PCR assays targeting rRNA genes were developed to identify species (genomovars) within the Burkholderia cepacia complex. Each assay was tested with 177 bacterial isolates that also underwent taxonomic analysis by whole-cell protein profile. These isolates were from clinical and environmental sources and included 107 B. cepacia complex strains, 23 Burkholderia gladiolistrains, 20 Ralstonia pickettii strains, 10Pseudomonas aeruginosa strains, 8 Stenotrophomonas maltophilia strains, and 9 isolates belonging to nine other species. The sensitivity and specificity of the 16S rRNA-based assay for Burkholderia multivorans (genomovar II) were 100 and 99%, respectively; for Burkholderia vietnamiensis(genomovar V), sensitivity and specificity were 87 and 92%, respectively. An assay based on 16S and 23S rRNA gene analysis ofB. cepacia ATCC 25416 (genomovar I) was useful in identifying genomovars I, III, and IV as a group (sensitivity, 100%, and specificity, 99%). Another assay, designed to be specific at the genus level, identified all but one of the Burkholderia andRalstonia isolates tested (sensitivity, 99%, and specificity, 96%). The combined use of these assays offers a significant improvement over previously published PCR assays forB. cepacia.

The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of A. xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of A. xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47 A. xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of A. xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobinbinding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Background: Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps in our knowledge remain. Unresolved issues include the identities of all genes exhibiting altered transcription in response to iron and heme availability, the fraction of such genes functioning in iron/heme acquisition, and the heterogeneity of this gene set among clinical isolates. Previously we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those observations by determining the iron/heme modulons of two clinical isolates, the type b isolate 10810 and the nontypeable isolate R2866. These data are used to begin to define the core iron/heme modulon of the species.

Haemophilus influenzae has an absolute growth requirement for a porphyrin source. This growth requirement can be satisfied in vitro by haem, haemoglobin or the haemoglobin-haptoglobin, haem-haemopexin and haem-albumin complexes. A family of proteins, termed the Hgp proteins, which are essential for utilization of the haemoglobin-haptoglobin complex, has previously been identified. A strain lacking the Hgp proteins also has a residual ability to utilize haemoglobin, indicating that additional moieties contribute to haemoglobin utilization. Using a haemoglobin affinity method an approximately 105 kDa protein was isolated. Mutation of the identified gene in an Hgp null background reduced the ability of the mutant strain to utilize haemoglobin in vitro. The mutation also resulted in a reduced ability to utilize haem, haem-haemopexin, haem-albumin and haemoglobin-haptoglobin, thus identifying a general haem-utilization protein (Hup) in Haemophilus influenzae.