Michael S. Gilmore

A molecular epidemiological analysis was undertaken to identify lineages ofStaphylococcus aureusthat may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405S. aureusclinical isolates collected from various infection types and geographic locations was performed. Five distinctS. aureuslineages (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 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.tstandcnawere 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 ofS. aureusare 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.

Enterococcus faecalis has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of E. faecalis is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids. E. faecalis strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3′ end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3′ to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn917 insertion.

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus,Enterococcus faecalis, andStaphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viableB. cereus,E. faecalis, orS. aureus, (ii) metabolically inactiveB. cereus,E. faecalis, orS. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology.B. cereusendophthalmitis followed a more rapid and virulent course thanE. faecalisorS. aureusendophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed thatB. cereusuniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection ofB. cereusorS. aureusculture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis ofB. cereus,S. aureus, andE. faecalisendophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.

Bacillus cereus is a rare cause of serious human infection but, paradoxically, causes one of the most severe posttraumatic or endogenous infections of the eye, endophthalmitis, which frequently results in blindness. The virulence of B. cereus endophthalmitis historically has been attributed to toxin production. We therefore sought to examine the contribution of the dermonecrotic toxin, hemolysin BL, to the pathogenesis of B. cereus infection in an endophthalmitis system that is highly amenable to study. The pathogenesis of infection resulting from intravitreal injection of 10 ² CFU of either a clinical ocular isolate of B. cereus producing hemolysin BL (HBL ⁺ ) or an isogenic mutant in this trait (HBL ⁻ ) was assessed bacteriologically and by slit lamp biomicroscopy, electroretinography, histology, and inflammatory cell enumeration. Both HBL ⁺ and HBL ⁻ strains evoked severe intraocular inflammatory responses as early as 12 h postinfection, with complete loss of retinal responsiveness by 12 h. The infections caused by both strains spread of the infection to adjacent tissues by 18 h. No significant differences in intraocular bacterial growth ( P ≥ 0.21) or inflammatory changes ( P ≥ 0.21) were observed in eyes infected with either HBL ⁺ or HBL ⁻ strains during the course of infection. The level of retinal responsiveness was greater in HBL ⁻ infected eyes than in HBL ⁺ -infected eyes at 6 h only ( P = 0.01). These results indicate that hemolysin BL makes no essential contribution to the severe and rapid course of infection in the endophthalmitis model.

Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis . Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes.

We report the identification of a new cell wall-associated protein of Enterococcus faecalis . Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant ( P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of ∼202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.