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A repeat multi-locus sequence analysis (MLSA) of concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequences of strains classified as Achromobacter marplatensis was performed. The results revealed that earlier reported sequence data of the proposed type strain were erroneous, and that the corrected concatenated sequence divergence between the A. marplatensis LMG 26219T (=CCUG 56371T) sequence type and that of strains of Achromobacter spiritinus was well below the 2.1% threshold value that delineates species of the genus Achromobacter. These results therefore demonstrated that strains which were classified as A. spiritinus should be reclassified as A. marplatensis and that the name Achromobacter spiritinus should no longer be used. An emendation of the description of Achromobacter marplatensis is warranted.

The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA-DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845(T) [=CCUG 62426(T)] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852(T) [=CCUG 62438(T)] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857(T) [=CCUG 62444(T)] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840(T) [=CCUG 62421(T)] as the type strain).

The phenotypic and genotypic characteristics of a historical collection of strains identified as Achromobacter denitrificanswere examined. Sequence analysis of a 765 bp nrdA gene fragment revealed that eight of these strains belonged to the recently described Achromobacter aegrifaciens, Achromobacter mucicolens, and Achromobacter insolitus, and that one strain belonged to Achromobacter xylosoxidans. The analysis also suggested the presence of four novel species of the genus Achromobacter among the remaining strains. The latter was confirmed by multilocus sequence analysis of concatenated nusA, eno, rpoB, gltB, lepA, nuoL andnrdA gene fragments and extensive genotypic and phenotypic characterization. We propose to name these novel species as Achromobacter agilis sp. nov., nom. rev. (type strain LMG 3411T=CCUG 62454T), Achromobacter pestifer sp. nov., nom. rev. (type strain LMG 3431T=CCUG 61959T) , Achromobacter kerstersii sp. nov. (type strain LMG 3441T=CCUG 62449T) and Achromobacter deleyi sp. nov. (type strain LMG 3458T=CCUG 62433T).

Nine Burkholderia cepacia complex (Bcc) bacteria were isolated during environmental surveys for the ecological niche of Burkholderia pseudomallei, the aetiological agent of melioidosis, in the Northern Territory of Australia. They represented two multi-locus sequence analysis-based clusters, referred to as Bcc B and Bcc L. Three additional environmental and clinical Bcc B isolates were identified upon deposition of the sequences in the PubMLST database. Analysis of the concatenated nucleotide sequence divergence levels within both groups (1.4 and 1.9 %, respectively) and towards established Bcc species (4.0 and 3.9 %, respectively) demonstrated that the two taxa represented novel Bcc species. All 12 isolates were further characterized using 16S rRNA and recA gene sequence analysis, RAPD analysis, DNA base content determination, fatty acid methyl ester analysis and biochemical profiling. Analysis of recA gene sequences revealed a remarkable diversity within each of these taxa, but, together, the results supported the affiliation of the two taxa to the Bcc. Bcc B strains can be differentiated from most other Bcc members by the assimilation of maltose. Bcc L strains can be differentiated from other Bcc members by the absence of assimilation of N-acetylglucosamine. The names Burkholderia stagnalis sp. nov. with type strain LMG 28156T ( = CCUG 65686T) and Burkholderia territorii sp. nov. with type strain LMG 28158T ( = CCUG 65687T) are proposed for Bcc B and Bcc L bacteria, respectively.

The phenotypic and genotypic characteristics of four Bordetella hinzii-like strains from human respiratory specimens and representing nrdA gene sequence based genogroups 3, 14 and 15 were examined. In a 16S rRNA gene sequence based phylogenetic tree, the four strains consistently formed a single coherent lineage but their assignment to the genus Bordetella was equivocal. The respiratory quinone, polar lipid and fatty acid profiles generally conformed to those of species of the genus Bordetella and were characterized by the presence of ubiquinone 8, of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several aminolipids, and of high percentages of C16 : 0, cyclo-C17 : 0 and summed feature 2, as major chemotaxonomic marker molecules, respectively. The DNA G+C content was about 66 mol%, which corresponded with that of the high-percentage DNA G+C content genera of the family Alcaligenaceae including the genus Bordetella. DNA–DNA hybridization experiments revealed the presence of three distinct genomospecies and thus confirmed phenotypic differences as revealed by means of extensive biochemical characterization. We therefore propose to formally classify Bordetella genogroups 3, 14 and 15 as Bordetella bronchialis sp. nov. (type strain LMG 28640T = AU3182T = CCUG 56828T), Bordetella sputigena sp. nov. (type strain LMG 28641T = CCUG 56478T) and Bordetella flabilis sp. nov. (type strain LMG 28642T = AU10664T = CCUG 56827T). In addition, we propose to reclassify Achromobacter sediminum into the novel genus Verticia, as Verticia sediminum, gen. nov., comb. nov., on the basis of its unique phylogenetic position, its marine origin and its distinctive phenotypic, fatty acid and polar lipid profile.

Two new PCR assays (for Ralstonia species and Ralstonia respiraculi ), together with previously published PCR assays, were used to assess Ralstonia isolates recovered from 111 cystic fibrosis patients. Ralstonia mannitolilytica accounted for 46% of isolates, while R. respiraculi and Ralstonia pickettii accounted for 19% and 18%, respectively. Ralstonia basilensis and Ralstonia metallidurans , species not previously recovered from human samples, were also identified.

Persons with cystic fibrosis (CF) are susceptible to chronic pulmonary infection due to certain Burkholderia species, but it is not clear whether this typically involves persistent infection with the same strain or sequential infection with distinct strains. We analyzed 1095 Burkholderia isolates recovered from serial sputum cultures from 379 patients with CF receiving care in 112 CF treatment centers in the United States. Genotyping was performed by random amplified polymorphic DNA typing or pulsed-field gel electrophoresis. Overall, a change in infecting strain was found in 24 (6.9%) of 347 patients infected with Burkholderia cepacia complex and in 3 (9%) of 32 patients infected with Burkholderia gladioli. Several patients were likely coinfected, at least transiently, with >1 B. cepacia complex strain. The potential for strain replacement during chronic infection may confound studies of the relationship between strain and clinical outcome and must be considered in designing effective infection-control practices.

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii , Burkholderia fungorum , Comamonas testosteroni , Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis , Pandoraea genomospecies 4, Ralstonia gilardii , Ralstonia mannitolilytica , Rhizobium radiobacter , and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the α- Proteobacteria . Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family Enterobacteriaceae . The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

Standard microbiology references describe Stenotrophomonas maltophilia as oxidase negative and variable with respect to utilization of lactose and sucrose. Analysis of a collection of 766 S. maltophilia isolates indicated that approximately 20% are oxidase positive and that this species should be reevaluated for other phenotypes, including oxidative fermentation of lactose and sucrose.

We analyzed Burkholderia cepacia complex isolates recovered from 1,218 cystic fibrosis (CF) patients and 90 patients without CF. Although all B. cepacia complex species were found, some were rarely identified. The distribution of species differed between the CF and non-CF populations and appears to be changing over time among CF patients.

Analysis of partial gyrB gene sequences revealed six taxa in a group of 17 Burkholderia glathei -like isolates which were further examined by (GTG)5-PCR fingerprinting, 16S rRNA gene sequence analysis, DNA–DNA hybridizations, determination of the DNA G+C content, whole-cell fatty acid analysis and an analysis of cell and colony morphology and more than 180 biochemical characteristics. The results demonstrated that one taxon consisting of three human clinical isolates represented Burkholderia zhejiangensis , a recently described methyl-parathion-degrading bacterium isolated from a wastewater-treatment system in China. The remaining taxa represented five novel species isolated from soil or rhizosphere soil samples, and could be distinguished by both genotypic and phenotypic characteristics. We therefore propose to formally classify these bacteria as Burkholderia humi sp. nov. (type strain, LMG 22934T = CCUG 63059T), Burkholderia choica sp. nov. (type strain, LMG 22940T = CCUG 63063T), Burkholderia telluris sp. nov. (type strain, LMG 22936T = CCUG 63060T), Burkholderia udeis sp. nov. (type strain, LMG 27134T = CCUG 63061T) and Burkholderia terrestris sp. nov. (type strain, LMG 22937T = CCUG 63062T).

Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883(T) (=CCUG 62895(T)). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42°C, acidification of sucrose and adonitol, lysine decarboxylase and β-galactosidase activity, and esculin hydrolysis.

The phenotypic and genotypic characteristics of fourteen human clinical Achromobacter strains representing four genogroups which were delineated by sequence analysis of nusA, eno, rpoB, gltB, lepA, nuoL and nrdA loci, demonstrated that they represent four novel Achromobacter species. The present study also characterized and provided two additional reference strains for Achromobacter ruhlandii and Achromobacter marplatensis, species for which, thus far, only single strains are publicly available, and further validated the use of 2.1% concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequence divergence as a threshold value for species delineation in this genus. Finally, although most Achromobacter species can be distinguished by biochemical characteristics, the present study also highlighted considerable phenotypic intraspecies variability and demonstrated that the type strains may be phenotypically poor representatives of the species. We propose to classify the fourteen human clinical strains as Achromobacter mucicolens sp. nov. (with strain LMG 26685(T) [=CCUG 61961(T)] as the type strain), Achromobacter animicus sp. nov. (with strain LMG 26690(T) [=CCUG 61966(T)] as the type strain), Achromobacter spiritinus sp. nov. (with strain LMG 26692(T) [=CCUG 61968(T)] as the type strain), and Achromobacter pulmonis sp. nov. (with strain LMG 26696(T) [=CCUG 61972(T)] as the type strain).

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.

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.

Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS 1363 , was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS 1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS 1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS 1363 was used to probe a collection of 943 B. cepacia complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS 1363 was not found in other genomovar III strains and, with the exception of B. ambifaria, was absent from other B. cepacia complex species. Genotyping analyses of all IS 1363 -positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.

We analyzed a collection of 97 well-characterized Burkholderia cepacia genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing. The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT). All methods showed excellent typeability. PFGE with Spe I was more reproducible than RAPD and BOX-PCR fingerprinting. The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting. In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement. Our data indicate that different genomic-based methods can be used to type B. cepacia genomovar III isolates depending on the situation and the epidemiologic question being addressed. PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology). In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT.

Objectives: We sought to determine whether the same Burkholderia cepacia complex strain has persisted as the dominant clonal lineage among patients in a large cystic fibrosis (CF) treatment center during the past 2 decades. Study design: The inter-city spread of B cepacia through transfer of a colonized patient and the impact of infection control measures in containing inter-patient transmission were investigated. We analyzed all available B cepacia complex isolates recovered from 1981 to 1987 and from 1996 to 2000 at one large CF treatment center (Center A) and from 1997 to 2000 at another center (Center B). Incidence of B cepacia complex infection and infection control measures in both centers were assessed. Results: Seventeen (81%) of 21 Center A patients from whom B cepacia complex bacteria were recovered between 1981 and 1987 and 40 (97%) of 41 patients culture-positive between 1996 and 2000 were infected with the same genomovar III strain. Transfer of a colonized patient from Center A to Center B was associated with an increase in B cepacia complex infection in Center B, all of which was with the Center A dominant strain. This strain, designated PHDC , lacks both B cepacia epidemic strain and cblA markers. Conclusions: B cepacia complex strains may remain endemic in CF treatment centers for many years. Responsible bacterial and host factors and optimal infection control measures to prevent inter-patient spread remain to be identified. (J Pediatr 2001;139:643–9)