molecular typing were performed. RESULTS. Thirty-one neonates with persistent CoNS bacteremia were compared with 60 randomly selected neonates from a group of 140 with nonpersistent CoNS bacteremia. The clinical manifestations at presentation, gestational ages, and birth weights were similar in the 2 groups. Thrombocytopenia was present in 26 (84%) neonates with persistent CoNS bacteremia but only in 8 (13%) neonates in the nonpersistent group. Central venous catheterization increased the risk for persistent CoNS bacteremia, but 42% of the persistent group was never catheterized. Staphylococcus epidermidis was the most common isolate in both groups. Molecular typing failed to identify a predominant clone. CONCLUSIONS. The syndrome of persistent CoNS septicemia is remarkable for thrombocytopenia and persistence in the absence of central venous catheterization. Clinical manifestations at presentation and demographic characteristics did not discriminate between the persistent and nonpersistent groups. We did not identify the emergence of a particularly virulent clone, but it is possible that some strains of CoNS have acquired the capacity to persist under different conditions.