Y. Jerold Gordon

A Review of Antimicrobial Peptides and Their Therapeutic Potential as Anti-Infective Drugs

Purpose: Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications. Methods: Literature review. Results: Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications. Conclusions: While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.

Emerging fluoroquinolone resistance in bacterial keratitis: A 5-year review1

OBJECTIVE To identify resistance patterns to the fluoroquinolones for patients with bacterial keratitis. DESIGN Retrospective observational case series. PARTICIPANTS All cases of bacterial keratitis presenting to the Charles T. Campbell Ophthalmic Microbiology Laboratory at the Eye and Ear Institute of Pittsburgh from January 1993 to December 1997 were reviewed. A total of 1053 ocular isolates from 825 cases of bacterial keratitis were identified. MAIN OUTCOME MEASURES In vitro laboratory susceptibility testing of ocular isolates to ciprofloxacin and ofloxacin was determined by the Kirby-Bauer disk diffusion method and interpreted using the National Committee for Clinical Laboratory Standards serum standards. RESULTS The number of cases of bacterial keratitis per year decreased from 284 in 1993 to 75 in 1997. The ratio of gram-positive to gram-negative organisms changed from 81.8%:18.2% in 1993 to 51.4%:48.6% in 1997 (chi-square, 66.00; degrees of freedom, 4; P < 0.000001). Resistance of Staphylococcus aureus to ciprofloxacin significantly increased annually from 5.8% in 1993 to 35.0% in 1997 (chi-square, 19.80; degrees of freedom, 4; P < 0.0001) and for ofloxacin from 4.7% to 35.0% over the same period (chi-square, 21.32; degrees of freedom, 4; P < 0.001). Streptococcus species and coagulase-negative Staphylococcus species showed significant resistance to both fluoroquinolones but no change in resistance over the study period. The gram-negative organisms showed good susceptibility to the fluoroquinolones. CONCLUSIONS This in vitro study shows a significant increased resistance of S. aureus to the fluoroquinolones from 1993 to 1997. In addition, gaps in fluoroquinolone coverage for Streptococcus and coagulase-negative Staphylococcus species raise concern for the use of monotherapy in treating bacterial keratitis. Contrary to what might be expected, the distribution of gram-positive to gram-negative organisms has shifted, with a decrease in the number of gram-positive organisms identified, while the number of gram-negative isolates has remained stable.

Gatifloxacin and moxifloxacin: an in vitro susceptibility comparison to levofloxacin, ciprofloxacin, and ofloxacin using bacterial keratitis isolates

PURPOSE We compared the in vitro susceptibility patterns and the minimum inhibitory concentrations (MICs) of gatifloxacin (GAT) and moxifloxacin (MOX) (fourth-generation fluoroquinolones) to ciprofloxacin (CIP) and ofloxacin (OFX) (second-generation fluoroquinolones) and levofloxacin (LEV; third-generation fluoroquinolone) using bacterial keratitis isolates. The goal was to determine whether the fourth-generation fluoroquinolones offer any advantages over the second- and third-generation fluoroquinolones. DESIGN Experimental laboratory investigation. In contrast to an epidemiologic prevalence study, this study was designed to compare the relative susceptibility of each bacterial group to different fluoroquinolones by deliberate selection of representative isolates that were both susceptible and resistant to second-generation fluoroquinolones. METHODS In retrospect, the MICs of 177 bacterial keratitis isolates were determined to CIP, OFX, LEV, GAT, and MOX using E tests. A relative susceptibility analysis was performed for each bacterial group that included separate bacterial groups that were resistant to second-generation fluoroquinolones. The NCCLS susceptibility patterns and the MICs were compared statistically. Comparing MICs, the antibiotic with the lower MICs has greater antibacterial activity. RESULTS For most keratitis isolates, there were no susceptibility differences among the five fluoroquinolones. The fourth-generation fluoroquinolones did, however, demonstrate increased susceptibility for Staphylococcus aureus isolates that were resistant to CIP, LEV and OFX. In general, CIP demonstrated the lowest MICs for gram-negative bacteria. The MICs for fourth-generation fluoroquinolones were statistically lower than the second-generation fluoroquinolones for all gram-positive bacteria tested. Comparing the two fourth-generation fluoroquinolones, MOX demonstrated lower MICs for most gram-positive bacteria, whereas GAT demonstrated lower MICs for most gram-negative bacteria. CONCLUSIONS Based on in vitro testing, the fourth-generation fluoroquinolones may offer some advantages over those currently available for the treatment of bacterial keratitis. Clinical studies will be required to confirm these results.

Human Cathelicidin (LL-37), a Multifunctional Peptide, is Expressed by Ocular Surface Epithelia and has Potent Antibacterial and Antiviral Activity

Purpose: This study determined whether LL-37 (cathelicidin) is expressed by conjunctival and corneal epithelia as part of ocular host defense. The antimicrobial activity of LL-37 was also assessed in vitro against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), herpes simplex virus type 1 (HSV-1), and adenovirus (Ad). Methods: Expression of LL-37/hCAP 18 mRNA and LL-37 protein was determined by reverse transcription–polymerase chain reaction (RT-PCR) and immunoblotting, respectively, in scraped human corneal epithelium and primary cultured human corneal and conjunctival epithelial cells. The EC50 values for three strains of PA and one each of SA and SE were determined for LL-37. LL-37 antiviral inhibition of HSV-1 and adenovirus was assessed by direct inactivation assays. Toxicity of LL-37 to A549 cells was evaluated by a MTT assay. Results: LL-37/hCAP18 mRNA and LL-37 peptide were expressed by human corneal and conjunctival epithelial cells. Antibacterial activity for LL-37 was demonstrated (EC50 values for the three PA strains were 2.8 ± 1.3, 1.9 ± 0.3, and 3.6 ± 2.1; for SA: 1.6 ± 1.5; for SE: 1.3 ± 1.9 μ g/ml). LL-37 produced a significant reduction (p < 0.001 ANOVA) in HSV-1 and Ad19 viral titers with distinctly different time-kill curves (p < 0.001). LL-37 (up to 111 μ M) produced no toxicity in A549 cells. Conclusions: Corneal and conjunctival epithelia express LL-37 as part of mucosal innate immunity to protect against bacterial and viral ocular infections.

Simvastatin vs Therapeutic Lifestyle Changes and Supplements: Randomized Primary Prevention Trial

OBJECTIVE To compare the lipid-lowering effects of an alternative regimen (lifestyle changes, red yeast rice, and fish oil) with a standard dose of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin). PATIENTS AND METHODS This randomized trial enrolled 74 patients with hypercholesterolemia who met Adult Treatment Panel III criteria for primary prevention using statin therapy. All participants were randomized to an alternative treatment group (AG) or to receive simvastatin (40 mg/d) in this open-label trial conducted between April 1, 2006, and June 30, 2006. The alternative treatment included therapeutic lifestyle changes, ingestion of red yeast rice, and fish oil supplements for 12 weeks. The simvastatin group received medication and traditional counseling. The primary outcome measure was the percentage change in low-density lipoprotein cholesterol (LDL-C). Secondary measures were changes in other lipoproteins and weight loss. RESULTS There was a statistically significant reduction in LDL-C levels in both the AG (-42.4%+/-15%) (P<.001) and the simvastatin group (-39.6%+/-20%) (P<.001). No significant differences were noted between groups. The AG also demonstrated significant reductions in triglycerides (-29% vs -9.3%; 95% confidence interval, -61 to -11.7; P=.003) and weight (-5.5% vs -0.4%; 95% confidence interval, -5.5 to -3.4; P<.001) compared with the simvastatin group. CONCLUSION Lifestyle changes combined with ingestion of red yeast rice and fish oil reduced LDL-C in proportions similar to standard therapy with simvastatin. Pending confirmation in larger trials, this multifactorial, alternative approach to lipid lowering has promise for a subset of patients unwilling or unable to take statins.

An in vitro resistance study of levofloxacin, ciprofloxacin, and ofloxacin using keratitis isolates of Staphylococcus aureus and Pseudomonas aeruginosa

PURPOSE We compared levofloxacin with ciprofloxacin and ofloxacin using the in vitro susceptibilities of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) keratitis isolates. DESIGN Retrospective, clinical laboratory study of antibiotic susceptibility among keratitis isolates. PARTICIPANTS Keratitis isolates from 200 patients with either SA or PA keratitis. METHODS Minimum inhibitory concentrations (MICs) were determined for levofloxacin, ofloxacin, and ciprofloxacin for 93 SA keratitis isolates (68 fluoroquinolone-resistant and 25 susceptible, as determined by disk diffusion) and 107 PA keratitis isolates (13 fluoroquinolone-resistant and 94 susceptible). National Committee for Clinical Laboratory Standards susceptibilities were determined and analyzed statistically. Time kill studies were determined for fluoroquinolone-susceptible and -resistant isolates to all antibiotics at 8 microg/ml. The killing rates were determined by regression, and the colony count decreases were analyzed. MAIN OUTCOME MEASURES The susceptibilities and potencies of levofloxacin, ciprofloxacin, and ofloxacin to SA and PA were determined from the MICs. Time kill studies determined the killing rates and decreases in colony counts. RESULTS The fluoroquinolone-resistant SA susceptibilities to levofloxacin, ofloxacin, and ciprofloxacin were only 22%, 10%, and 3%, respectively. The fluoroquinolone-susceptible SA were 100% susceptible to all antibiotics, with levofloxacin demonstrating the best potency. The fluoroquinolone-resistant PA were resistant to all antibiotics. The fluoroquinolone-susceptible PA isolates were highly susceptible to levofloxacin, ofloxacin, and ciprofloxacin, with ciprofloxacin demonstrating the highest potency. For fluoroquinolone-susceptible SA and PA, the time kill studies determined that the killing rates and decreases in colony counts were equivalent for all three antibiotics tested. The time kill studies demonstrated no colony count decreases for the fluoroquinolone-resistant SA and PA. CONCLUSIONS Taken together, our susceptibility and time kill data failed to demonstrate convincing differences in the susceptibility of SA and PA keratitis isolates to levofloxacin, ciprofloxacin, and ofloxacin. In general, bacterial isolates that were resistant to ciprofloxacin and ofloxacin were also resistant to levofloxacin.

Prolonged Recoverability of Desiccated Adenovirus Type 19 from Various Surfaces

Epidemic keratoconjunctivitis is a highly contagious disease whose transmission has been linked to the ophthalmologists office. The authors studied the ability of adenovirus 19 (ADV 19) to survive on surfaces commonly found in the office setting. An initial in vitro laboratory experiment demonstrated that ADV 19 in a desiccated state could be recovered up to 8 days from paper, and up to 10 days from cloth, metal, and plastic. The amount of recovered ADV 19 was significantly greater (analysis of variance, P less than 0.0001) from nonporous surfaces (plastic, metal) compared with porous surfaces (cloth, paper). A second experiment demonstrated that 35 days was the maximum length of time that desiccated ADV 19 could be recovered from a nonporous surface (plastic). The authors conclude that despite drying, ADV 19 is a hearty virus that remains potentially infectious for a long time on various surfaces that may be found in an ophthalmologists office.

Validation of Real-Time PCR for Laboratory Diagnosis of Acanthamoeba Keratitis

ABSTRACT Confirmation of Acanthamoeba keratitis by laboratory diagnosis is the first step in the treatment of this vision-threatening disease. Two real-time PCR TaqMan protocols (the Rivière and Qvarnstrom assays) were developed for the detection of genus-specific Acanthamoeba DNA but lacked clinical validation. We have adapted these assays for the Cepheid SmartCycler II system (i) by determining their real-time PCR limits of detection and amplification efficiencies, (ii) by determining their ability to detect trophozoites and cysts, and (iii) by testing a battery of positive and negative samples. We also examined the inhibitory effects of a number of commonly used topical ophthalmic drugs on real-time PCR. The results of the real-time PCR limit of detection and amplification efficiency of the Rivière and Qvarnstrom assays were 11.3 DNA copies/10 μl and 94% and 43.8 DNA copies/10 μl and 92%, respectively. Our extraction protocol enabled us to detect 0.7 Acanthamoeba cysts/10 μl and 2.3 Acanthamoeba trophozoites/10 μl by both real-time PCR assays. The overall agreement between the assays was 97.0%. The clinical sensitivity and specificity of both real-time PCR assays based on culture were 100% (7 of 7) and 100% (37 of 37), respectively. Polyhexamethylene biguanide was the only topical drug that demonstrated PCR inhibition, with a minimal inhibitory dilution of 1/640 and an amplification efficiency of 72.7%. Four clinical samples were Acanthamoeba culture negative and real-time PCR positive. Our results indicate that both real-time PCR assays could be used to diagnose Acanthamoeba keratitis. Polyhexamethylene biguanide can inhibit PCR, and we suggest that specimen collection occur prior to topical treatment to avoid possible false-negative results.

Topical corticosteroids of limited potency promote adenovirus replication in the Ad5/NZW rabbit ocular model

Purpose. To determine the effect of topical therapy with several corticosteroids with limited potency on viral clearance in the adenovirus type 5 (Ad5) rabbit ocular model. Methods. Sixty rabbits were inoculated in both eyes with Ad5. On the first day, the rabbits were equally divided into four topical treatment groups: 0.12% prednisolone acetate (PA), 0.1% fluorometholone (FM), 1% rimexolone (RMX), and control. Treatment was administered four times daily, in both eyes, for 3 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, 14, 16, 18, and 21. Results. Compared with the control group, treatment with PA, FM, and RMX significantly increased the number of Ad5 positive eye cultures from days 7–21. Fluorometholone and RMX prolonged the duration of Ad5 shedding, and FM increased the mean combined Ad5 titer from days 1–5 and 7–21. Conclusions. Treatment of an experimental ocular adenovirus infection with PA, FM, and RMX for 3 days significantly enhanced adenovirus replication compared with the control group. Short-term treatment of EKC with several commercially available topical corticosteroids with limited potency may offer symptomatic relief, but may also delay viral clearance and promote office and community epidemics.

Host species and strain differences affect the ability of an HSV-1 ICP0 deletion mutant to establish latency and spontaneously reactivate in vivo

HSV-1 latency and reactivation were studied in vivo by spontaneous and iontophoresis-induced ocular viral shedding in New Zealand rabbits, Balb/c and A/J mice latently infected with wild-type KOS, and dl x 3.1, a progeny ICP0 deletion mutant. The presence of trigeminal ganglionic latency was demonstrated by the in vitro methods of cocultivation and in situ hybridization. Although the efficiency of ganglionic latency was significantly less (P less than .0001) for dl x 3.1 than for KOS in both mice and rabbits, only dl x 3.1 shed spontaneously in the NZ rabbit. Iontophoresis of adrenergic agents failed to induce reactivation and ocular viral shedding of KOS or dl x 3.1 in mice or rabbits. The establishment of latency and reactivation of KOS and dl x 3.1 was dependent on the host animal. We conclude that host factors as exemplified by host species and host strain differences significantly affected the ability of KOS and dl x 3.1 to establish latency, to reactivate, and to shed spontaneously. ICP0 expression was not required for the establishment or maintenance of latency, nor was it required for the reactivation of latent HSV-1. Furthermore, the biological activity of KOS and dl x 3.1 during latency in vivo did not correlate with latency studies based on in vitro methods.