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.

Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven ''central aromatic'' and twenty ''peripheral aromatic'' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.genomics ͉ niche adaptation ͉ evolution ͉ biodegradation ͉ redundancy

Several distinct species (genomovars) comprise bacteria previously identified merely as Burkholderia cepacia. Understanding how these species, collectively referred to as the B. cepacia complex, differ in their epidemiology and pathogenic potential in cystic fibrosis (CF) is important in efforts to refine management strategies. B. cepacia isolates recovered from 606 CF patients receiving care at 132 treatment centers in 105 cities in the United States were assessed to determine species within the B. cepacia complex and examined for the presence of putative transmissibility markers (B. cepacia epidemic strain marker [BCESM] and cable pilin subunit gene [cblA]). Fifty percent of patients were infected with B. cepacia complex genomovar III, 38% with B. multivorans (formerly genomovar II), and 5% with B. vietnamiensis (formerly genomovar V); fewer than 5% of patients were infected with either genomovar I, B. stabilis (formerly genomovar IV), genomovar VI, or genomovar VII. BCESM was found in 46% of genomovar III isolates and not in any other species. Only one isolate, from a patient infected with the ET12 epidemic lineage, contained the complete cblA pilin subunit gene. Our data indicate a differential capacity for human infection among the phylogenetically closely related species of the B. cepacia complex. The low frequency of BCESM and cblA suggests that they are not sufficient markers of B. cepacia virulence or transmissibility.

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 ␣-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.Cystic fibrosis (CF), an autosomal-recessive hereditary disease caused by mutations in the CF transmembrane conductance regulator gene, is characterized by the production of abnormally thickened, viscous mucus and a disturbance in electrolyte transport across epithelial membranes (34). Defects in the CF transmembrane conductance regulator gene mainly affect the respiratory tract and the pancreas, and exacerbations of pulmonary infections cause significant morbidity and mortality in persons with CF (16). Typical CF pathogens include Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex organisms (16), but other glucose nonfermenters such as Ralstonia pickettii, Alcaligenes xylosoxidans, Stenotrophomonas maltophilia, and Burkholderia gladioli can be recovered from CF sputum culture as well (3,16). Correct identification of CF pathogens is important, since it underlies effective infection control measures and therapuetic intervention (24,25,36); however, several studies have shown that the identification of CF pathogens, including B. cepacia complex organisms, is far from straightforward (3,8,22,28).Here we report the characterization of 51 bacterial isolates recovered from respiratory secretions of CF patients. The majority of these isolates were identified by referring microbiology laboratories as B. ...

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