ARTICLE

A New Target for Staphylococcus aureus Associated With Keratitis

Takashi Suzuki, MD, PhD

Abstract: Staphylococcus aureus is a leading cause of keratitis,

with an increased number of isolates exhibiting antibiotic resistance. Therefore, we need to understand the present situation regarding drug-resistant S. aureus in the ocular site. It has been shown that 35% of S. aureus isolates from ocular sites are methicillin-resistant Staphylococcus aureus (MRSA). MRSA isolates from ocular sites have a high rate of multiple mutations and high levels of resistance against uoroquinolones. Wall teichoic acids (WTAs) are major polyanionic polymers in the cell wall of S. aureus and are likely to be important in the pathogenesis of eye infection. A new compound, targocil, was recently shown to function as a bacteriostatic inhibitor of WTA biosynthesis in S. aureus. The minimum inhibitory concentration (MIC) at which 90% of the keratitis isolates are inhibited (MIC90) by targocil was 2 mg/mL for both MRSA and methicillinsensitive Staphylococcus aureus. Targocil exhibited little toxicity at concentrations near the MIC, with increased toxicity at higher concentrations and longer exposure times. Targocil inhibited intracellular bacteria in the presence of human corneal epithelial cells to a greater extent than vancomycin. Targocil-resistant strains exhibited a signicantly reduced ability to adhere to human corneal epithelial cells (P , 0.001). The WTA biosynthesis pathway of S. aureus appears to be a viable target for preventing keratitis caused by strains of this bacterium. Key Words: drug resistance, uoroquinolone, keratitis, MRSA, new antibiotics, Staphylococcus aureus, wall teichoic acid (Cornea 2011;30(Suppl. 1):S34S40)

aeruginosa are the leading causes of bacterial keratitis.1 In particular, Staphylococcus aureus is the predominant pathogen of keratitis and community- and hospital-acquired infection of the skin, soft tissue, bloodstream and other sites.1 Compounding the problem is antibiotic resistance, which initially developed in hospitals, but has since spread to the community where rates of methicillin-resistant Staphylococcus aureus (MRSA) are now approaching those of hospitals.2 Fluoroquinolones and cephalosporins are often used empirically for the treatment or prevention of keratitis. However, along with increased levels of methicillin resistance among isolates, resistance to cephalosporins and uoroquinolones is increasing in parallel.3,4 Furthermore, S. aureus has recently been reported to have acquired resistance to vancomycin, a drug of last resort.5,6 Because of the advance of antibiotic resistance among S. aureus isolates from cases of keratitis, and other anatomical sites, it was of interest to explore the potential utility of novel antibiotics. We review the current state of knowledge regarding drug-resistant S. aureus isolated from the ocular site, including the cornea, and describe a new therapy targeting S. aureus.

DRUG-RESISTANT STAPHYLOCOCCUS AUREUS ISOLATED FROM THE OCULAR FIELD

To understand the current status of Japanese drugresistant S. aureus in ocular sites, we checked the prevalence of mecA, which is the staphylococcal methicillin resistance determinant, in 46 S. aureus strains isolated from the external eye at Ehime University Hospital. Nearly 35% of the S. aureus isolates possessed the mecA gene and showed resistance against methicillin. These data could indicate that MRSA have spread to the ocular eld (unpublished data, Suzuki T, MD, July 2005). In subsequent experiments, we investigated the uoroquinolone resistance of MRSA. Fluoroquinolone antibacterial agents have potent activities against gram-positive and gram-negative bacteria. In the ophthalmologic eld, their low toxicity, safety, good ocular surface penetration, prolonged tear lm concentration, stability at room temperature, and easy availability have made uoroquinolone ophthalmic solutions an excellent initial choice in treatment. They are extensively used for the topical therapy of bacterial infection. In addition, corneal concentrations of uoroquinolones after infrequent topical application (12 doses) have been demonstrated to be higher than those achieved in the serum after systemic administration.7
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nfectious keratitis is characterized by corneal inammation and defects caused by replicating bacteria, fungi, or protozoa. These infections can progress rapidly with devastating consequences, including corneal scarring and loss of vision. Thus, it is imperative to identify this condition promptly and begin an aggressive course of therapy to limit tissue damage. The principal risk factors of infectious keratitis include trauma, contact or orthokeratology lens wear, ocular surface disease, ocular surgery, and systemic disease. Staphylococcus spp, Streptococcus spp, and Pseudomonas

From the Department of Ophthalmology, Ehime University School of Medicine, Toon, Japan. The author declares no nancial disclosures or conicts of interest. Reprints: Takashi Suzuki, Department of Ophthalmology, Ehime University School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan (e-mail: t-suzuki@m.ehime-u.ac.jp). Copyright 2011 by Lippincott Williams & Wilkins

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Mechanisms of uoroquinolone resistance in S. aureus have been extensively studied and are mainly related to mutations in the genes encoding the target enzymes. For any uoroquinolone, the primary target is DNA topoisomerase IV, which is composed of the GrlA and GrlB subunits, encoded by grlA and grlB, respectively.812 The secondary target is DNA gyrase, which is composed of the GyrA and GyrB subunits, encoded by gyrA and gyrB, respectively.13 After exposure to uoroquinolones, overexpression of norA, which encodes the multidrug efux pump, has been observed.14 In most cases, mutations are located in the highly conserved quinolone resistancedetermining regions of grlA and gyrA. A combination of mutations in both genes can cause high levels of uoroquinolone resistance, even for the newer uoroquinolones.15,16 The prevalence of mutations in gyrA, gyrB, grlA, and grlB was investigated in 21 clinical isolates of MRSA recovered from individuals with conjunctivitis or keratitis. These isolates were distributed among 16 different pulsed-eld gel electrophoresis patterns.17 Mutations identied in the QRDRs of the gyrA, gyrB, grlA, and grlB genes are summarized in Table 1. Nine different combinations of mutations were detected in the 21 isolates, with the isolates recovered from identical patients exhibiting the same types of mutation. Removing these overlapping isolates from the analysis, the fourth type of mutation, involving substitution of an amino acid, was found in 10 mutants (62.5%). Double mutations were observed in 5 mutants (31.25%). Only 1 isolate had a single mutation. In gyrA, 2 single mutations (43.75%) at codons 84 [TCA(Ser) ! TTA(Leu)] and 88 [GAA(Glu) ! AAA(Lys)] and 3 double mutations (50%) at codons 84 and 85 [TCA(Ser) ! TTA(Leu) and TCT(Ser) ! CCT(Pro), respectively] or codons 84 and 88 [TCA(Ser) ! TTA(Leu) and GAA(Glu) ! GGA(Gly), respectively, or TCA(Ser) ! TTA(Leu) and GAA(Glu) ! AAA(Lys), respectively] were

observed.17 All nucleic acid alterations were identical to those previously reported.15,16 No mutations were found in gyrB. In grlA, 3 single mutations (50%) at codons 80 [TCC(Ser) ! TTC(Phe)], 84 [GAA(Glu) ! AAA(Lys)], and 108 [AGT(Ser) ! AAT(Asn)] and 4 double mutations (50%) at codons 84 and 85 [TCC(Ser) ! TAC(Tyr) and GAA(Glu) ! AAA(Lys), respectively, or TCC(Ser) ! TAC(Tyr) and GAA(Glu) ! GGA(Gly), respectively, or TCC(Ser) ! TTC(Phe) and GAA(Glu) ! AAA(Lys), respectively] and codons 84 and 108 [TCC(Ser) ! TTC(Phe) and AGT(Ser) ! AAT(Asn), respectively] were observed.17 Among these mutations, the one at position 108 was novel to grlA. In grlB, 3 novel point mutations were found. These were located at codons 443 [GAC(Asp) ! GAA(Glu)], 444 [GGC(Arg) ! AGC(Ser)], and 471 [GAA(Lys) ! AAA (silent)]. One single (6.25%) and 1 double (12.5%) mutation were observed.17 The minimum inhibitory concentrations (MICs) of gatioxacin (GFLX) and levooxacin (LVFX) were measured. The MIC values ranged from 0.5 mg/mL to greater than 256 mg/mL for LVFX and from 0.125 mg/mL to greater than 64 mg/mL for GFLX (Table 1).17 One isolate was susceptible to LVFX, and 4 were susceptible to GFLX. These results suggest that MRSA isolates from ocular sites have high rates of multiple mutations and high levels of resistance against uoroquinolones.

A NEW TARGET FOR STAPHYLOCOCCUS AUREUS KERATITIS

The occurrence of drug-resistant S. aureus in ocular sites is increasing; therefore, development of new treatments is desperately needed. One target for new antibiotics is wall teichoic acid (WTA). WTAs are phosphate-rich highly anionic polymers that are covalently linked to the peptidoglycan cell

TABLE 1. Mutations in the QRDR of gyrA, gyrB, grlA, and grlB and Susceptibility to Fluoroquinolones
Mutation Mutation Type 1 Isolates (n) 1 1 1 1 4 1 2 1 2 1 1 1 1 1 1 1 gyrA S84L + S84L + S84L + S84L + S84L + S84L + S84L + S84L + S84L S84L S84L S84L S84L S84L E88 K S85P S85P S85P S85P E88 G E88 G E88 G E88 K gyrB grlA S80F + E84 K S80F + E84 K S80F + E84 K S80F + S108 N S80Y + E84 K S80Y + E84 K S80Y + E84 K S80Y + E84 G S80F S80F S80F E84 K E84 K E84 K S80F S108 N grlB K471 (silent) D443E + R444S D443E + R444S MIC (mg/mL) LVFX .256 .256 .256 .128 .256 to .128 .128 .256 .128 8 8 8 8 8 8 8 0.5 GFLX .64 .64 .64 64 64 64 6432 64 42 4 4 4 4 2 2 0.125

2 3

4 5

6 7 8 9

QRDR, quinolone resistancedetermining region. Reproduced with permission from Iihara et al.17

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FIGURE 1. Depiction of the primary Staphylococcus aureus wall teichoic acid (L-WTA) biosynthetic pathway. Deletion of nonessential WTA pathway enzymes (green) leads to an avirulent phenotype. Deletion of conditionally essential enzymes (red) is lethal on the wild-type background but not on the DtarO or DtarA backgrounds. After intracellular assembly, the poly(ribitol phosphate) polymer is transported to the outside by a 2-component ABC transporter, TarGH, and then covalently linked through a phosphodiester bond to the MurNAc sugars of peptidoglycan. Reproduced with permission from Swoboda et al.21

wall of most gram-positive pathogens. They affect the cell envelope by contributing to charge/cation binding, tensile strength, rigidity, and permeability.18 The S. aureus WTA polymer is composed of repeating units of ribitol phosphate, and its biosynthesis is facilitated by enzymes encoded by the teichoic acid ribitol (tar) pathway (Fig. 1).18,19 The primary S. aureus WTA polymer is synthesized on a bactoprenol carrier lipid embedded in the cytoplasmic membrane. WTA assembly begins with the addition of 2 sugars by TarO and TarA, followed by 2 glycerol-3-phosphate units, mediated by TarB and TarF and, nally, the addition of a polyribitol phosphate repeat by TarL. They are then exported through an ATPbinding cassette transporter (TarGH) to the external surface of the membrane where the polymer is covalently linked to peptidoglycan through an unknown mechanism.19Staphylococcus aureus tarO- or tarA-null mutants are viable in vitro; however, genes downstream of tarA in the WTA pathway cannot be deleted unless tarO or tarA is deleted rst.20 This difference in the dispensability of different components of the WTA pathway implies that blocking late-acting WTA biosynthetic enzymes after ux into the pathway has been initiated is deleterious to bacterial growth, likely through the accumulation of toxic intermediates or the depletion of the peptidoglycan carrier lipid, bactoprenol.

WTAs have been suggested to play key roles in S. aureus colonization of epithelial and endothelial cells22,23 and contribute to virulence by promoting the establishment and spread of infection. To check if WTA plays a role in adhesion to corneal epithelial cells, we performed a bacterial adhesion assay using tarO-null mutants, which are not able to produce WTA.24 Figure 2 shows the adhesion of tarO-null mutants to Simian virus 40immortalized human corneal epithelial cells (HCECs). Both laboratory (RN4220) and clinical (Newman) strains of DtarO mutants were attenuated in their ability to attach to HCECs compared with the parental strains, and this was independent of growth phase.25 Confocal microscopy also showed that laboratory strain (RN6390) tarO-null mutants could not attach to HCECs (Fig. 3). Therefore, targeting WTA biosynthesis could result in antimicrobials that, in addition to inhibiting microbial growth, inhibit host colonization and tissue invasion. A cell-based, pathway-specic, high-throughput screen exploiting this mixed gene dispensability was used to identify the rst small molecule to target WTA biosynthesis, 1835F03.21 A structureactivity relationship study of 1835F03 led to the discovery of a second-generation analog, targocil, that is 10-fold more potent.26 The target of targocil was identied to be tarG, the transmembrane component of the

FIGURE 2. Adhesion to HCECs by WTA-decient bacteria. Data represent the mean 6 SEs (n = 10). ***P , 0.001 (comparison with parental strain). Modied with permission from Suzuki et al.25.

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FIGURE 3. Adhesion of Staphylococcus aureus to HCECs. HCEC monolayers were incubated for 1 hour in the presence of S. aureus RN6390 (wild type) or RN6390 DtarO. Bacteria were immunostained for protein A (green). Nuclei were stained with TO-PRO-3 iodide (blue). Magnication, 320.

ATP-binding cassette transporter that exports WTA to the cell surface.26 The MIC of targocil against S. aureus keratitis isolates, including MRSA, has been investigated. Targocil showed excellent activity against S. aureus isolates from suspected cases of bacterial keratitis, including both methicillin-sensitive Staphylococcus aureus and MRSA, with MICs of 12 mg/mL (Table 2).25 This compound inhibits the growth of a number of S. aureus isolates, including MRSA strains. Furthermore, a standard broth microplate checkerboard synergy assay was conducted to determine the extent to which targocil and antibiotics of other classes, including vancomycin, ciprooxacin, methicillin, and gentamicin, interact with S. aureus strains. Targocil showed synergistic activity with methicillin.25 No drug interactions were observed between targocil and representatives of the other classes of antibiotics tested. The compounds were further tested to assess for toxicity against corneal epithelial cells.25 HCECs in 96-well culture plates were exposed to targocil at various concentrations (5, 10, 20, and 40 mg/mL) for 6, 15, and 25 hours. Cell culture media controls with or without 1% DMSO were also examined. Viable epithelial cells were quantied using MTS assays, and the toxicity of targocil against corneal epithelial
TABLE 2. In Vitro Activities of WTA Inhibitors Against Keratitis Isolates
MIC (mg/mL)* Targocil Organism (No. Isolates) and Growth Condition Staphylococcusaureus, methicillin susceptible (12) CAMHB Staphylococcusaureus, methicillin resistant (6) CAMHB 50% 1 1 90% 2 2 Range 12 12

*50% and 90%, MICs for 50 and 90% of isolates tested, respectively. CAMHB, cation-adjusted Mueller-Hinton broth. Reproduced with permission from Suzuki et al.25

cells was determined (Fig. 4). Compared with vehicle, targocil exhibited little toxicity against HCECs at 5 mg/mL, even after an exposure time of 24 hours.25 Targocil exhibits detectable HCEC toxicity in a concentration-dependent and exposure timedependent manner. Targocil was tested further to assess activity in the presence of HCECs.25 The efcacy of targocil against internalized bacteria in HCECs was examined. The S. aureus Newman (clinical), MG2375 (methicillin-sensitive Staphylococcus aureus keratitis), and MG2389 (MRSA keratitis) strains were inoculated into wells containing HCEC monolayers. After incubation for 1 hour, monolayers were covered with 1 mL of medium containing 100 mg of gentamicin and incubated for an additional hour to kill any residual extracellular bacteria. The efcacy of targocil against internalized bacteria was tested by measuring the number of viable internalized bacteria after treatment with 1% DMSO, vancomycin, or targocil at a 10-fold increase of the MIC. Figure 5 shows the change in log10 colony-forming units per milliliter of internalized bacteria after a 6-hour incubation.25 Newman, MG2375, and MG2389 strains treated with medium containing 1% DMSO persisted within corneal epithelial cells. Vancomycin inhibited the intracellular growth of the MG2375 and MG2389 strains but not the Newman strain. It was found that targocil was more effective in blocking intracellular S. aureus growth than vancomycin. Because the rst 2 genes in the WTA biosynthesis pathway are not essential, and inactivating them confers resistance to the WTA inhibitor, it seemed likely that a targocil-resistant mutant population would consist largely of mutations in tarO or tarA. It was also possible that mutations occurred elsewhere, such as in the molecular target of the WTA inhibitor (tarG), generating strains still capable of synthesizing WTA. To determine whether targocil-resistant S. aureus mutants exhibited altered HCEC adherence, approximately 105 colony-forming units of each of 2 keratitis isolates (MG2375 and MG2389) were treated with 5 mg/mL targocil to select for the development of resistance. The
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FIGURE 4. Epithelial cell toxicity of targocil in HCECs. Data represent the mean 6 SEs (n = 10). *P , 0.05; **P , 0.01 (comparison with 1% DMSO). Reproduced with permission from Suzuki et al.25

resistant population was then cultured and tested in adherence assays. Mixed populations of targocil-resistant mutants from both S. aureus strains were found to be greatly attenuated in their ability to attach to HCECs compared with the parent wild-type strains (Fig. 6A).25 To determine whether targocil resistance resulted in a loss of WTA biosynthesis, we puried resistant colonies of MG2375 and tested for targocil sensitivity, WTA production using polyacrylamide gel electrophoresis, and tar gene mutations. The MICs of targocil for all mutants were .32 mg/mL. Mutants 1 and 2 did not produce WTA, but mutant 3 was WTA+ (Fig. 6B).25 We observed that mutants 1 and 2 contained a unique point mutation in tarA (TarA: R229C), whereas mutant 3 contained a unique point mutation in tarG (TarG: S204 T).25 Mutants with a point mutation in tarA (m1 and m2) exhibited a signicant reduction in HCEC adherence (P , 0.01).25 The mutant containing a point mutation in tarG (m3) behaved similarly to the original strain (Fig. 6C).25 Resistant mutants that arise on selection with these compounds are highly attenuated and show reduced epithelial cell binding.

DISCUSSION
Keratitis associated with contact lens wear or ocular injury is generally a community-acquired infection, reecting the increasing antibiotic-resistant phenotypes observed in the

community. Worldwide, 125 million people use contact lenses for corrective, cosmetic, or therapeutic reasons.27 Additionally, keratoplasty and refractive surgery create surgical alterations to the cornea, mostly in the form of laser-assisted in situ keratomileusis surgery. Staphylococcus aureus is one the leading causative pathogens of infectious keratitis associated with contact lens wear, trauma, and corneal surgery. This is because it is a commensal agent of the adjacent skin and mucosa and can easily contaminate the surface of the eye.1 Topical antibiotics are sometimes used prophylactically in corneal surgery, but there is concern that this practice can lead to the appearance of drug-resistant S. aureus on external surfaces, and infections with these strains are more difcult to treat. Based on our unpublished data, methicillin resistance in S. aureus seems to be on the rise in ocular sites. A population review conducted in 3 communities showed the annual incidence of community-acquired MRSA during 20012002 to be 1825.7 per 100,0002; most community-acquired MRSA isolates were associated with clinically relevant infections, and 23% of patients required hospitalization. In the ocular eld, levels of methicillin resistance are increasing.4 Along with methicillin resistance, increasing uoroquinolone resistance is also an emerging problem in the eld of ophthalmology. In our study, 62.5% of isolates had 4 or 5 mutations, 31.3% of isolates had a double mutation, and only 1 isolate contained a single mutation.17 Previous studies have shown that

FIGURE 5. Growth change (2Dlog CFU/mL) of internalized bacteria (Newman, MG2375, and MG2389 strains) treated with 1% DMSO, 10 mg/mL vancomycin, and 10 mg/mL targocil for 6 hours are shown. Data represent the mean values 6 SEs of the mean (n = 10). CFU, colonyforming units. Modied with permission from Suzuki et al.25

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FIGURE 6. Adhesion of targocil-resistant bacteria to HCECs. A, The relative adhesion to HCECs of mixed populations of mutants that arise after prolonged incubation with targocil for MG2375 and MG2389. B, WTA PAGE analysis of the targocilresistant mutants and wild-type bacteria. C, The relative adhesion of puried targocil-resistant mutants generated from MG2375 to HCECs is shown. Data represent means 6 SEs (n = 10). A, B, ***P , 0.001 (in comparison with wild type). Reproduced with permission from Suzuki et al.25

S. aureus, including MRSA, with 4 mutations in gyrA and grlA, composed less than 23.5% of the population15,16,2831; however, our results indicate that this is closer to 50.0% when overlapping isolates are excluded. This result may explain why a previous study reported that 57.1% of MRSA isolates derived from ocular infections were resistant to GFLX.32 It is hypothesized that such a high prevalence of multiple mutations generates greater resistance to newer uoroquinolones. A new strategy for addressing the problem of antibiotic resistance is to develop antimicrobials that select for mutants that exhibit a profound attenuation. The biosynthesis of WTA in S. aureus presents an opportunity to explore this unconventional strategy.18,19 WTA biosynthetic genes downstream of tarO and tarA are essential, except in tarO or tarA mutants.20 The WTA biosynthesis inhibitors tested targeted the conditionally essential WTA enzyme, TarG.21 In our study, the MICs of targocil were 12 mg/mL, even against MRSA strains. Because of these promising results, it was of interest to evaluate other characteristics of targocil. We tested its synergistic interactions with other antibiotics and observed a synergistic effect with methicillin against the tested strains. Little is known about the relationship between WTAs and penicillin-binding proteins, the targets of methicillin; however, inhibition of WTA production increased the afnity of methicillin for its targets. These data suggest that combination therapies with a WTA inhibitor and methicillin may be benecial. The toxicity of this new class of drugs has yet to be examined. In our study, toxicity of targocil against corneal epithelial cells was found to increase in a concentrationdependent and exposure timedependent manner. Toxic effects were only seen at concentrations 5-fold greater than the MIC values. It was further noted that targocil effectively
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inhibited the growth of internalized bacteria. This indicates that the drug can penetrate into eukaryotic cells by an undetermined mechanism, without being inhibited by cytoplasmic proteins. Interestingly, the number of bacteria present after treatment with targocil (which is a bacteriostatic agent in vitro) decreases in HCECs. Shiratsuchi et al33 demonstrated that concentrations of superoxide, which can kill engulfed bacteria, were increased in macrophages incubated with WTAdecient S. aureus but not wild-type S. aureus. Thus, one explanation for the observed decrease in S. aureus viability may be that bacteria in which WTA production is inhibited and also treated with targocil are weakened against the effects of superoxide produced in epithelial cells. In contrast, vancomycina drug of last resort for MRSA infectiondid little to inhibit the growth of the internalized S. aureus Newman strain, consistent with previous observations.34 Bacterial invasion may contribute to persistence of bacteria at the site of infection and treatment failure. Targocil may represent an option for treating infections such as keratitis, where other treatments less capable of penetrating the epithelial cell have failed. Resistant mutants can still give rise to chronic infections in a clinical setting, partly because compensatory mutations would arise. Targocil treatment generates mutants that lack normal representation of WTA in the cell wall and are highly attenuated. Because bacterial adhesion to the epithelial surface is a prerequisite for skin and soft-tissue infections including keratitis, and WTAs are necessary for the colonization of epithelial tissues in animal models,22,25 WTA biosynthesis, in addition to being a conditionally required activity, also represents a virulence target. We found that targocil-resistant populations adhered poorly to HCECs. Bacteria that survive after targocil treatment belong to 1 of at least 2 classes: mutants that have unique mutations in tarA or in tarG.21 The WTA-null tarA mutants showed less adhesion ability to HCECs; however,
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the tarG mutants did not exhibit reduced adhesion. Little is known about the exact ratio of mutant populations in the targocil-resistant bacteria, but the adhesion ability of mixed populations of mutant bacteria was also decreased. Irrespective of the actual mechanisms, S. aureus WTAs directly or indirectly play a critical role in bacterial adhesion and invasion. Targocil treatment elicits mutants that lack a virulence factor critical for adhesion and invasion; it is an example of a new class of compounds that inhibit bacterial growth and virulence. This combination of inhibitory effects channels the evolution of a pathogen toward a nonpathogenic strain. It is not known whether compensatory mutations that allow infection will arise in mutant strains that no longer express a critical virulence factor such as WTA. This issue is of considerable interest because current arguments in favor of virulence factors as antimicrobial targets rest on the unproven idea that there will be minimal pressure for the development of pathogenic drug-resistant mutants. Although targocil itself is specic for S. aureus, targeting essential enzymes linked to virulence pathways with antibiotics represents a previously unexplored strategy. This may possibly assist in combating bacterial resistance to antibiotics. These compounds may be viable options for treating S. aureus keratitis infections and other infections involving S. aureus adherence and/or internalization. Further investigation will be necessary to determine the efcacy of these compounds in vivo and their potential for use in clinical cases. REFERENCES
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