Despite advances in diagnosis and treatment, bacterial keratitis continues to be a sight-threatening condition. In India, 15.4% of blindness is attributed to corneal diseases. Successful management of bacterial keratitis requires appropriate antibiotic therapy, which is either based on microbiological studies, such as direct smear examination and culture of corneal scrapings, or on microbial prevalence data in a given area, or both. Efficacy of ciprofloxacin (0.3%) ophthalmic solution monotherapy, in the treatment of bacterial corneal ulcers, was shown to be clinically and statistically equivalent to the standard therapy with fortified tobramycin and cefazolin. Several other studies reported similar efficacy of ofloxacin ophthalmic solution monotherapy in bacterial keratitis. But it has not taken long for bacteria to develop resistance to fluroquinolones despite the fact that the mechanism of resistance is chromosomal (modification of target enzymes such as DNA gyrase and topoisomerase IV or mutations in regulatory genes mexR and nfxB). Reports of resistance of bacterial keratitis isolates to ciproflxacin have cautioned against continued dependence of ophthalmologists on ciprofloxacin monotherapy. 
In this communication, we attempted to determine the in vitro ciprofloxacin susceptibility of Staphylococcus aureus isolates cultured from patients with microbial keratitis and to analyse the clinical features and treatment outcome of patients with ciprofloxacin resistant S.aureus keratitis.
Materials and Methods
Patients who met the criteria of having: 1) corneal stromal infiltrate, and 2) microbiological processing of the corneal scraping showing significant growth of S. aureus, were identified from the prospectively collected corneal ulcer database of LV Prasad Eye Institute, Hyderabad, India. Significant growth in culture was defined as confluent growth in any solid medium, or growth in one medium with consistent result in Gram-stained smear, or repeat isolation of the organism.
The technique and microbiological processing of corneal scrapings and interpretation of results have been described earlier.
The culture isolates were identified using standard microbiology procedures and subjected to in vitro antimicrobial susceptibility testing against several antibiotics including ciprofloxacin, using the Kirby-Bauer disk diffusion method. The isolates were considered resistant, intermediate or susceptible to an antibiotic based on the zone of inhibition (HiMedia Laboratories Pvt. Ltd., Bombay, India). With a disc containing 5µg of ciprofloxacin, the zone size of 15mm was considered resistant, 16-20mm intermediate and >20mm susceptible.
Topical antibiotic therapy of the patients was based on initial smear examination results and it was modified, if required, based on clinical response and antibiotic susceptibility profile of the culture isolate. A combination therapy with fortified gentamicin (1.4%) and fortified cefazolin (5%) was selected only if the smears showed gram-positive cocci. In cases where the smears were negative, commercially available 0.3% ciprofloxacin was started, subject to modification, if required. The topical antibiotics were applied every minute for 5 minutes and then every half hour for 48-72 hours. Clinical assessment for symptomatic relief, size and density of the infiltrate, depth of involvement, associated corneal thinning and anterior chamber reaction, was done daily and clinical impression of resolving, unchanged, or worsening was noted. Resolving status was determined based on reduction in epithelial defect, density and size of the infiltrate, clearing of the surrounding cornea and anterior chamber reaction. Worsening was defined as an increase in all these parameters on two consecutive visits. A change in antibiotic was considered only in cases that were unchanged or worsened after three days of intensive topical therapy. Penetrating keratoplasty was done whenever there was progressive increase in size of the infiltrate despite intensive medical therapy, or the infiltrate was very large with risk of limbal involvement, or there was more than 2mm perforation. Evisceration was performed in patients with extensive infection with poor visual potential.
The initially determined in vitro resistance of the S.aureus isolates to ciprofloxacin by Kirby-Bauer disk diffusion method, was further confirmed by minimum inhibitory concentration (MIC) of ciprofloxacin using commercially available sensititre® plates (Trek Diagnostic Systems Ltd., UK). The MIC of oxacillin was also determined using the same system for ciprofloxacin resistant isolates of S.aureus. The range of ciprofloxacin concentrations tested was 0.004-8µg/mL, and it was 0.008-8µg/mL, for oxacillin. An MIC of ž4µg/mL was considered resistant for ciprofloxacin as well as oxacillin, while MIC of 2µg/mL was considered intermediate sensitivity to ciprofloxacin.
The mean age of the patients was 40 years [Figure - 1] and the male to female ratio was 7:2. An analysis of the predisposing factors in these cases is shown in [Figure 2].
[Table - 1] shows the results of antibiotic susceptibility of 121 isolates of S.aureus based on the disk-diffusion method. In descending order, the highest proportion of susceptibility was to vancomycin, cefazolin, chloramphenicol, ciprofloxacin and gentamicin. Twenty-five patients had ciprofloxacin resistant S.aureus keratitis. The clinical details of these patients are given in [Table - 2]. Sixteen patients had consulted a local ophthalmologist prior to visiting us, four of whom were treated with antifungal and rest with antibacterial antibiotics. The large variation in the usage of the prior antibiotics precludes their enumeration.
As shown in [Table - 2], medical therapy was begun with 0.3% ciprofloxacin in 15 (60%) of 25 patients. Twelve (80%) of these patients showed no clinical improvement after 3-8 days of intensive topical therapy. Based on susceptibility results the antibiotic treatment was modified in all these patients to fortified cefazolin and gentamicin in 7 (58.3%), 5% vancomycin in one (8.3%), and 4% chloramphenicol in four (33.3%). Three patients continued on ciprofloxacin therapy as they were improving on initial therapy with ciprofloxacin. Eventually the corneal infiltrate healed in 11 (73.3%) of 15 eyes while two patients required penetrating keratoplasty, (PK) one required evisceration and one was lost to follow up.
Four (44.4%) of the nine patients who were initially started on fortified cefazolin and gentamicin, responded after 3-7days of therapy and required no change of antibiotics. One of these patients was lost to follow up. No improvement was seen in the remaining four patients, two of whom responded to vancomycin, one to chloramphenicol and one required PK. Patient # 25 was started and continued on chloramphenicol as the isolate was resistant to other antibiotics. He ultimately required PK due to extensive corneal involvement.
Antibiotic susceptibility results of 25 ciprofloxacin resistant (by disk-diffusion) isolates of S.aureus showed variable sensitivity to other antibiotics [Table - 3]. While 13 (76.4%) of 17 isolates were sensitive to vancomycin, the sensitivity to cefazolin was 48% (12/25), to chloramphenicol was 44% (11/25), and to gentamicin was 12% (3/25). Resistance or intermediate sensitivity to these antibiotics could not confirmed by testing for MIC. However, MICs of ciprofloxacin and oxacillin could be determined in 16 isolates of S.aureus that were available. Ciprofloxacin resistance was confirmed in 15 (93.8%) of 16 isolates and 13 (81.2%) of 16 isolates were found resistant to oxacillin indicating methicillin resistance [Table - 3].
Year-wise distribution of S.aureus isolates and the proportion of ciprofloxacin resistant S.aureus are shown in [Table - 4].
Introduced in 1980s by modification of nalidixic acid, fluoroquinolones are known to have a much wider spectrum of antibacterial activity compared to several other antimicrobials. The bactericidal activity is known to be more towards gram-negative rather than gram-positive bacteria. Not surprisingly, infectious disease literature first reported resistance in gram-positive bacteria, notably S.aureus. Very high levels of resistance of non-ocular isolates of S.aureus to ciprofloxacin, reaching up to 76-82%, have been reported.
Such reports of development of resistance to ciprofloxacin among ocular isolates are relatively recent.  Nevertheless, they have caused discomfiture among ophthalmologists who were satisfied with the various advantages of ciprofloxacin monotherapy over dual therapy with fortified antibiotics for bacterial keratitis. In an earlier publication from our institute we reported a general trend of increased insensitivity to ciprofloxacin in most organisms except Streptococcuspneumoniae and S.aureus. These organisms had shown a significant level of resistance but without a trend. The findings of the present study, with respect to yearly distribution of ciprofloxacin resistant S.aureus isolates [Table - 4], confirm our earlier observation of lacking a trend. There is no significant linear trend showing either an increase or decrease in levels of resistance. The overall ciprofloxacin resistance of S.aureus isolates from bacterial keratitis was 20.6% with a range of 10-35% over the years. Our findings are in contrast to those of others who have reported a steady rise in S.aureus resistance to ciprofloxacin. While Goldstein et al reported an annual increase from 5.8% in 1993 to 35.0% in 1997, Alexandrakis et al reported an increase from 11% in 1990 to 28% in 1998. Although we recognise the limitations of results from a single tertiary care center; the patients seen at our institute presented an alarming situation right from 1993 (35%). The clinical utility and effectiveness of ciprofloxacin in the treartment of staphylococal keratitis is therefore seriously questionable and newer antibiotics with enhanced activity against gram-positive bacteria seem overdue.
While considering a reasonable second-line therapy for ciprofloxacin-insensitive organisms in our earlier publication, we had reported that cefazolin, a first generation cepholosporin, had the greatest (88.4%) coverage against gram-positive cocci. Quite close (81.6%) to the earlier finding was the overall in vitro cefazolin susceptibility of 121 isolates of S.aureus in this study. But, only 48% (12/25) of the ciprofloxacin resistant S.aureus were found sensitive to cefazolin [Table - 3]. This finding was also reflected in the treatment outcome. Resolution of corneal infiltrate was achieved in only 44-70 % of cases of ciprofloxacin resistant S.aureus keratitis irrespective of whether they were started on topical fortified cefazolin (44.4%) or changed over to topical fortified cefazolin after initial treatment with ciprofloxacin (5/7, 71.4%). These results point towards existence of multi-drug resistance among our patients with S.aureus keratitis. The finding of methicillin resistance among our isolates further highlights this. A concomitant methicillin resistance (indicated by oxacillin resistance) was noted in 13 of 16 isolates of S.aureus tested in this study. Such a correlation has not been shown earlier with ocular isolates although it was reported long back in systemic infections. On the contrary there have been reports of ciprofloxacin being effective in keratitis due to methicillin-resistant S.aureus (MRSA).
Infections caused by MRSA are common in India and we believe that prevalence of MRSA in the community coupled with ciprofloxacin resistance, account for the high level of resistance to ciprofloxacin and other antibiotics seen among S.aureus isolates in this study. These findings have added credence to our earlier observations on the debatable role of ciprofloxacin monotherapy in bacterial keratitis.
Presently, vancomycin is a highly active antibiotic against gram-positive bacteria including MRSA and it can be a final choice for treatment of S.aureus keratitis resistant to all other antibiotics. A 5% eye drop of vancomycin was used in three patients in this study (Patient # 12, 16, 18) with good results; however, we do not recommend its usage without first trying other options. Being the most important drug available today against methicillin-resistant staphylococci, vancomycin must be sparingly used. Moreover, added to the inconvenience of reconstitution, the topical application is often painful to the patient owing to low pH (2.5-4.5). Of the four methods applied to combat the appearance and spread of antibiotic resistance, namely, avoidance of indiscriminate use of the antibiotic, use of combination therapy, use of increased concentrations of the drug and continued development of new antibiotics; clearly a need for the latter has come, as far as treatment of bacterial keratitis is concerned. Newer fluoro-quinolones with broader spectrum of activity may hold a promise for such a future, especially if we prefer to stay with the convenience of monotherapy in bacterial keratitis.
Source of Support:
Conflict of Interest:
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