Isolation and characterization of the dnaA gene of Rickettsia prowazekii.

The dnaA gene encoding the initiator protein of DNA replication was isolated from the obligate intracellular bacterium, Rickettsia prowazekii. Comparison of the deduced amino acid sequence of R. prowazekii DnaA with other bacterial DnaA proteins revealed extensive similarity. However, the rickettsial sequence is unique in the number of basic lysine residues found within a highly conserved portion of the putative DNA binding region, suggesting that the rickettsial protein may recognize a DNA sequence that differs from the consensus DnaA box sequence identified in other bacteria. Consensus DnaA box sequences, found upstream of many bacterial dnaA genes, were not identified upstream of rickettsial dnaA gene. In addition, gene organization within this region differed from that of other bacteria. The putative start of transcription of the rickettsial dnaA gene was localized to a site 522 nucleotides (nt) upstream of the DnaA start codon.

Evaluation of BACTEC MYCO/F lytic medium for recovery of mycobacteria and fungi from blood.

The reliability of MYCO/F Lytic medium in the BACTEC 9240 blood culture system was evaluated by comparing its performance to that of the Isolator system for the recovery of fungi and to that of the ESP II system for the recovery of mycobacteria. Of 717 specimens of blood cultured for fungi, 24 were positive; 12 samples were positive with both systems, 7 samples were positive with the Isolator system only, and 5 samples were positive with MYCO/F Lytic medium only. Fourteen samples grew Histoplasma capsulatum; both systems detected H. capsulatum in seven samples but the Isolator system alone detected H. capsulatum in seven samples. The mean times to the detection of H. capsulatum were 8 days (range, 4 to 13 days) for MYCO/F Lytic medium and 9 days (range, 6 to 18 days) for the Isolator system; the mean times to identification were 20 days (range, 15 to 24 days) for isolates recovered with MYCO/F Lytic medium and 11 days (range, 6 to 18 days) for those recovered with the Isolator system (P < 0.05). Cryptococcus neoformans was isolated from 10 fungal cultures; five isolates grew in both systems, and five isolates grew in MYCO/F Lytic medium only. The mean times to detection of C. neoformans were 4 days (range, 2 to 6 days) for MYCO/F Lytic medium and 7 days (range, 5 to 7 days) for the Isolator system (P < 0.05); the mean times to identification were 15 days (range, 7 to 27 days) for isolates recovered with MYCO/F Lytic medium and 8 days (range, 7 to 11 days) for those recovered with the Isolator system. Of the 687 samples of blood cultured for mycobacteria, 64 blood samples from 42 patients grew mycobacteria (58 grew Mycobacterium avium complex, 4 grew Mycobacterium kansasii, and 2 grew Mycobacterium tuberculosis); 42 isolates were recovered with both systems, 18 were isolated with MYCO/F medium only, and 4 were isolated with the ESP II system only alone (P < 0.05). The mean time to detection of mycobacteria with MYCO/F Lytic medium was 14 days, whereas it was 17 days with the ESP II system (P < 0.05). In summary, the combination of MYCO/F Lytic medium and the BACTEC 9240 instrument is an excellent blood culture system for the growth and detection of mycobacteria. A valid assessment of MYCO/F Lytic medium with regard to fungal isolation, however, was not possible due to the small number of isolates recovered.

Use of the Treponema pallidum-specific captia syphilis IgG assay in conjunction with the rapid plasma reagin to test for syphilis.

The Captia Syphilis IgG enzyme immunoassay (EIA) was evaluated for use in conjunction with the rapid plasma reagin test (RPR) as a method to test for syphilis. A total of 1,288 serum specimens were tested by the routine laboratory protocol of the RPR followed by microhemagluttination assay for Treponema pallidum (MHA-TP) testing of RPR-reactive sera as well as the EIA-RPR protocol in which the automated EIA followed by a manual RPR test for EIA-positive specimens is used. When using the routine protocol, 131 specimens were initially reactive by the RPR, and 113 of these were reactive by MHA-TP. When using the EIA-RPR protocol, 170 specimens were initially positive by EIA, and of these, 112 were RPR reactive, indicating active disease. When compared to the routine protocol, the EIA-RPR protocol had sensitivity, specificity, and positive and negative predictive values of 96.5, 99.7, 97.3, and 99.7%, respectively. After resolution of discrepancies by additional testing, the adjusted sensitivity, specificity, and positive and negative predictive values were 100, 99.8, 98.3, and 100%, respectively. This evaluation demonstrates that when used in conjunction with the RPR, the Captia Syphilis EIA is a reliable method by which to test for syphilis.

Sequence analysis of the Rickettsia prowazekii gyrA gene.

The Rickettsia prowazekii (Rp) gyrA gene, which codes for a subunit of DNA gyrase in this obligate intracellular bacterium, has been isolated and characterized. Nucleotide sequence analysis revealed an open reading frame (ORF), initiating with a GTG start codon, of 2718 bp that could encode a protein of 905 amino acids (aa) with a calculated M(r) of 101,048. The Rp gyrase subunit A (GyrA), when compared to GyrA analogs of other bacterial species, exhibited 43 to 50% identity. Alignment of the Rp GyrA aa sequence with the other analogs revealed the presence of a span of additional aa within the putative DNA-binding domain. The lack of an ORF within 865 bp upstream from the Rp gyrA demonstrates a Rp gene organization different from that of characterized gyrA from other species. Despite the similarity to Escherichia coli GyrA, Rp GyrA did not complement an E. coli gyrA temperature-sensitive mutant. However, Rp gyrA was dominant to an E. coli gyrA96 nalidixic-acid-resistant (NalR) mutant, conferring Nal sensitivity when introduced into the NalR E. coli strain.