Identification of members of the Burkholderia cepacia complex by species-specific PCR.

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans and B. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13 B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis.

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.

Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.