Henri Sueke

Minimum Inhibitory Concentrations of Standard and Novel Antimicrobials for Isolates from Bacterial Keratitis

PURPOSE To determine the minimum inhibitory concentrations (MICs) of 12 antimicrobials in current ophthalmic use and 4 potentially new alternatives against isolates from bacterial keratitis. METHODS Bacteria were collected from cases of bacterial keratitis in six centers in the United Kingdom between 2003 and 2006. MICs were measured by using susceptibility strips containing a concentration gradient of the antimicrobials penicillin, cefuroxime, ceftazidime, chloramphenicol, gentamicin, amikacin, vancomycin, teicoplanin, ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, meropenem, linezolid, tigecycline, and daptomycin. RESULTS Isolates (n = 772) were collected including coagulase negative Staphylococcus (CNS) (30%), Pseudomonas aeruginosa (23%), Staphylococcus aureus (14%), Enterobacteriaceae (14%), and streptococci (13%). Meropenem had low MICs for most isolates. All isolates except P. aeruginosa were susceptible to tigecycline. Linezolid was active against the majority of Gram-positive pathogens. Ten percent of S. aureus and 20% of CNS isolates were methicillin resistant. When systemic breakpoints were used, 84% of S. aureus isolates were susceptible to ciprofloxacin and 98% to moxifloxacin. Of the P. aeruginosa isolates, 99% were susceptible to ceftazidime, 96% to gentamicin, 99% to ciprofloxacin and 100% to moxifloxacin. More than 97% of Enterobacteriaceae isolates were susceptible to ceftazidime, gentamicin, ciprofloxacin, and moxifloxacin. CONCLUSIONS Based on systemic breakpoint data, resistance to commonly used antimicrobials was apparent. Meropenem is a potentially effective agent for ophthalmic use, with low MICs throughout all the bacterial subgroups. Tigecycline and linezolid showed good activity against particular groups and may be useful for treating bacterial keratitis resistant to current antimicrobials. Of the fluoroquinolones, moxifloxacin showed the lowest MICs and resistance for both Gram-positive and -negative bacteria.

Imaging and Evaluation of Corneal Vascularization Using Fluorescein and Indocyanine Green Angiography

PURPOSE To evaluate indocyanine green angiography (ICGA) and fluorescein angiography (FA) in imaging and quantifying corneal neovascularization (CNV). METHODS Patients with CNV were studied using a standardized protocol of color digital photography, FA, and ICGA. Images were graded independently by two observers and assessed for quality, phases of fluorescence, and leakage. Areas of CNV and vasculature geometric properties were analyzed and quantified by an automated program. RESULTS Twenty-three patients with good quality images were included. Mean times to appearance of ICG and fluorescein were 17 and 20 seconds (P = 0.10). Best images for analysis were obtained at 64 seconds for ICGA and 47 seconds for FA. CNV not apparent on color or FA, particularly in the presence of scarring, was well delineated by ICGA. Leakage of ICGA did not occur. Fluorescein leakage from apical CNV images occurred significantly earlier (32 seconds) in patients with CNV of <6-month duration than those of >1-year (50 seconds) duration (P = 0.04). Mean area of CNV and vessel diameter were similar with ICGA (8.79 mm(2), 0.058 mm) or FA (7.74 mm(2), 0.054 mm) but significantly larger than on color (1.94 mm(2), 0.026 mm) images (P < 0.01). Vessel tortuosity was similar on ICGA (1.16), FA (1.17), and color (1.15) (P = 0.27). CONCLUSIONS Combined use of FA and ICGA are valuable tools with which to assess CNV and provide better vessel delineation than can be obtained with only color images. Parameters used to assess CNV, such as leakage, area, diameter, and tortuosity, may be useful measures for evaluating treatment. Videography is useful for detecting early leakage.

An In Vitro Investigation of Synergy or Antagonism between Antimicrobial Combinations against Isolates from Bacterial Keratitis

PURPOSE To investigate antimicrobial combinations for synergy or antagonism against isolates of Staphylococcus aureus and Pseudomonas aeruginosa. METHODS Isolates were collected from cases of microbial keratitis from six centers in the United Kingdom. Minimum inhibitory concentrations (MICs) were determined by using E-test strips for 16 antimicrobials, including both current and potentially available agents. E-test strips were used to test selected antimicrobials in combination against a representative set of 10 S. aureus and 10 P. aeruginosa isolates. E-tests of the two antimicrobials were placed on sensitivity agar at right angles intersecting at their respective MICs. Antimicrobial combinations were classified as synergistic, additive, indifferent, or antagonistic, according to their fractional inhibitory concentration (FIC). RESULTS The combinations meropenem and ciprofloxacin, meropenem and teicoplanin, moxifloxacin and teicoplanin, and ciprofloxacin and teicoplanin, gave the lowest mean FICs for S. aureus, with synergy or additivity being seen in 60% to 80% of isolates. The meropenem/ciprofloxacin combination gave the lowest mean FIC for P. aeruginosa isolates, with 90% showing an additive or synergistic effect. The other combinations elicited a predominantly indifferent response. No consistent antagonistic effect was observed with the combinations used. CONCLUSIONS The combination of meropenem and ciprofloxacin was predominantly additive or synergistic for both S. aureus and P. aeruginosa. Teicoplanin combined with meropenem, ciprofloxacin, or moxifloxacin was also predominantly additive or synergistic against S. aureus.

Quantifying changes in corneal neovascularization using fluorescein and indocyanine green angiography

PURPOSE To quantify changes in corneal neovascularization in patients with active keratitis after treatment using color imaging, fluorescein angiography (FA), and indocyanine green angiography (ICGA). DESIGN Prospective, interventional case series. METHODS Twelve consecutive patients were studied. A comparison of corneal neovascularization parameters was undertaken before and after resolution of the keratitis. A slit-lamp digital camera acquired images of the neovascularization using color imaging, FA, and ICGA. The best-quality images were selected using a grading system, and the neovascular regions of interest were analyzed using automated in-house software. The parameters of analysis were vessel area, diameter, tortuosity, and FA dye leakage. RESULTS There was a significant reduction in the area of neovascularization after treatment on color imaging (0.78 mm(2); P < .05), FA (2.33 mm(2); P < .01), and ICGA (2.07 mm(2); P < .01). There was also a significant reduction in mean vessel diameter across the region of interest for each patient, more marked on FA (42.74 to 32.52 μm; P < .01) and ICGA (44.77 to 33.29 μm; P < .01) than on color imaging (29.10 to 25.17 μm; P < .01). A significant change in vessel tortuosity was not observed. There was a significant increase in FA dye leakage time (12.41 seconds; P < .05) after treatment. CONCLUSIONS We demonstrate application of an objective method for analyzing changes in corneal neovascularization. The excellent vessel delineation with ICGA even in the presence of stromal scars makes it an ideal agent for measurement of vessel parameters. FA is useful at detecting vessel leakage, and the time to leakage provides a possible measure of vessel staging.

lukSF-PV in Staphylococcus aureus keratitis isolates and association with clinical outcome.

PURPOSE To determine the prevalence, genetic diversity, and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton-Valentine leukocidin, in Staphylococcus aureus isolates from cases of bacterial keratitis in the United Kingdom. METHODS Multiplex PCRs investigating carriage of lukSF-PV and mecA were performed on S. aureus isolates from patients. The lukSF-PV operon was sequenced to investigate its diversity, and multilocus sequence typing to test for a clonal relationship between lukSF-PV isolates. Antimicrobial minimum inhibitory concentrations (MICs) and clinical outcome data were compared for isolates characterized as lukSF-PV+ve, mecA+ve, and lukSF-PV/mecA-ve. RESULTS Of 95 isolates, 9 (9.5%) were lukSF-PV+ve, 9 (9.5%) mecA+ve, and 1 was positive for both. Five single nucleotide polymorphisms were found in lukSF-PV genes of seven strains. There was no significant difference between the MICs of lukSF-PV/mecA-ve and lukSF-PV+ve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecA+ve isolates had significantly higher mean MICs to meropenem and fluoroquinolones (P < 0.05). There were nonsignificant trends for healing and treatment times, ulcer and scar size, and overall clinical score to be greater in the lukSF-PV+ve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PV+ve isolates with an odds ratio of 7.8 (95% CI 1-42, P = 0.018) for patients requiring surgery. CONCLUSIONS lukSF-PV+ve isolates were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus-associated keratitis.

Recurrent bacterial keratitis.

PURPOSE To investigate the rate of recurrent bacterial keratitis, associated bacteria, and surgical intervention. METHODS Patients with suspected bacterial keratitis were identified from microbiological requests over a 16-year period between 1995 and 2010. Recurrences and number of surgical interventions were analyzed according to bacterial type. RESULTS A total of 2418 patients were included, of whom 2124 (87.84%) had only one episode of keratitis, 294 (12.15%) at least two, 88 (3.63%) at least three, 40 (1.65%) at least four, and 22 (0.91%) five or more episodes. The bacterial isolation rate was 35.74% (SD 9.41%), increasing to 56.01% in patients with two or more episodes. There was an increase in the isolation of Staphylococcus aureus with increasing number of episodes (P = 0.008), and S. aureus occurred more commonly in patients with recurrent disease due to the same bacterial group (P = 0.04). Patients whose recurrent keratitis was associated with S. aureus had a higher rate of requiring subsequent corneal transplantation (7 of 10) compared to those with Enterobacteriaceae (2 of 7), Pseudomonas aeruginosa (2 of 4), streptococci (2 of 5), or coagulase-negative staphylococci (none of 8) (P = 0.02). CONCLUSIONS S. aureus is particularly associated with recurrent keratitis. Identification and treatment of the possible source of the infection may be necessary to reduce the risk of recurrent disease. The potential for the autocthonous S. aureus colonizing the nasopharynx or conjunctiva or lid margin to be a reservoir for recurrent keratitis suggests that decolonization of S. aureus could be considered as a potential intervention in those patients with recurrent disease.

Impression membrane for the diagnosis of microbial keratitis

Purpose To evaluate a corneal impression membrane (CIM) for isolation of bacteria, fungi and acanthamoeba in suspected microbial keratitis. Methods Consecutive patients presenting with suspected microbial keratitis were included. For each patient, samples were collected in a random order using a surgical blade and a 4-mm-diameter polytetrafluoroethylene CIM disc, and transported in brain heart infusion broth. Risk factors, best corrected visual acuity (BCVA), size, location, depth and healing time of the ulcer were recorded. The microbial isolation rate was used to compare sampling methods. Results 130 patients were included (mean age 62.6 years, SD 19.0). An antimicrobial had been used prior to presentation in 36 (27.7%) patients. Mean major and minor ulcer diameters were 2.1 mm (SD 2.0) and 1.6 mm (SD 1.7). Mean healing time was 12.4 days (SD 13.6). BCVA at presentation and following healing was 0.7 (SD 0.7) and 0.62 (SD 0.7) (p=0.34). There were 66 isolates (50.8%); 53 (40.8%) using a CIM and 35 (26.9%) using a blade (p=0.02). Staphylococcus aureus and coagulase-negative staphylococci were the commonest isolates. Isolation rate was not influenced by organism type, although in four cases Acanthamoeba spp. were isolated; three using CIM and one a blade. Conclusions In this study, the isolation of microorganisms from cases of suspected microbial keratitis was significantly higher using a CIM than a surgical blade. A CIM may be a useful alternative or addition for sample collection in microbial keratitis.

Pharmacokinetics of Meropenem for Use in Bacterial Keratitis

PURPOSE To investigate the toxicity and corneal pharmacokinetics of meropenem as a potential antimicrobial for bacterial keratitis. METHODS Corneal epithelial cell and keratocyte toxicity was investigated using methyl thiazolyl tetrazolium (MTT) and LIVE/DEAD assays. The penetration of meropenem through the human cornea was measured using an artificial anterior chamber. In one group of corneas, the epithelial and endothelial layers were removed and in a second group these layers were left intact. We applied 50 μL (10 mg/mL) meropenem to the corneal surface and collected samples in the anterior chamber from 45 minutes up to 24 hours. Meropenem concentrations were estimated with a bioassay and HPLC. RESULTS Meropenem had significantly higher cellular metabolic activity (MTT assay) at both 5 mg/mL and 2.5 mg/mL compared with moxifloxacin (P = 0.029 and P = 0.018, respectively), with 96% cell viability (LIVE/DEAD assay). The measured values for meropenem concentrations in corneal and aqueous samples were significantly higher using a bioassay than with HPLC (P = 0.004). For both intact and denuded corneas, the concentrations in the anterior chamber increased from 0.48 μg/mL (SD 0.89) and 0.89 μg/mL (SD 0.81) to 6.35 μg/mL (SD 0.81) and 13.48 μg/mL (SD 14.82) using HPLC, and from 0.68 μg/mL (SD 1.50) and 1.31 μg/mL (SD 1.55) to 47.03 μg/mL (SD 5.51) and 43.69 μg/mL (SD 27.22) measured with a bioassay. CONCLUSIONS Meropenem has very low toxicity in vitro. It has good corneal penetration, achieving anterior chamber concentrations above MIC90 for bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, streptococci, coagulase-negative staphylococci, and the Enterobacteriaceae.

New Developments in Antibacterial Chemotherapy for Bacterial Keratitis

The treatment of bacterial keratitis depends on identifying the causative agent and selecting an appropriate antimicrobial.

Orlans HO, Hornby SJ, Bowler IC (2011) In vitro antibiotic susceptibility patterns of bacterial keratitis isolates in Oxford, UK: a 10-year review. Eye Jan 21 (Epub ahead of print) PMID 21252952

Bacterial keratitis remains a major cause of corneal opacity and loss of vision worldwide. Topical antimicrobial therapy is critical to its management. A number of antimicrobials are available to treat bacterial keratitis, with new antimicrobials occasionally introduced to provide an improved spectrum of activity against emerging or resistant pathogens. For example, cephalosporins were introduced in the 1960s as resistance of Staphylococcus aureus to penicillin became common, while gentamicin was introduced in the 1970s mainly due to an upsurge in Pseudomonas aeruginosa infections, associated with increasing contact lens wear use [1]. The firstand second-generation fluoroquinolones (ciprofloxacin and ofloxacin) have excellent potency against Gram-negative bacteria and reasonably good activity against the Gram-positive bacteria, with little corneal toxicity. When the thirdand fourth-generation fluoroquinolones (levofloxacin, moxifloxacin and gatifloxacin) became available in the early 2000s, they offered a broader Gram-positive cover [1–3]. Despite the success of the firstand second-generation fluoroquinolones, there has been a trend in increased resistance from both S. aureus [4] and P. aeruginosa [5]. The fourth-generation fluoroquinolones, moxifloxacin and gatifloxacin, have not unfortunately been a treatment panacea because of the emergence of resistance [6]. Deciding which antimicrobials to use for the treatment of bacterial keratitis can be challenging. It is important to be aware of the contemporaneous spectrum of likely causative bacteria, as this can vary according to the latitude and the degree of urbanisation of the population studied. Information regarding causative bacteria can only be attained by the collection of samples from the corneal ulcer and their subsequent analysis. Over time, a collection of such isolates provides knowledge not only on the likely bacteria, but also on their changing profiles against antimicrobial agents. This is also important, as treating physicians are often faced with an increasing choice of newer antimicrobial agents. The normal management strategy, if microbial infection is suspected, is to collect samples of corneal tissue for culture and then to initiate antimicrobial treatment empirically. The initial choice of treatment is either a single antimicrobial agent or a combination of antimicrobials providing a broad range of activity against both Gram-positive and Gramnegative bacteria. Once an isolate has been obtained, however, it is then possible to modify the treatment according to the in vitro susceptibility pattern of the isolate. Orlans et al. [7] report on the spectrum of bacteria isolated from cases of keratitis in Oxford and the in vitro susceptibility data over a 10-year period. This type of study provides valuable information for ophthalmologists treating suspected bacterial keratitis in the clinical setting. In this study, they found that the most commonly isolated bacteria were Staphylococci (40.1%), followed by Pseudomonas species (28.5%), other Gram-negative species (17.2%), Streptococci (7.1%), and Corynebacteriacea (6.0%), which S. Kaye (*) :R. Gilbert :H. Sueke Department of Ophthalmology, Royal Liverpool University Hospital, Prescott Street, Liverpool L7 8XP, UK e-mail: s.b.kaye@liv.ac.uk