Robust asymmetric synthesis of unnatural alkenyl amino acids for conformationally constrained α-helix peptides.

The efficient asymmetric synthesis of unnatural alkenyl amino acids required for peptide 'stapling' has been achieved using alkylation of a fluorine-modified Ni(II) Schiff base complex as the key step.

Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria.

Bacteria from the Burkholderia cepacia complex (Bcc) are encountered as industrial contaminants, and little is known about the species involved or their mechanisms of preservative resistance. Multilocus sequence typing (MLST) revealed that multiple Bcc species may cause contamination, with B. lata (n = 17) and B. cenocepacia (n = 11) dominant within the collection examined. At the strain level, 11 of the 31 industrial sequence types identified had also been recovered from either natural environments or clinical infections. Minimal inhibitory (MIC) and minimum bactericidal (MBC) preservative concentrations varied across 83 selected Bcc strains, with industrial strains demonstrating increased tolerance for dimethylol dimethyl hydantoin (DMDMH). Benzisothiazolinone (BIT), DMDMH, methylisothiazolinone (MIT), a blend of 3:1 methylisothiazolinone-chloromethylisothiazolinone (M-CMIT), methyl paraben (MP), and phenoxyethanol (PH), were all effective anti-Bcc preservatives; benzethonium chloride (BC) and sodium benzoate (SB) were least effective. Since B. lata was the dominant industrial Bcc species, the type strain, 383(T) (LMG 22485(T)), was used to study preservative tolerance. Strain 383 developed stable preservative tolerance for M-CMIT, MIT, BIT, and BC, which resulted in preservative cross-resistance and altered antibiotic susceptibility, motility, and biofilm formation. Transcriptomic analysis of the B. lata 383 M-CMIT-adapted strain demonstrated that efflux played a key role in its M-CMIT tolerance and elevated fluoroquinolone resistance. The role of efflux was corroborated using the inhibitor l-Phe-Arg-ß-napthylamide, which reduced the MICs of M-CMIT and ciprofloxacin. In summary, intrinsic preservative tolerance and stable adaptive changes, such as enhanced efflux, play a role in the ability of Bcc bacteria to cause industrial contamination.

Multilocus sequence typing identifies evidence for recombination and two distinct lineages of Corynebacterium diphtheriae.

We describe the development of a multilocus sequence typing (MLST) scheme for Corynebacterium diphtheriae, the causative agent of the potentially fatal upper respiratory disease diphtheria. Global changes in diphtheria epidemiology are highlighted by the recent epidemic in the former Soviet Union (FSU) and also by the emergence of nontoxigenic strains causing atypical disease. Although numerous techniques have been developed to characterize C. diphtheriae, their use is hindered by limited portability and, in some instances, poor reproducibility. One hundred fifty isolates from 18 countries and encompassing a period of 50 years were analyzed by multilocus sequence typing (MLST). Strain discrimination was in accordance with previous ribotyping data, and clonal complexes associated with disease outbreaks were clearly identified by MLST. The data produced are portable, reproducible, and unambiguous. The MLST scheme described provides a valuable tool for monitoring and characterizing endemic and epidemic C. diphtheriae strains. Furthermore, multilocus sequence analysis of the nucleotide data reveals two distinct lineages within the population of C. diphtheriae examined, one of which is composed exclusively of biotype belfanti isolates and the other of multiple biotypes.

Novel Corynebacterium diphtheriae in domestic cats.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

Oxidative stress of Burkholderia cenocepacia induces insertion sequence-mediated genomic rearrangements that interfere with macrorestriction-based genotyping.

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.

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes.

BACKGROUND: The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East. RESULTS: This scheme uses 7 loci; atpD, fusA, glnS, gltB, gyrB, infB, and pps. There were 12 sequence types (ST) identified in C. sakazakii, and 3 in C. malonaticus. A third (22/60) of C. sakazakii strains were in ST4, which had almost equal numbers of clinical and infant formula isolates from 1951 to 2008. ST8 may represent a particularly virulent grouping of C. sakazakii as 7/8 strains were clinical in origin which had been isolated between 1977 - 2006, from four countries. C. malonaticus divided into three STs. The previous Cronobacter biotyping scheme did not clearly correspond with STs nor with species. CONCLUSION: In conclusion, MLST is a more robust means of identifying and discriminating between C. sakazakii and C. malonaticus than biotyping. The MLST database for these organisms is available online at http://pubmlst.org/cronobacter/.

Expanded multilocus sequence typing for burkholderia species.

PCR primers targeting loci in the current Burkholderia cepacia complex multilocus sequence typing scheme were redesigned to (i) more reliably amplify these loci from B. cepacia complex species, (ii) amplify these same loci from additional Burkholderia species, and (iii) enable the use of a single primer set per locus for both amplification and DNA sequencing.

Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex.

Members of the genus Burkholderia are known for their ability to suppress soil-borne fungal pathogens by the production of various antibiotic compounds. In this study we investigated the role of N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) in the expression of antifungal traits. Using a quorum quenching approach, that is, by heterologous expression of the Bacillus sp. AiiA lactonase, we show that expression of antifungal activities is AHL-dependent in the large majority of the investigated strains belonging to various Burkholderia species. We demonstrate that in certain strains of Burkholderia ambifaria, Burkholderia pyrrocinia and Burkholderia lata, one of the QS-regulated antifungal agents is pyrrolnitrin (prn), a common broad-spectrum antibiotic that is also produced by some Pseudomonas and Serratia species. To investigate the underlying molecular mechanisms of AHL-dependent prn production in better detail, we inactivated the AHL synthase cepI as well as cepR, which encodes the cognate AHL receptor protein, in B. lata 383. Both QS mutants no longer produced prn as assessed by gas chromatography-mass spectrometry analysis and as a consequence were unable to inhibit growth of Rhizoctonia solani. Using fusions of the lacZ gene to the promoter of the prnABCD operon, which directs the synthesis of prn, we demonstrate that expression of prn is positively regulated by CepR at the level of transcription.

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.

Biocide susceptibility of the Burkholderia cepacia complex.

OBJECTIVES: The Burkholderia cepacia complex (Bcc) species are important opportunistic pathogens with intrinsic antibiotic resistance. They are also well known as contaminants of disinfectants, yet their biocide susceptibility has not been studied in detail. We investigated Bcc biocide susceptibility and correlated it to their taxonomy, antibiotic susceptibility and ability to form biofilms. METHODS: Genetically distinct Bcc strains belonging to 12 of the defined species were examined. Biocide susceptibility was assessed by (i) broth dilution MIC assays, (ii) agar growth-based MBC screens and (iii) suspension tests. Antibiotic MIC was determined by Etest strips, and the ability to form biofilms was examined in a 96-well plate assay. RESULTS: Biocide susceptibility varied across the Bcc complex with high MIC recorded for chlorhexidine (>100 mg/L), cetylpyridinium chloride (>200 mg/L), triclosan (>500 mg/L), benzalkonium chloride (>400 mg/L) and povidone (>50 000 mg/L). Species-dependent differences were apparent only for cetylpyridinium chloride. There was no correlation between biocide susceptibility and (i) antibiotic susceptibility or (ii) the ability to form biofilms. Biocide MBC was considerably higher than the MIC (chlorhexidine, 6-fold greater; cetylpyridinium chloride, 20-fold greater). Cystic fibrosis outbreak strains (Burkholderia multivorans Glasgow strain and Burkholderia cenocepacia ET12) possessed elevated chlorhexidine resistance, and Bcc bacteria were also shown to remain viable in current commercial biocide formulations. CONCLUSIONS: Bcc bacteria are resistant to a wide range of biocides and further representatives of this group should be included as reference strains in the development of new anti-infectives and commercial formulations.

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients.

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.

Bayesian modeling of recombination events in bacterial populations.

BACKGROUND: We consider the discovery of recombinant segments jointly with their origins within multilocus DNA sequences from bacteria representing heterogeneous populations of fairly closely related species. The currently available methods for recombination detection capable of probabilistic characterization of uncertainty have a limited applicability in practice as the number of strains in a data set increases. RESULTS: We introduce a Bayesian spatial structural model representing the continuum of origins over sites within the observed sequences, including a probabilistic characterization of uncertainty related to the origin of any particular site. To enable a statistically accurate and practically feasible approach to the analysis of large-scale data sets representing a single genus, we have developed a novel software tool (BRAT, Bayesian Recombination Tracker) implementing the model and the corresponding learning algorithm, which is capable of identifying the posterior optimal structure and to estimate the marginal posterior probabilities of putative origins over the sites. CONCLUSION: A multitude of challenging simulation scenarios and an analysis of real data from seven housekeeping genes of 120 strains of genus Burkholderia are used to illustrate the possibilities offered by our approach. The software is freely available for download at URL http://web.abo.fi/fak/mnf//mate/jc/software/brat.html.

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.

Diversity of the parB and repA genes of the Burkholderia cepacia complex and their utility for rapid identification of Burkholderia cenocepacia.

BACKGROUND: Burkholderia cenocepacia is the most prominent species of the B. cepacia complex (Bcc), a group of nine closely related and difficult to identify bacteria that cause serious infections in patients with cystic fibrosis. Despite its clinical relevance, identification of B. cenocepacia as a single species is unavailable, as it splits by a widely used recA gene-based PCR identification method into discrete phylogenetic subgroups IIIA, IIIB, IIIC and IIID. With the aim of identifying gene targets suitable for unified detection of B. cenocepacia strains, we examined sequence polymorphisms in the repA and parB genes. These essential genes are involved in the replication and partitioning of bacterial replicons, hence we also had the opportunity for the first time to investigate the evolution of the multireplicon (three chromosome) structure of Bcc genomes. RESULTS: Alignment of the repA and parB genes from publicly available Bcc genome sequences enabled the design of primers for their amplification and sequence analysis. Multilocus sequencing typing, a highly discriminatory method for Bcc species and strain discrimination, was used to select strains of unique sequence types (STs) that spanned the known Bcc genetic diversity. Sequence datasets of repA (83 isolates, 67 STs) and parB (120 isolates, 95 STs) genes from the second chromosome were aligned and examined phylogenetically to identify polymorphisms suitable for identification of B. cenocepacia. In contrast to parB, the Bcc repA sequences demonstrated distinct clustering of B. cenocepacia from other species, which enabled the design a species-specific multiplex PCR. The novel single-reaction B. cenocepacia detection method was tested on a panel of 142 different Bcc strains (142 STs) and distinguished recA groups IIIA, IIIB and IIID, from all other Bcc members with 100% sensitivity and 93% specificity. CONCLUSION: The repA-based multiplex PCR is a useful aid to the rapid identification of the most clinically relevant B. cenocepacia recA subgroups IIIA, IIIB and IIID. Phylogenetic analysis of repA and parB genes demonstrated that acquisition of the second and third replicons of Bcc genomes occurred prior to their differentiation into discrete species and that the sharing of replicons across species had not occurred.

Elucidating global epidemiology of Burkholderia multivorans in cases of cystic fibrosis by multilocus sequence typing.

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.

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing.

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.

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.

Reliability of multilocus sequence typing of the Burkholderia cepacia complex in cystic fibrosis.

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 2.36x10(-6) per nucleotide per year (50% confidence limit) and 1.02x10(-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.

Multilocus sequence typing breathes life into a microbial metagenome.

Shot-gun sequencing of DNA isolated from the environment and the assembly of metagenomes from the resulting data has considerably advanced the study of microbial diversity. However, the subsequent matching of these hypothetical metagenomes to cultivable microorganisms is a limitation of such cultivation-independent methods of population analysis. Using a nucleotide sequence-based genetic typing method, multilocus sequence typing, we were able for the first time to match clonal cultivable isolates to a published and controversial bacterial metagenome, Burkholderia SAR-1, which derived from analysis of the Sargasso Sea. The matching cultivable isolates were all associated with infection and geographically widely distributed; taxonomic analysis demonstrated they were members of Burkholderia cepacia complex Group K. Comparison of the Burkholderia SAR-1 metagenome to closely related B. cepacia complex genomes indicated that it was greater than 98% intact in terms of conserved genes, and it also shared complete sequence identity with the cultivable isolates at random loci beyond the genes sampled by the multilocus sequence typing. Two features of the extant cultivable clones support the argument that the Burkholderia SAR-1 sequence may have been a contaminant in the original metagenomic survey: (i) their growth in conditions reflective of sea water was poor, suggesting the ocean was not their preferred habitat, and (ii) several of the matching isolates were epidemiologically linked to outbreaks of infection that resulted from contaminated medical devices or products, indicating an adaptive fitness of this bacterial strain towards contamination-associated environments. The ability to match identical cultivable strains of bacteria to a hypothetical metagenome is a unique feature of nucleotide sequence-based microbial typing methods; such matching would not have been possible with more traditional methods of genetic typing, such as those based on pattern matching of genomic restriction fragments or amplified DNA fragments. Overall, we have taken the first steps in moving the status of the Burkholderia SAR-1 metagenome from a hypothetical entity towards the basis for life of cultivable strains that may now be analysed in conjunction with the assembled metagenomic sequence data by the wider scientific community.

Multilocus sequence typing scheme that provides both species and strain differentiation for the Burkholderia cepacia complex.

A single multilocus sequence typing (MLST) scheme was developed for precise characterization of the opportunistic pathogens of Burkholderia cepacia complex (BCC), a group composed of at least nine closely related species. Seven conserved housekeeping genes were selected after a comparison of five Burkholderia species, and a collection of strains was subjected to nucleotide sequence analysis using a nested PCR amplification approach for each gene. MLST differentiated all nine current BCC species and identified 114 sequence types within a collection of 119 strains. No differentiation was found between strains recovered from environmental or clinical sources. The improved resolution in strain identification offered by MLST was able to identify previously characterized epidemic strain lineages and also demonstrated the presence of four novel potential species groups within the complex. There was also evidence for recombination having an important role in the recent evolution of individual BCC species. This highly transferable, validated, MLST scheme provides a new means to assist in species identification as well as unambiguous strain discrimination of the BCC by a single approach. It is also the first MLST scheme designed at the outset to incorporate multiple species and should facilitate global epidemiological investigations of the BCC.