Mary C. Booth

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.

Clonal Associations among Staphylococcus aureus Isolates from Various Sites of Infection

ABSTRACT A molecular epidemiological analysis was undertaken to identify lineages of Staphylococcus aureus that may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405 S. aureus clinical isolates collected from various infection types and geographic locations was performed. Five distinct S. aureus lineages (SALs 1, 2, 4, 5, and 6) were identified, which accounted for 19.01, 9.14, 22.72, 10.12, and 4.69% of isolates, respectively. In addition, 85 lineages which occurred with frequencies of <2.5% were identified and were termed “sporadic.” The most prevalent lineage was methicillin-resistant S. aureus (SAL 4). The second most prevalent lineage, SAL 1, was also isolated at a high frequency from the anterior nares of healthy volunteers, suggesting that its prevalence among clinical isolates may be a consequence of high carriage rates in humans. Gene-specific PCR was carried out to detect genes for a number of staphylococcal virulence traits. tstand cna were found to be significantly associated with prevalent lineages compared to sporadic lineages. When specific infection sites were examined, SAL 4 was significantly associated with respiratory tract infection, while SAL 2 was enriched among blood isolates. SAL 1 and SAL 5 were clonally related to SALs shown by others to be widespread in the clinical isolate population. We conclude from this study that at least five phylogenetic lineages of S. aureus are highly prevalent and widely distributed among clinical isolates. The traits that confer on these lineages a propensity to infect may suggest novel approaches to antistaphylococcal therapy.

In vitro pharmacodynamics of ofloxacin and ciprofloxacin against common ocular pathogens.

Purpose. Time-kill curve methodology was used to assess the pharmacodynamics of two fluoroquinolones, ofloxacin and ciprofloxacin, against six strains representing the most common ocular pathogens:Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Serratia marcescens, and Haemophilus influenzae. Methods. For time-kill studies, ofloxacin and ciprofloxacin solutions were prepared at concentrations of 0.5 ×, 1.0 ×, 2.0 ×, and 3.0 × the MIC (minimal inhibitory concentration) for each respective strain. Inocula were prepared by diluting overnight cultures to final concentrations of 102, 103, 104, and 105 cfu (colony-forming units)/mL in each antibiotic solution. Growth controls were included. Viability counts of antibiotic-containing and control bacterial suspensions were performed at 0, 10, 20, 30, 60, 90, 120, and 180 minutes. Results. In general, the kill rates of ofloxacin at 1.0 ×, 2.0 ×, and 3.0 × the MIC were significantly faster than the kill rates of ciprofloxacin by approximately 30 minutes, regardless of the bacterial concentration tested. At 0.5 × MIC, the kill kinetics of ciprofloxacin and ofloxacin were similar, regardless of the strain tested. At 1.0 × MIC, ofloxacin achieved 99.9% killing of P. aeruginosa and S. aureus within 30 minutes, and S. epidermidis and S. marcescens within 90 minutes. Overall, the kill kinetics of both quinolones for H. influenzae were similar, while neither quinolone achieved 99.9% killing of S. pneumoniae, regardless of the antibiotic concentration tested. Conclusion. Time-kill curve analyses in the present study demonstrate that ofloxacin achieved killing of the majority of ocular pathogens tested at rates equivalent to or faster than that of ciprofloxacin. Both fluoroquinolones were more effective against nonencapsulated bacteria than against encapsulated bacteria.

Host/Parasite Interactions in Bacterial Endophthalmitis

Bacterial infections within the eye arise as complications of intraocular surgery, penetrating injury, or hematogenous spread from distant anatomical sites. Because: 1) the interior surfaces of the eye are lined with sensitive, nonregenerating tissues, 2) the inner chambers of the eye are relatively sequestered from circulating immunological components, 3) the integrity of blood-ocular barriers provides poor penetration of systemically administered antibiotics, and 4) aqueous and vitreous humor represent rich, relatively acellular culture media; endophthalmitis often progresses rapidly and total loss of vision frequently results. Years of clinical experience have shown that current therapies for endophthalmitis, including antimicrobials, antiinflammatory agents, and vitrectomy, are frequently unsuccessful in ameliorating destruction of intraocular tissues. While bacterial and host factors were thought to play key roles in the course and severity of endophthalmitis, it is only recently that their contributions have been experimentally defined. Molecular-based techniques are gaining increased use in the study of infectious eye diseases. Current findings regarding the host/parasite interactions within the eye are reviewed, and a resulting integrative model of the natural course of endophthalmitis proposed. A molecular-level understanding of the roles of both bacterial and host factors during endophthalmitis will likely reveal potential targets for therapeutic intervention aimed at salvaging vision.