Michelle C. Callegan

Bacterial Endophthalmitis: Epidemiology, Therapeutics, and Bacterium-Host Interactions

SUMMARY Endophthalmitis is a severe inflammation of the interior of the eye caused by the introduction of contaminating microorganisms following trauma, surgery, or hematogenous spread from a distant infection site. Despite appropriate therapeutic intervention, bacterial endophthalmitis frequently results in visual loss, if not loss of the eye itself. Although the pathogenicity of bacterial endophthalmitis has historically been linked with toxin production during infection, a paucity of information exists as to the exact mechanisms of retinal toxicity and the triggers for induction of the intraocular immune response. Recently, research has begun to examine the bacterial and host molecular and cellular events that contribute to ocular damage during endophthalmitis. This review focuses on the causative agents and therapeutic challenges of bacterial endophthalmitis and provides current data from the analysis of the role of bacterial virulence factors and host inflammatory interactions in the pathogenesis of eye infections. Based on these and related studies, a hypothetical model for the molecular pathogenesis of bacterial endopthalmitis is proposed. Identifying and understanding the basic mechanisms of these bacterium-host interactions will provide the foundation for which novel, information-based therapeutic agents are developed in order to prevent vision loss during endophthalmitis.

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.

Bacterial endophthalmitis: Therapeutic challenges and host–pathogen interactions

Endophthalmitis is an infection of the posterior segment of the eye that frequently results in loss of vision. This devastating result occurs despite prompt and often aggressive therapeutic and surgical intervention. Over the past decade, research has centered on determining the bacterial and host factors involved in this potentially blinding disease. The initial focus on the bacterial factors responsible for intraocular virulence has recently expanded into analysis the inflammatory response to infection, including the molecular and cellular interactions between the pathogen and host. This review discusses the epidemiology and therapeutic challenges posed by endophthalmitis, as well as recent findings from the analysis of interactions between the host and pathogen. Based on these findings, a model for the pathogenesis of endophthalmitis is presented. A more comprehensive understanding of the molecular and cellular interactions taking place between pathogen and host during endophthalmitis will expose possible therapeutic targets designed to arrest the infection and prevent vision loss.

Relationship of plcR-regulated factors to Bacillus endophthalmitis virulence.

ABSTRACT The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B. cereus or B. thuringiensis or a plcR-deficient mutant. The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis. The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis.

Antibacterial activity of the fourth-generation fluoroquinolones gatifloxacin and moxifloxacin against ocular pathogens

The ideal ophthalmic anti-infective exhibits broad-spectrum activity against grampositive, gram-negative, and atypical bacterial species. These pathogens can cause potentially blinding infections such as keratitis and endophthalmitis, both of which are associated with ophthalmic surgery or traumatic injury. These infections often require aggressive antibacterial therapy, preferably with newer generations of antibiotics. In this study, minimal inhibitory concentration (MIC) values for gatifloxacin and moxifloxacin were determined in vitro against bacterial strains that were isolated from suspected cases of bacterial keratitis and endophthalmitis. The ocular isolates included 7 gram-positive, 4 gram-negative, and 3 atypical bacterial species. Catifloxacin and moxifloxacin exhibited similar activity against 6 grampositive organisms:Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus cereus, andEnterococcus faecalis. MIC90 values for the drugs against these isolates ranged from 0.08 mg/mL to 0.57 mg/mL and were comparable to previously published values against isolates from patients with systemic infections. The MIC90 for gatifloxacin againstStreptococcus viridans was 0.22 mg/mL compared with 0.73 mg/mL for moxifloxacin (P=.011). Among the gram-negative isolates, the mean MIC90 for gatifloxacin againstPseudomonas aeruginosa was 1.28 mg/mL compared with 2.60 mg/ mL for moxifloxacin (P=.023). MIC90 values for gatifloxacin againstKlebsiella pneumoniae andEnterobacter aerogenes were one fourth to one fifth the values for rnoxifloxacin. For the atypicals, the MIC90 values for gatifloxacin againstNocardia asteroides andMycobacterium chelonae were one fourth the corresponding values for rnoxifloxacin. Gatifloxacin demonstrated a broad spectrum of activity against several key ocular pathogens tested in this study and was at least as effective as moxifloxacin against these pathogens.

Acute inflammation and loss of retinal architecture and function during experimental Bacillus endophthalmitis.

Rapid vision loss and explosive inflammation are devastating consequences of Bacillus endophthalmitis that have not been well defined. We therefore analyzed the evolution of intraocular inflammation and loss of retinal architecture and function during experimental Bacillus endophthalmitis. Mice were intravitreally injected with 100 CFU of B. cereus, and eyes were analyzed for bacterial growth, retinal function, architectural changes and retinal cellular stress, inflammatory cytokines, and infiltrating cells. Retinal electrophysiologic and structural changes began as early as 4 to 6 hr postinfection. Significant declines in retinal function paralleled the loss of retinal architecture. Glial fibrillary acidic protein (GFAP) was detected in retina, indicating potential stress. Polymorphonuclear leukocyte (PMN) infiltration into the vitreous began as early as 4 hr postinfection, coinciding with a significant increase in TNF-α in the eye. These results indicated that acute inflammation and detrimental architectural and electrophysiologic changes in the retina began earlier than once thought, suggesting that therapeutic intervention should be given at the earliest possible time to avoid vision loss during Bacillus endophthalmitis.

Hypermucoviscosity as a Virulence Factor in Experimental Klebsiella pneumoniae Endophthalmitis

PURPOSE Klebsiella pneumoniae is a common cause of endogenous bacterial endophthalmitis, a disease that frequently results in a poor visual outcome. Hypermucoviscosity has been identified as a virulence factor among clinical bacteremia isolates of K. pneumoniae. In this study, an experimental murine model of K. pneumoniae endophthalmitis was established, and the role of hypermucoviscosity in its pathogenesis was analyzed. METHODS C57BL/6J mice were intravitreously injected with 100 CFU of hypermucoviscous (HMV+) or nonhypermucoviscous (HMV-) K. pneumoniae. Intraocular bacterial growth, retinal function, gross pathology, and inflammatory responses were monitored every 3 hours until the eyes lost significant (>90%) retinal function, or the infection appeared to clear. RESULTS The HMV+ strain grew logarithmically in eyes until approximately 15 hours postinfection (PI), reaching a stationary phase of growth at approximately 8.0 log(10) CFU/eye. The HMV- strain grew logarithmically to approximately 7.6 log(10) by 18 hours, but bacterial count declined to approximately 6.4 log(10) CFU/eye by 21 hours PI. Eyes infected with the HMV+ strain retained approximately 35% a-wave and <10% b-wave function by 18 hours PI. These eyes also had a cumulative clinical score of 14+ by 18 hours and underwent phthisis between 21 and 24 hours. Eyes infected with the HMV- strain had a cumulative clinical score of <6 and retained >60% a-wave and >50% b-wave function throughout 21 hours. Five of 7 eyes had <100 CFU HMV- K. pneumoniae at 27 hours PI. CONCLUSIONS The findings demonstrate the site-threatening consequences of K. pneumoniae endophthalmitis and the importance of the hypermucoviscosity phenotype in the pathogenesis of experimental K. pneumoniae endophthalmitis.

Contribution of Membrane-Damaging Toxins to Bacillus Endophthalmitis Pathogenesis

ABSTRACT Membrane-damaging toxins are thought to be responsible for the explosive clinical course of Bacillus endophthalmitis. This study analyzed the contribution of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) to the pathogenesis of experimental Bacillus endophthalmitis. Isogenic mutants were constructed by insertion of lacZ into Bacillus thuringiensis genes encoding PI-PLC (plcA) and PC-PLC (plcB). Rabbit eyes were injected intravitreally with 2 log10 CFU of strain BT407 (wild type), the PI-PLC mutant (BTplcA::lacZ), or the PC-PLC mutant (BTplcB::lacZ). The rates of decrease in retinal responses of eyes infected with the isogenic mutants were similar to that of wild type, with all infections resulting in elimination of retinal function by 18 h. Strain BT407 caused a significant increase in the latency of retinal responses at 6 h, but strains BTplcA::lacZ and BTplcB::lacZ did not. All strains elicited significant inflammatory cell influx into the anterior chamber by 12 h. Histologically, eyes infected with each strain were indistinguishable throughout the infection course. In this model, neither PI-PLC nor PC-PLC had an effect on the course or severity of experimental Bacillus endophthalmitis. Alterations in retinal responses early in infection may mark the beginnings of specific photoreceptor or glial cell dysfunction.

Virulence Factor Profiles and Antimicrobial Susceptibilities of Ocular Bacillus Isolates

Bacillus causes one of the most rapidly blinding intraocular infections: endophthalmitis. In this study, Bacillus spp. were isolated from ocular infection cases, taxonomically characterized by riboprint analysis, and screened for the presence of putative virulence factors. The ability of these isolates to kill retinal and corneal cells was examined, as were antibiotic susceptibility profiles. The majority of isolates belonged to the B. cereus taxonomic group of microorganisms and were identified as B. cereus (53%) or B. thuringiensis (26%). Toxins were identified in most B. thuringiensis and B. cereus isolates. Most B. cereus and B. thuringiensis killed corneal and retinal cells within 6 h. All isolates were susceptible to most antibiotics tested, with quinolones and vancomycin being the most potent. These findings represent the first report of B. thuringiensis as an important ocular pathogen, demonstrates the potential ocular toxicity of B. cereus and B. thuringiensis isolates, and identifies antibiotics whose efficacy against Bacillus were superior to those used clinically.

Methicillin resistance of Staphylococcus species among health care and nonhealth care workers undergoing cataract surgery

Purpose: The purpose of this study is to characterize the bacterial flora of the ocular and periocular surface in cataract surgery patients and to determine the prevalence of methicillin resistance among staphylococcal isolates obtained from health care workers (HCWs) and non-HCWs. Methods: In this prospective, multicenter, case series study, eyelid and conjunctival cultures were obtained from the nonoperative eye of 399 consecutive cataract patients on the day of surgery prior to application of topical anesthetics, antibiotics, or antiseptics. Speciation and susceptibility testing were performed at the Dean A. McGee Eye Institute. Logistic regression was utilized to evaluate whether any factors were significant in predicting the presence of methicillin-resistant staphylococcal isolates. Results: Staphylococcus epidermidis (62.9%), followed by S. aureus (14.0%), was the most frequently isolated organism. Methicillin-resistant S. epidermidis accounted for 47.1% (178/378) of S. epidermidis isolates, and methicillin-resistant S. aureus accounted for 29.5% (26/88) of S. aureus isolates. Methicillin-resistant staphylococcal isolates were found in 157 of 399 (39.3%) patients, the majority (89.2%) of whom were non-HCWs. The likelihood of being colonized with methicillin-resistant organisms increased with age (odds ratio [OR], 1.27; 95% confidence interval [CI]: 1.02–1.58; P = 0.04) but decreased with diabetes (OR, 0.51; 95% CI: 0.29–0.89; P = 0.02). Being a HCW (OR, 1.25; 95% CI: 0.61–2.58; P = 0.54) was not a risk factor for colonization with methicillin-resistant organisms. Conclusion: Patients without exposure to health care environments are as likely as HCWs to be colonized with methicillin-resistant organisms. Increasing methicillin resistance with age may partially explain the increased risk of endophthalmitis reported with older age.