Antibiotic Susceptibility of Conjunctival Bacterial Isolates from Refractive Surgery Patients

Transcription

1 Antibiotic Susceptibility of Conjunctival Bacterial Isolates from Refractive Surgery Patients Jae Lim Chung, MD, 1 Kyoung Yul Seo, MD, 1 Dong Eun Yong, MD, 2 Francis S. Mah, MD, 3 Tae-im Kim, MD, 1 Eung Kweon Kim, MD, 1 Jin Kook Kim, MD 4 Purpose: To determine the in vitro antibiotic susceptibility patterns of conjunctival bacterial flora isolated before surgery from patients undergoing refractive surgery. Design: In vitro laboratory investigation. Participants: One hundred five eyes from 105 patients scheduled for refractive surgery at Balgensesang Ophthalmology Clinic between September 2005 and January 2006 were studied. Among 105 patients, 71 (67.6%) underwent LASIK using a femtosecond laser, 24 (22.9%) underwent LASIK using an automated microkeratome, 8 (7.6%) underwent LASEK, and 2 (1.9%) patients underwent phakic intraocular lens implantation. Methods: Preoperative conjunctival swab samples were inoculated directly in culture media at the bedside before topical anesthetic or antibiotic application. Blood agar, chocolate agar, thioglycolate broth, Sabouraud dextrose agar, and Ogawa media were used for bacterial, fungal, and mycobacterial cultures. Main Outcome Measures: Minimum inhibitory concentrations (MICs) of ofloxacin (OFX), levofloxacin (LEV), gatifloxacin (GAT), moxifloxacin (MOX), gemifloxacin (GEM), and other commonly used antibiotics were determined using an E test. Results: From 105 patients, 73 (85%) coagulase-negative staphylococci (CNS), 2 (2.3%) Staphylococcus aureus, 1 (1.2%) Streptococcus pneumoniae, and 5 (4.8%) gram-negative bacilli were isolated. No fungi or mycobacteria were isolated. The MIC that would inhibit the growth of 90% of the tested bacterial isolates (MIC 90 ) of OFX, LEV, GAT, MOX, and GEM for methicillin-susceptible CNS (n 46) were 0.5 g/ml, 0.19 g/ml, g/ml, g/ml, and g/ml, respectively. The MIC 90 values for methicillin-resistant CNS (n 27) were 32 g/ml, 4 g/ml, 1 g/ml, 0.5 g/ml, and 0.25 g/ml, respectively (P 0.001). Conclusions: The most effective against conjunctival bacteria isolated from refractive surgery patients were GEM, MOX, and GAT; however, resistance to earlier-generation fluoroquinolones (OFX and LEV) is increasing among methicillin-resistant CNS. It may be a therapeutic option to use newer fluoroquinolones in patients undergoing refractive eye surgery to reduce such infections as methicillin-resistant CNS. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2009;116: by the American Academy of Ophthalmology. The surface of the eye is rich in nutrients and, consequently, supports a diverse range of microorganisms that constitute the normal ocular flora. 1 These microorganisms interact with each other and with the immune system of the host. The consequence of these interactions is that, under normal conditions, the growth of individual microorganisms is regulated and eye infection is prevented. Infection of the eye results from either the acquisition of a virulent microorganism or uncontrolled growth of an existing organism because of lowered host resistance. LASIK is the most commonly performed refractive surgical procedure for the correction of ametropia worldwide. Infectious keratitis, although rare, is a potentially devastating complication of LASIK and can lead to horrifying outcomes. The incidence of infectious keratitis is difficult to estimate and can vary widely depending on the source of information. In a comprehensive review and analysis of the literature, Chang et al 2 stated that the incidence of infections after LASIK can vary widely (0% 1.5%); however, reported cases have become increasingly more common in recent years. 3,4 Karp et al 3 reported an estimated incidence between 0.02% and 0.1% of LASIK-associated infections at a single institution in Florida. More recently, Moshirfar et al 4 reported a 0.31% incidence in eyes in the Utah area. Resistance of bacteria to antimicrobial agents is a worldwide concern. In Korea, resistant bacteria are more prevalent than in other industrialized countries. 5 According to the Korean Nationwide Surveillance of Antimicrobial Resistance program, methicillin-resistant staphylococci, penicillin nonsusceptible pneumococci, -lactamase producing gonococci, extended-spectrum -lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, fluoroquinolone-resistant E. coli, aminoglycoside-resistant Acinetobacter species, and imipenem-resistant Pseudomonas aeruginosa are examples of resistant bacteria prevalent in Korea. 6 Ciprofloxacin by the American Academy of Ophthalmology ISSN /09/$ see front matter Published by Elsevier Inc. doi: /j.ophtha

2 Ophthalmology Volume 116, Number 6, June 2009 resistance rates of E. coli, Acinetobacter species, and P. aeruginosa were 27%, 70%, 42%, respectively, in a Korean Nationwide Surveillance of Antimicrobial Resistance 2000 study 7 versus 5.5%, 52.4%, 26.5%, respectively, in a Tracking Resistance in the United States Today surveillance 2000 study. 8 According to data from the global Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin surveillance program, levofloxacin resistance in pneumococci was low overall (1% worldwide), with higher rates in Hong Kong (14.3%) and South Korea (2.9%) and a very low incidence in European countries. 9 These differences between countries suggest a high level of antimicrobial selective pressure because of high systemic antimicrobial use, use of antibiotics in animal feed, and the nosocomial spread of resistant bacteria. The resistance rates of nosocomially acquired bacteria are generally higher than those of community-acquired ones. Most reports on antimicrobial resistance are based on systemic isolates (not ocular isolates) from large, tertiary care hospitals; therefore, these reported resistance rates are influenced by the prevalence of nosocomial infections. The analysis of resistance of isolates from outpatients also may fail to show the true resistance rates of community-acquired pathogens that comprise most ophthalmologic infectious diseases. Currently, most refractive surgeries are performed at outpatient clinics; however, the Korean Nationwide Surveillance of Antimicrobial Resistance 2003 study revealed similar fluoroquinolone-resistance rates of E. coli in commercial laboratories (33%); that study tested a large number of specimens submitted mostly from primary care clinics and in tertiary care hospitals (32%). This suggests a spread of fluoroquinolone resistance in the community. 10 A Taiwanese surveillance study 11 in 2000 and the Tracking Resistance in the United States Today 2000 study 8 showed similar results. A number of recent studies have reported emerging fluoroquinolone resistance among ocular isolates, particularly among gram-positive organisms Goldstein et al 13 reported that resistance of staphylococcus aureus to ciprofloxacin and ofloxacin increased 7-fold from 1993 to Significant resistance to these older fluoroquinolones among streptococci and Coagulase-negative staphylococci (CNS) also was found. Emerging ciprofloxacin resistance among ocular gram-negative isolates such as P. aeruginosa also has been reported. 15,16 This study evaluated 5 fluoroquinolones, including gemifloxacin (7-pyrrolidinyl fluoroquinolone). Gemifloxacin is the most recently Food and Drug Administration-approved fourth-generation fluoroquinolone with enhanced affinity for the topoisomerase IV and DNA gyrase of gram-positive organisms. Currently, its main indication is for the treatment of community-acquired pneumonia; a topical ophthalmic agent has not yet been developed. 17 The aim of this study was to determine the in vitro antibiotic susceptibility patterns of conjunctival bacterial flora isolated before surgery from patients undergoing refractive surgery at an outpatient laser surgery clinic. Patients and Methods One hundred five eyes from 105 patients scheduled for refractive surgery at Balgensesang Ophthalmology Clinic between September 2005 and January 2006 were studied. The approval of the institutional review board and informed consent from each patient were obtained. Preoperative demographic characteristics and details of refractive surgeries performed are shown in Table 1. Seventy-five (71.4%) patients were female with a mean age of years (range, years). Seventy-six (72.4%) patients were previous contact lens wearers. All contact lens wear was discontinued at least 3 weeks before surgery. Any recent (at least 3 months) user of antibiotics that could alter the status of conjunctival flora was excluded. The most common refractive surgical procedure in this study was LASIK using a femtosecond laser, in 71 (67.6%) patients, followed by 24 (22.9%) who underwent LASIK using an automated microkeratome, 8 (7.6%) LASEK (laser epithelial keratomileusis) procedures, and 2 (1.9%) phakic intraocular lens implantations. Preoperative conjunctival cultures were obtained on the day of surgery before application of topical antibiotics, anesthetics, and povidone iodine. The patient was asked to look up, and the inferior cul de sac was swabbed using a sterile cotton tip without touching the eyelid margin or lashes. To improve culture yields, conjunctival swab samples were inoculated directly in culture media at the bedside without using transport media. Five percent sheep blood agar, chocolate agar, thioglycolate broth, Sabouraud dextrose agar, and 3% Ogawa media (equivalent to Lowenstein-Jensen media) were used for bacterial, fungal, and mycobacterial culture. Blood agar plates were incubated with 5% carbon dioxide to encourage aerobic and microaerophilic bacterial growth. The chocolate agar plates were incubated in an anaerobic chamber for isolation of anaerobic bacteria. All cultures were incubated at 37 C for at least 14 days. If Staphylococci grew, CNS were differentiated from S. aureus by coagulase production. Methicillin resistance was confirmed by the 30- g cefoxitin disc diffusion method, 18 which is currently recommended by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) standard for the detection of methicillin resistance in CNS. Minimum inhibitory concentrations (MICs) of ofloxacin (OFX), levofloxacin (LEV), gatifloxacin (GAT), moxifloxacin (MOX), gemifloxacin (GEM), and other commonly used antibiotics Table 1. Preoperative Demographics and Details of Refractive Surgeries Performed Demographic or Clinical Variable Descriptive Statistic Gender Male 30 (28.6%) Female 75 (71.4%) Age (yrs) Mean SD (minimum maximum) (19 48) Contact lens wear None 29 (27.6%) Soft lens 66 (62.9%) Hard lens 4 (3.8%) Both 6 (5.7%) Refractive surgery performed LASIK using femtosecond laser 71 (67.6%) LASIK using automated microkeratome 24 (22.9%) LASEK 8 (7.6%) Phakic intraocular lens implantation 2 (1.9%) LASEK laser epithelial keratomileusis; SD standard deviation. 1068

3 Chung et al Antibiotic Susceptibility of Flora from Refractive Surgery Fluoroquinolones Ofloxacin Levofloxacin Gatifloxacin Moxifloxacin Gemifloxacin Cephalosporins Cephalothin Cefotaxime Carbapenems Imipenem Table 2. List of Antibiotics Tested (Table 2) were determined using the E test (AB Biodisk, Piscataway, NJ). The E test is a method for measuring MICs of antimicrobial agents against microorganisms and is based on diffusion of a preformed antibiotic gradient from a plastic strip. Correlation between MICs by the agar dilution and E test methods was more than 95%. 19,20 The versatility and ease of use of the E test make the method an attractive alternative to conventional dilution tests. Test inoculums for each isolate and quality control strains were prepared using a 0.5 McFarland standard and then swabbed onto a 150-mm Mueller-Hinton agar plate. E test strips were dropped on a plate for each isolate. Plates were then incubated for 18 to 24 hours in a non-co 2 incubator. The MIC value (E test range, g/ml) was read at 18 to 24 hours as the point where the edge of the growing culture intersected the strip. Classification of isolates as sensitive, intermediate, and resistant were in accordance with Clinical and Laboratory Standards Institute break points for MICs. 21 These standards are based on safe, achievable concentrations of antibiotics in the serum. There are no standards for topical ocular therapy that represent the concentrations of antibiotics in the ocular tissue, but the serum standards can be used to interpret susceptibility if it is assumed the antibiotic concentrations in the ocular tissues are equal to or more than the antibiotic concentrations in the serum. When comparing MICs, antibiotics with lower MICs have greater antibacterial activity. The MIC values of more than 2 g/ml were recorded as resistant for GAT, MOX, and GEM. For OFX and LEV, the resistance cutoff value was more than 4 g/ml. The MIC values were analyzed nonparametrically as discrete data. The antibiotic potencies of all fluoroquinolones against CNS were compared using the Kruskal-Wallis test (MedCalc software version 9.2; MedCalc, Mariakerke, Belgium). The analysis ranked all MICs from the lowest to highest and compared the antibiotics by analysis of variance analysis of ranks (not the actual MICs) using the Duncan multiple comparisons at P 0.05 significance (SPSS software version 12.0; SPSS, Inc., Chicago, IL). Table 3. Conjunctival Flora Culture Results from Refractive Surgery Patients According to Contact Lens-Wearing History Contact Lens History Nonwearer Wearer Aminoglycosides Tobramycin Amikacin Glycopeptides Vancomycin Macrolides Erythromycin Clarithromycin Others Chloramphenicol Total No growth 7 (24.1%) 13 (17.1%) 19 (18.1%) Growth 22 (75.9%) 63 (82.9%) 86 (81.9%) Total 29 (27.6%) 76 (72.4%) 105 There were no statistical differences between groups (P 0.587, Fisher exact test). Table 4. Conjunctival Flora Culture Results from Refractive Surgery Patients Results Bacterial Culture No. of Samples (n 105) Gram positive 87 (82.9%) Coagulase-negative staphylococci Methicillin susceptible 46 (53.5%) Methicillin resistant 27 (31.4%) Staphylococcus aureus Methicillin susceptible 2 (2.3%) Methicillin resistant 0 (0) Streptococcus pneumoniae 1 (1.2%) -Streptococcus 3 (3.5%) Diphtheroid 8 (9.3%) Gram negative 5 (4.8%) Pseudomonas putida 1 (1.2%) Comamonas acidovorans 1 (1.2%) Enterobacter cloacae 1 (1.2%) Klebsiella oxytoca 1 (1.2%) Serratia marcescens 1 (1.2%) Fungi 0 (0) Atypical mycobacterial species 0 (0) Several patients showed growth of multiple organisms. Among the 105 patients whose conjunctival specimens were examined, 86 (81.9%) patients had positive bacterial growth (Table 3). When the growth rates were compared according to contact lens wearing history, there were no statistical differences between groups (P 0.587, Fisher exact test). These results agreed with those of previous reports, 22,23 which may be because all contact lens wear was discontinued at least 3 weeks before refractive surgery. The identified organisms are summarized in Table 4. Coagulase-negative staphylococci was the most commonly isolated bacterial species and was found in 73 (84.9%) patients. Among the 73 subjects with CNS, 46 (53.5%) organisms were methicillin-susceptible CNS and 27 (31.4%) were methicillinresistant CNS. Other gram-positive microorganisms included methicillin-susceptible S. aureus (n 2; 2.3%), S. pneumoniae (n 1; 1.2%), -streptococci (n 3; 3.5%), and diphtheroids (n 3; 3.5%). Isolated gram-negative rods were identified as Pseudomonas putida, Comamonas acidovorans, Enterobacter cloacae, Klebsiella oxytoca, and Serratia marcescens from 1 (1.2%) patient. Isolates from several patients grew multiple organisms. No fungal or mycobacterial species were isolated. The MICs that would inhibit the growth of 90% of the tested bacterial isolates (MIC 90 ) of OFX, LEV, GAT, MOX, and GEM for methicillin-susceptible CNS (n 46) were 0.5 g/ml, 0.19 g/ml, g/ml, g/ml, and g/ml, respectively, and those for methicillin-resistant CNS (n 27) were 32 g/ml, 4 g/ml, 1 g/ml, 0.5 g/ml, and 0.25 g/ml, respectively (P 0.001, Kruskal-Wallis test; Table 5). These results suggest that the MICs of methicillin-resistant CNS were statistically higher than susceptible CNS in all 5 fluoroquinolones. In the methicillinresistant CNS group, OFX demonstrated high-level resistance (32 g/ml), and LEV showed low-level resistance (4 g/ml). In the methicillin-susceptible CNS group, all isolates showed 100% susceptibility to all 5 fluoroquinolones; however, in the methicillinresistant CNS group, 11.1% of isolates were resistant to OFX and LEV and 11.1% and 3.7% of isolates showed intermediate resistance to GAT and MOX, respectively, whereas no isolates dem- 1069

4 Ophthalmology Volume 116, Number 6, June 2009 Table 5. Comparative In Vitro Activities of Antibiotics against Organism (No.) MIC Ofloxacin Levofloxacin Gatifloxacin Moxifloxacin Gemifloxacin Cephalothin Methicillin-susceptible MIC CNS (46) MIC Range S/I/R 100/0/0 100/0/0 100/0/0 100/0/0 100/0/0 100/0/0 Methicillin-resistant MIC CNS (27) MIC Range S/I/R 88.9/0/ /0/ /11.1/0 96.3/3.7/0 100/0/0 96.3/3.7/0 S. aureus A (1) MIC and 0.5 S 0.19 S S S S 0.38 S S. aureus B (1) Susceptibility 0.5 S S S 0.32 S S 0.19 S S. pneumoniae (1) 2 S 1 S 0.1 S S S 1.5 I K. oxytoca (1) 0.25 S S S S S 3 S S. marcescens (1) 1 S 0.25 S 0.25 S 0.25 S 0.25 S 256 R E. cloacae (1) 0.19 S 0.94 S S S S 256 R P. putida (1) 2 S 1 S 0.38 S 0.5 S 0.38 S 256 R C. acidovorans (1) 0.38 S 0.19 S S S S 24 I C. acidovorans Comamonas acidovorans; CNS coagulase-negative staphylococci; E. cloacae Enterobacter cloacae; I intermediate; K. oxytoca bacterial isolates; MIC 90 antibiotic concentration that would inhibit the growth of 90% of the tested bacterial isolates; P. putida Pseudomonas putida; *Measured by E test strips; range, g/ml. Susceptibility percentage of isolates. MIC value and susceptibility of each microorganism against each antibiotic. onstrated either complete or intermediate resistance to GEM. Other gram-positive (except CNS) and gram-negative organisms (n 8) showed all susceptible MIC results against 5 fluoroquinolone agents. Methicillin-susceptible CNS isolates showed 100% susceptibility to cephalosporins, vancomycin, and imipenem, but in the methicillin-resistant CNS group, 3.7% of isolates showed intermediate resistance to cephalothin and vancomycin, 29.6% of isolates demonstrated intermediate resistance to cefotaxime, and 11.1% of isolates were resistant to imipenem (Table 5). In the methicillinsusceptible CNS group, the MIC that would inhibit the growth of 50% of the tested bacterial isolates and the MIC 90 values for imipenem were similar to those for GEM. Isolates from the methicillin-susceptible CNS group showed 100% susceptibility to amikacin, erythromycin, and clarithromycin, but 41.3% and 34.8% of isolates had intermediate resistance to tobramycin and chloramphenicol, respectively. In methicillinresistant CNS isolates, only amikacin showed 96.6% susceptibility. Tobramycin, erythromycin, clarithromycin, and chloramphenicol showed 59.3%, 59.3%, 59.3%, and 18.5% resistance, respectively, and all the MIC 90 values were more than 32 g/ml (high-level resistance). Gram-negative organisms demonstrated resistance to erythromycin, clarithromycin, and chloramphenicols (Table 5). A statistical comparison of MICs of 5 fluoroquinolones against methicillin-susceptible CNS demonstrated that the potency of newer fluoroquinolones were statistically superior compared with older fluoroquinolones (GEM MOX GAT LEV OFX; P 0.001). In methicillin-resistant CNS, there was also a statistically significant superiority of newer fluoroquinolones to older fluoroquinolones, but there were no statistical differences among fourth-generation fluoroquinolones (GEM MOX GAT LEV OFX; P 0.04; Table 6). Discussion Previous studies of antibiotic susceptibility of preoperative ocular bacterial flora focused primarily on intraocular surgeries, with particular interests in endophthalmitis This study was aimed at refractive surgeries with special interest in conjunctival bacterial flora that could predispose patients to infectious keratitis after LASIK. Intraocular surgeries for cataract, glaucoma, or retinal disease are performed mostly on aged ( 50 years) patients at a large hospital on an inpatient or outpatient basis; however, most refractive surgeries are performed on young (20 40 years), healthy people in private clinics on an outpatient basis. Refractive surgeries should not be performed on patients younger than age 20. The patient populations therefore are quite different between these 2 types of surgery. In this study, refractive surgery patients were mostly young, female adults, and most of them were previous contact lens wearers. The most prevalent microorganism isolated from these patients was CNS. This finding is consistent with those of previously published studies CNS are among the most frequently isolated bacteria in clinical microbiology laboratories and are becoming increasingly important, especially as causes of opportunistic and hospital-acquired infections. These bacteria are normal inhabitants of human skin and mucous membranes. CNS and other gram-positive bacteria, including Bacillus subtilis Table 6. Statistical Comparison of Antibacterial Potencies of 5 Fluoroquinolones for Coagulase-Negative Staphylococcal Isolates Coagulase-Negative staphylococci Antibacterial Potency P Value Methicillin susceptible GEM MOX GAT LEV OFX Methicillin resistant GEM MOX GAT LEV OFX 0.04 GAT gatifloxacin; GEM gemifloxacin; LEV levofloxacin; MOX moxifloxacin; OFX ofloxacin. 1070

5 Chung et al Antibiotic Susceptibility of Flora from Refractive Surgery Conjunctival Bacterial Isolates from Refractive Surgery Patients Vancomycin Cefotaxime Imipenem Tobramycin Amikacin Erythromycin Clarithromycin Chloramphenicol /0/0 100/0/0 100/0/0 58.7/41.3/0 100/0/0 100/0/0 100/0/0 65.2/34.8/ /3.7/0 70.4/29.6/0 88.9/0/11.1 0/40.7/ /3.7/0 33.3/7.4/ /0/59.3 0/81.5/ S 3 S S 0.38 S 3 S 0.25 S 4 I 0.25 S 2 S 1 S S S 1.5 S S 3 I S 3.8 I 0.38 S S 64 R 96 R 256 R 3 R 256 R 256 R 0.47 S 0.25 S 1 S 2 S 64 R 3 S 96 R 256 R 0.25 S 0.38 S 4 S 3 S 64 R 12 I 128 R 256 R 0.19 S 0.5 S 0.75 S 2 S 64 R 8 I 48 R 256 R 24 I 0.5 S 0.38 S 2 S 16 R 256 R 32 R 256 R S S 8 I 12 S 64 R 24 R 128 R Klebsiella oxytoca; MIC minimal inhibitory concentration; MIC 50 antibiotic concentration that would inhibit the growth of 50% of the tested R resistant; S susceptible; S. aureus Staphylococcus aureus; S. marcescens Serratia marcescens; S. pneumoniae Streptococcus pneumoniae. and lactobacilli but not S. aureus, can produce antibiotic peptides called lantibiotics. 28 These peptides may explain their effective colonization of skin. Lantibiotic production also may play a substantial role in bacterial interference in skin and mucous membranes by excluding competing organisms that are sensitive to their bactericidal activities. An important factor in the pathogenesis of foreign body associated infections of CNS is the ability of these bacteria to colonize the polymer surface (e.g., intraocular lens) by formation of a thick, multilayered biofilm. 28 Biofilm formation takes place in 2 phases. The first phase involves the attachment of bacteria to polymer surfaces of invasive medical instruments with host extracellular matrix proteins (e.g., along the LASIK interface or cataract incision wound). In the second phase, the bacteria proliferate and accumulate into multilayered cell clusters. Treatment of CNS infection is difficult because many CNS carry multiple antibiotic resistances naturally and after a biofilm is formed, the penetration of antibiotics is hampered. Based on a study conducted in Japan, Kato and Hayasaka 27 reported the incidence of methicillin-resistant CNS isolated from clinically healthy conjunctiva as 1.6% in Watanabe et al 29 also reported a 0.5% incidence of methicillin-resistant CNS in In this study, the frequency of methicillin-resistant CNS was much higher than previous reported. CNS were isolated in 84.9% patients, among which 53.5% were methicillin-susceptible CNS and 31.4% were methicillin-resistant CNS, whereas the 2 S. aureus isolates were both methicillin susceptible. These findings suggest that methicillin-resistant CNS strains have disseminated in the community. Methicillin-resistant CNS isolates, as well as methicillinresistant S. aureus (MRSA) isolates carry the methicillin-resistance gene meca, encoded by a mobile genetic element designated staphylococcal cassette chromosome mec (SCCmec). The meca gene encodes an additional penicillin-binding protein (PBP), PBP2a, which has low affinity for all lactam antibiotics. 30 PBP2a is a high molecular-weight class B transpeptidase that catalyzes the formation of crossbridges in the bacterial cell wall peptidoglycan, and it can confer multiple drug resistance. Methicillin-resistant CNS may serve as a reservoir for antibiotic-resistant genes that can be transferred to other gram-positive organisms, including strains of S. aureus. 31 Although it was not isolated in this study, emergence of community-associated MRSA (CA-MRSA) is another growing threat throughout the world. 32 CA-MRSA strains are commonly associated with skin and soft tissue infections; however, they can cause unusually severe disease including necrotizing fasciitis, sepsis, endocarditis, and pneumonia. 33 Infections occur predominantly among healthy, community-dwelling persons who lack traditional risk factors for MRSA. Healthcareassociated MRSA typically is resistant to clindamycin and other non -lactam antimicrobials. Large type II-III SCCmec elements are responsible for this multidrugresistant phenotype of healthcare-associated MRSA. Strains of CA-MRSA carry smaller SCCmec type IV or V and Panton-Valentine leukocidin (PVL) gene. They are resistant to -lactams and 1 or 2 other drug classes but susceptible to clindamycin and other non -lactam antibiotics. Both CA-MRSA and healthcare-associated MRSA strains now circulate in the community. 34 The natural habitat of staphylococci as a common colonizer of skin and mucous membranes probably contributed in part to opportunities for staphylococci exposure to fluoroquinolones that are used for conventional treatment indications. Fluoroquinolone resistance in staphylococci is chromosomally mediated and commonly is associated with 1071

6 Ophthalmology Volume 116, Number 6, June 2009 point mutations in the subunits of the 2 intracellular target enzymes of fluoroquinolones, topoisomerase IV (ParC, ParE mutation) and DNA gyrase (GyrA, GyrB mutation), and less commonly with overexpression of NorA, a multidrug efflux protein. 35 Exposure to subinhibitory concentrations of fluoroquinolones may select sequentially for mutations that can raise the MIC of fluoroquinolones for CNS to resistant levels in vitro and is one possible mechanism promoting the emergence of resistance in vivo. Venezia et al 36 reported that growth in the presence of various fluoroquinolones increased the proportion of the meca-positive heteroresistant S. aureus that developed methicillin resistance. Ciprofloxacin exhibited a more than 1000-fold greater effect of developing methicillin resistance than the newer quinolones. The resultant methicillin-resistant strains also showed a 1.5- fold to 3-fold increase in fluoroquinolone MICs. Resistance to methicillin emerged as early as 8 hours after exposure to this fluoroquinolone. This phenomenon is not caused by the fluoroquinolone acting as a mutagen, but rather is the result of selection of more fluoroquinolone-resistant subpopulations, which also were predominantly more resistant to methicillin. The mechanism of resistance to the 2 drug classes (fluoroquinolone and methicillin) are unrelated, but their selective emergence tends to be associated. 37 In this study, MIC 90 s of fluoroquinolones for methicillinresistant CNS were higher than for methicillin-susceptible CNS. In the methicillin-resistant CNS group, OFX demonstrated high-level resistance (32 g/ml) and LEV showed low-level resistance (4 g/ml). Low-level in vitro resistance (single-step mutants) may not necessarily translate into a clinical treatment failure because the tissue levels that can be achieved with topical dosing may be much higher than that typically achieved after systemic dosing. By contrast, high-level resistant isolates (multistep mutants) are more likely to be associated with treatment failure because the MICs of the isolate may not be achievable even with a topical route of delivery. 14 Preventing the development of resistance, primary use of the newer fluoroquinolones actually may be a better strategy than initial use of an older fluoroquinolone. The use of newer fluoroquinolones may help to avoid selection of resistant mutants. The conventional strategy of reserving newer antimicrobial agents for use only when initial treatment with the older antimicrobial fails may not be a wise strategy if applied to the fluoroquinolone class of antibiotics. Primary use of an older fluoroquinolone is more likely to lead to acquisition of a first-step mutation. After a bacterial population is already preenriched for first-step mutants, subsequent mutations can be acquired readily, thus impairing the value of newer fluoroquinolones. In this study, GEM was statistically superior to MOX that was superior to GAT that was superior to LEV and OFX in the methicillin-susceptible CNS group. Regarding methicillin-resistant CNS, GEM, MOX, and GAT were equivalents in terms of susceptibility, but were superior statistically to LEV and OFX. Because there were no resistant isolates against fourth-generation quinolones (GEM, MOX, GAT) among 2 CNS groups, all 3 agents may be used effectively. GEM is the most recent Food and Drug Administration-approved fourth-generation quinolone for the treatment of community-acquired pneumonia and chronic bronchitis. It is freely soluble at a neutral ph and is highly lipophilic; hence, its tissue penetration is good. 38 The reported adverse events in clinical trials were diarrhea, nausea, rash, and mild phototoxicity. 39 In this study, no mycobacterium species or fungi were detected, and staphylococci were the major isolates. Chang et al 2 reported that the most common causative organisms of infectious keratitis after LASIK, from 1993 to 2003, were atypical mycobacteria (28%) and staphylococci (20%). A recent American Society of Cataract and Refractive Surgery survey for the year 2004) revealed a significant decrease in atypical mycobacteria, with only 2 cases reported. 40 Sixty-one percent of cases reported in this survey were the result of staphylococci. Forty-eight infections were reported by 46 surgeons who had performed an estimated procedures. It is likely that the widespread prophylactic use of fourthgeneration fluoroquinolones has altered significantly the occurrence patterns of infectious keratitis. 41 In conclusion, the most frequently isolated microorganism from refractive surgery patients was CNS. The frequency of methicillin-resistant CNS in the community was substantially higher than that reported in previous studies. GEM, MOX, and GAT were most active against conjunctival bacteria isolated from refractive surgery patients; however, resistance against lower-generation fluoroquinolones (OFX and LEV) is increasing among methicillin-resistant CNS. If GEM can be developed as an ophthalmic agent, it will provide another powerful weapon against ophthalmic infectious disease. It may be a therapeutic option to use newer fluoroquinolones after refractive surgery to reduce such infections as methicillin-resistant CNS. References 1. Armstrong RA. The microbiology of the eye. Ophthalmic Physiol Opt 2000;20: Chang MA, Jain S, Azar DT. Infections following laser in situ keratomileusis: an integration of the published literature. Surv Ophthalmol 2004;49: Karp CL, Tuli SS, Yoo SH, et al. Infectious keratitis after LASIK. Ophthalmology 2003;110: Moshirfar M, Welling JD, Feiz V, et al. Infectious and noninfectious keratitis after laser in situ keratomileusis: occurrence, management, and visual outcomes. 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8 Footnotes and Financial Disclosures Ophthalmology Volume 116, Number 6, June 2009 Originally received: May 28, Final revision: December 26, Accepted: December 29, Available online: April 22, Manuscript no Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. 2 Research Institute of Bacterial Resistance, Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea. 3 Charles T. Campbell Ophthalmic Microbiology Laboratory, University of Pittsburgh, Pittsburgh, Pennsylvania. 4 Balgensesang Ophthalmology Clinic, Seoul, Korea. Presented as a poster at: Association for Research in Vision and Ophthalmology Annual Meeting, May 2007, Fort Lauderdale, Florida. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Supported by Yonsei University College of Medicine, Seoul, Korea (faculty research grant no.: ). Correspondence: Kyoung Yul Seo, MD, Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul , Korea. seoky@yuhs.ac. 1074