Peter Vandamme

Re-evaluating prokaryotic species

There is no widely accepted concept of species for prokaryotes, and assignment of isolates to species is based on measures of phenotypic or genome similarity. The current methods for defining prokaryotic species are inadequate and incapable of keeping pace with the levels of diversity that are being uncovered in nature. Prokaryotic taxonomy is being influenced by advances in microbial population genetics, ecology and genomics, and by the ease with which sequence data can be obtained. Here, we review the classical approaches to prokaryotic species definition and discuss the current and future impact of multilocus nucleotide-sequence-based approaches to prokaryotic systematics. We also consider the potential, and difficulties, of assigning species status to biologically or ecologically meaningful sequence clusters.

Cutting a gordian knot: Emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom nov (basonym, Cytophaga aquatilis Strohl and Tait 1978).

The phylogenetic positions and G+C contents of most species belonging to the genera Flavobacterium, Cytophaga, and Flexibacter and several related taxa were determined. Most of the strains included in this study belong to rRNA superfamily V, as shown by DNA-rRNA hybridization data, but the three main genera are highly polyphyletic. Several so-called Cytophaga and Flexibacter species isolated from soil and freshwater cluster with the type species of the genus Flavobacterium, Flavobacterium aquatile, and with Flavobacterium branchiophilum. The fatty acid and protein profiles of members of this group of organisms were determined. We provide an emended description of the genus Flavobacterium and propose new combinations for the following 7 of the 10 validly described species included in this genus: Flavobacterium columnare, Flavobacterium flevense, Flavobacterium johnsoniae (we also correct the specific epithet of this taxon), Flavobacterium pectinovarum, Flavobacterium psychrophilum, Flavobacterium saccharophilum, and Flavobacterium succinicans. A new name, Flavobacterium hydatis, is proposed for [Cytophaga] aquatilis Strohl and Tait 1978. The emended genus Flavobacterium contains bacteria that have the following main characteristics: gram-negative rods that are motile by gliding, produce yellow colonies on agar, are chemoorganotrophs and aerobes, decompose several polysaccharides but not cellulose, and are widely distributed in soil and freshwater habitats. Three Flavobacterium species are pathogenic for fish. The G+C contents of Flavobacterium DNAs range from 32 to 37 mol%. An emended description of the family Flavobacteriaceae is also provided.

Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov.

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Ralstonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relationships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which was identified as Burkholderia vietnamiensis. This group of five phenotypically similar species is referred to as the B. cepacia complex. The name Burkholderia multivorans is proposed for one of these genomic species, which was formerly referred to as B. cepacia genomovar II; the remaining B. cepacia groups are referred to as genomovars I, III, and IV, pending additional differential phenotypic tests. The role and pathogenic potential of each of these taxa, particularly in view of the potentially fatal infections in cystic fibrosis patients, need further evaluation. The data presented also demonstrate that Pseudomonas glathei and Pseudomonas pyrrocinia should be reclassified as Burkholderia species.

Dysbiosis of the faecal microbiota in patients with Crohn's disease and their unaffected relatives

Background and aims A general dysbiosis of the intestinal microbiota has been established in patients with Crohns disease (CD), but a systematic characterisation of this dysbiosis is lacking. Therefore the composition of the predominant faecal microbiota of patients with CD was studied in comparison with the predominant composition in unaffected controls. Whether dysbiosis is present in relatives of patients CD was also examined. Methods Focusing on families with at least three members affected with CD, faecal samples of 68 patients with CD, 84 of their unaffected relatives and 55 matched controls were subjected to community fingerprinting of the predominant microbiota using denaturing gradient gel electrophoresis (DGGE). To analyse the DGGE profiles, BioNumerics software and non-parametric statistical analyses (SPSS V.17.0) were used. Observed differences in the predominant microbiota were subsequently confirmed and quantified with real-time PCR. Results Five bacterial species characterised dysbiosis in CD, namely a decrease in Dialister invisus (p=0.04), an uncharacterised species of Clostridium cluster XIVa (p=0.03), Faecalibacterium prausnitzii (p<1.3×10−5) and Bifidobacterium adolescentis (p=5.4×10−6), and an increase in Ruminococcus gnavus (p=2.1×10−7). Unaffected relatives of patients with CD had less Collinsella aerofaciens (p=0.004) and a member of the Escherichia coli–Shigella group (p=0.01) and more Ruminococcus torques (p=0.02) in their predominant microbiota as compared with healthy subjects. Conclusion Unaffected relatives of patients with CD have a different composition of their microbiota compared with healthy controls. This dysbiosis is not characterised by lack of butyrate producing-bacteria as observed in CD but suggests a role for microorganisms with mucin degradation capacity.

New perspectives in the classification of the flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen-nov, and Empedobacter nom-rev.

Our present knowledge concerning the genotypic, chemotaxonomic, and phenotypic characteristics of members of the genus Flavobacterium and some related genera, including the genus Weeksella, was used to revise the classification of these organisms. The generically misclassified organisms Flavobacterium balustinum, Flavobacterium gleum, Flavobacterium indologenes, Flavobacterium indoltheticum, Flavobacterium meningosepticum, and Flavobacterium scophthalmum are included in a new genus, Chryseobacterium, with Chryseobacterium gleum as the type species. The generically misclassified organism Flavobacterium breve is included in the revived genus Empedobacter as Empedobacter brevis, whereas the generically misclassified organism Weeksella zoohelcum is included in the new genus Bergeyella as Bergeyella zoohelcum.

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.

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.

PCR-Based Assay for Differentiation of Pseudomonas aeruginosa from Other Pseudomonas Species Recovered from Cystic Fibrosis Patients

ABSTRACT Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.

Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy

Abstract A standardized method for amplified ribosomal DNA restriction analysis (ARDRA) is described. The first step involves selection of five tetracutter restriction enzymes on the basis of theoretical digestions of known 16S rDNA (rRNA) sequences. In the second step, the experimentally obtained restriction patterns are normalized and combined by means of the pattern recognition and analysis software GelCompar. Finally, numerical analysis allows the strains to be grouped according to the similarities in their combined ARDRA patterns. Results obtained with representatives of two phylogenetic lineages, the genera Alcaligenes and Bordetella and the genera Bacillus and Paenibacillus , are presented. In general, the clustering of the strains corresponded well with known species delineations and topology of phylogenetic groupings except for the discrepant position of the Bacillus lautus type strain, which can probably be explained by non-authenticity of this strain in one of the analyses. The effect of using less than five restriction enzymes on the clustering was evaluated. The frequent occurrence of interoperon variability of the 16S rRNA gene in Bacillus and Paenibacillus was also demonstrated. Because ARDRA detects interspecies and interstrain as well as interoperon variability and enables a relatively fast multiple strain analysis per taxon, this technique is appropriate to obtain indicative phylogenetic and taxonomic information. This information can be used to select strains for further detailed taxonomic studies. ARDRA fingerprinting also allows the construction of a database for indentification purposes.

Burkholderia cenocepacia sp. nov.--a new twist to an old story.

DNA-DNA hybridisation experiments between isolates representing Burkholderia cepacia genomovar III recA lineages IIIA and IIIB reinforced the classification of both phylogenetic subgroups as a single genospecies, distinct from B. cepacia (genomovar I). A formal classification of B. cepacia genomovar III encompassing the recA lineages IIIA and IIIB, and the new recA lineages IIIC and IIID, as B. cenocepacia sp. nov., with LMG 16656 as the type strain, is proposed.