During the mid-1940s, articles appeared in the medical literature describing strains of Escherichia coli and Staphylococcus aureus that were resistant to penicillin.^3,4 A growing list of reports during the 1950s and 1960s extended these observations to other antibiotics: streptomycin,^5–7 chloramphenicol,^8–12 tetracycline,^13,14 actinomycin,¹⁵ erythromycin,^16–18 aureomycin,^19–20 and methicillin.²¹ Several microorganisms were found to be resistant to one or more antibiotics, including Enterobacteriaceae,^22,23 pneumococci,^16,24 Haemophilus,²⁵ Pseudomonas,¹⁰ and Bacillus.^13,15,17 However, the potential clinical importance of acquired antibiotic resistance among bacterial species was initially ignored by the medical community. The Surgeon General proclaimed to Congress in 1969 that it was time to “close the book on infectious disease.”²⁵

As an increasing list of pathogenic bacteria acquired resistance to multiple antibiotics, the world entered a postantibiotic era.^26–28 Examples of this phenomenon include the following: (1) S. aureus is now invariably resistant to penicillin and increasingly resistant to methicillin and other semisynthetic penicillinase-resistant penicillins, (2) 15% to 25% of the strains of Streptococcus pneumoniae are relatively resistant to penicillin and an increasing percentage are resistant to many fluoroquinolones, (3) a high percentage of enterococci are resistant to ampicillin and aminoglycosides and some are resistant to vancomycin, and (4) 30% to 40% of Haemophilus influenzae strains and almost all Moraxella are resistant to the β-lactam antibiotics.^2,29 Multidrug-resistant strains have become commonplace—Shigella, Salmonella, Escherichia, Enterobacter, Klebsiella, Proteus, Serratia, Pseudomonas, Streptococcus, and mycobacteria are often resistant to multiple.^26,30–37

The economic costs of antibiotic resistance are difficult to determine,³⁸ but the Centers for Disease Control and Prevention (CDC) has estimated that the costs related to treatment of infections caused by antibiotic-resistant organisms in the United States is more than $4 billion annually.³⁹ Antimicrobial resistance among ocular infections contributes to substantial economic expenses.

Table 1. Mechanisms of Antimicrobial Resistance

TARGET MODIFICATION

Many bacteria acquire resistance to antibiotics through modification of an antibiotic-binding protein or nucleic acid target.

• β-Lactam antibiotics (penicillins and cephalosporins) bind to transpeptidases and transcarboxypeptidases located on the cell membrane of susceptible bacteria, thereby preventing cross-linking of linear glycopeptides into the peptidoglycan complex and final cell wall assembly.¹ In some resistant bacteria (e.g., methicillin-resistant S. aureus), the penicillin-binding proteins are altered, and the β-lactam antibiotic is unable to bind to its target.
  
• The binding of quinolones to deoxyribonucleic acid (DNA) gyrase and topoisomerase IV inhibits normal bacterial replicative and transcriptional activity. Modification of the α (and perhaps β) subunit of DNA gyrase and/or topoisomerase IV through chromosomal mutation prevents quinolone binding and the attendant expression of antibacterial activity.⁴⁰
  
• Other examples of a modified target and the corresponding antibiotic include ribonucleic acid (RNA) polymerase and rifampin, methylated 23S RNA and erythromycin/clindamycin, dihydropterate synthetase and sulfonamides, and dihydrofolate reductase and trimethoprim.¹
  

REDUCTION IN TARGET EXPOSURE

Target exposure can be reduced through mechanisms that either decrease the uptake of antibiotic and/or increase its rate of efflux. Such mechanisms of resistance, because they may affect permeation or efflux of multiple different antibiotic substrates, may contribute to the phenomenon of multi-drug resistance that has been increasingly observed in recent years.

• Gram-negative bacteria have channels (called porins) in their outer lipid membrane through which β-lactam antibiotics pass to reach the penicillin-binding proteins.¹ Modification or loss of an outer membrane protein can prevent antibiotic ingress into the bacterium, thus, eliminating binding to the target and averting the bacteriostatic and bactericidal effects. Pseudomonas aeruginosa becomes resistant to imipenem through this mechanism.
  
• Some bacteria (e.g., Haemophilus, Vibrio, Aeromonas, and Moraxella) become resistant to tetracycline by acquiring a gene that encodes for a cytoplasmic membrane efflux protein that can pump a number of antibiotic substrates out of the cell at a rate equal to or greater than its uptake.⁴¹ Other species (e.g., Bacillus subtilis) have a single copy of an efflux protein gene but only become resistant when the copy number is increased. Fluoroquinolone resistance in S. aureus can occur by a similar mechanism of increased expression of the NorA efflux pump.
  

ANTIBIOTIC INACTIVATION

Some bacteria produce enzymes and other products that neutralize the activity of antimicrobial agents.

• Within a few years of widespread penicillin G use, most strains of S. aureus were penicillin-resistant, with resistance being mediated by an exoenzyme (β-lactamase) that catalyzes the hydrolysis of the β-lactam ring to an inactive form.¹ β-lactamase producing bacteria can also inactivate many of cephalosporins.
  
• Chloramphenicol is susceptible to enzymatic inactivation by certain gram-negative and gram-positive organisms that synthesize chloramphenicol acetyl-transferase.¹ Once it has been acetylated, chloramphenicol exhibits substantially less affinity for bacterial ribosomes, and intracellular protein synthesis is no longer suppressed.
  

CHROMOSOMAL MUTATION

Spontaneous chromosomal mutations occur relatively infrequently (10^-5 to 10^-9/generation),⁴² but the mutation rate is strikingly increased following bacterial exposure to various external factors such as irradiation, heat, or chemical agents.⁴³ The probability of an induced mutational event is dependent on the strength and duration of exposure of the external factor, with a greater number of events occurring whenever the inducing factor is applied at a suboptimal level over a protracted period.

One example of antibiotic resistance resulting from one or more chromosomal gene mutation is acquired bacterial resistance to the fluoroquinolones through mutation of the gyrA or parC genes encoding for the α subunits of DNA gyrase and topoisomerase IV, respectively. Mutations in the gyrA or parC gene have been detected in E. coli, Salmonella typhimurium, P. aeruginosa, Enterobacter cloacae, Campylobacter jejuni, Mycobacterium tuberculosis, and S. aureus.⁴⁰ In E. coli, the quinolone-resistance determining region of gyrA has been located to an area between nucleotides 199 and 318, encoding the amino acids at positions 67 to 106 in the α subunit of DNA gyrase, which is close to its active site at tyrosine-122. The most common mutation sites in E. coli are at nucleotides 247 to 249, where serine in position 83 is usually substituted for leucine, which results in a moderate increase in the in vitro minimum inhibitory concentration (MIC) value from 0.015 to 0.5 mg/L for ciprofloxacin. If an additional mutation were to occur in this region (substitutions at both Ser-83 and Asp-87), then the ciprofloxacin MIC value is markedly increased to 64 mg/L.

GENETIC TRANSFERENCE

Most bacteria become resistant to antibiotics not through a mutation in the chromosomal genome but by acquiring a gene residing in extrachromosomal genetic material (plasmid or transposon) from another organism.² Plasmids are double-stranded circular DNA molecules containing 7 to 200 genes that replicate autonomously, integrate into chromosomal DNA, and encode for special phenotypic traits, the most clinically important of which are antibiotic resistance and pathogen virulence.⁴² Transposons are smaller DNA-containing elements that carry various genes including those associated with antibiotic resistance. Although not autonomously replicating, transposons are capable of inserting into chromosomal, plasmid, and bacteriophage genomes.

The mechanisms by which plasmid and transposon antibiotic-resistant-encoding DNA can be transferred from one bacteria to another include transformation (naked DNA transfer), transduction (transfer via a viral vector), and conjugation (unidirectional transfer through cell-cell contact).⁴² Of the three, the most important is the latter that occurs in five distinct stages: (1) specific pair formation—initial interaction between the donor's conjugation pilus and the recipient's cell surface to form a stable mating pair, (2) effective pair formation—joining of donor and recipient cellular envelopes, (3) chromosomal mobilization—initial processing of plasmid/transposon DNA, (4) chromosomal transfer—transfer and simultaneous replication of plasmid/transposon and chromosomal DNA, and (5) recombination—pairing of homologous segments of donor and recipient DNA.

Plasmids and transposons can confer resistance to either a single antibiotic or multiple antibiotics. Bacteria that are resistant to three or more antibiotics, each with a different mechanism of action, are said to be multidrug resistant.² The list of microorganisms that have acquired plasmids that mediate antibiotic resistance is extensive and includes virtually all bacteria of ophthalmic interest (e.g., S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Clostridium species, Bacteroides species, H. influenzae, Neisseria gonorrhoeae, Acinetobacter species, Aeromonas species, E. coli, Klebsiella species. Proteus species, Providencia species, Serratia marcescens, and P. aeruginosa).¹

A similar scenario, from widespread susceptibility to the development of resistance, occurred with ciprofloxacin and other fluoroquinolones. In 1990, quinolones were introduced into ophthalmology and were proposed as an alternative for resistant infections.⁴⁴ These agents were highly active against a broad array of gram-positive and gram-negative bacteria and were effective against aminoglycoside-resistant strains.⁴⁸ Within 2 years, however, the first cases of quinolone-resistant bacterial keratitis were reported.⁴⁹ Fluoroquinolone-resistant ocular infections are now recognized worldwide.^50–52 A trend toward progressively higher frequencies of fluoroquinolone resistance has been observed both in gram-positive pathogens (e.g., S. epidermidis endophthalmitis isolates) and gram-negative bacteria (e.g., P. aeruginosa keratitis isolates). Newer fluoroquinolones have improved activity against gram-positive cocci resistant to earlier generation fluoroquinolones, but unfortunately they appear to have no activity against P. aeruginosa resistant to earlier generation quinolones.

β-Lactam resistance has also become prevalent among ocular isolates. Longitudinal surveillance data indicate a progressively increasing prevalence of methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae, and β-lactamose producing H. influenzae. Among pneumococci isolated from 1988 to 1995 in Florida,⁵³ 14% were resistant to penicillin, with the highest proportion found in intraocular fluid (20%), followed by blepharoconjunctival (15%) and corneal (9%) lesions. In a series from Japan between 1992 and 1995,⁵⁴ penicillin resistance was found in 15% of pneumococci. A 20-year longitudinal study of penicillin-resistant S. pneumoniae demonstrated the emergent nature of bacterial resistance in Texas⁵⁵; only 2% of pneumococci recovered between 1976 through 1991 were penicillin-resistant in contrast to 20% obtained between 1992 and 1995 (Fig. 1).

A consortium of 12 laboratories in the United States, Canada, Mexico, and Argentina provided an overview of antibiotic susceptibility among ocular infections.⁵⁶ Using both disk-diffusion and broth-dilution methods to assess the in vitro antibiotic sensitivity of 1,291 bacterial strains isolated from primarily the conjunctiva (42%) and cornea (24%), eight antibiotics were tested. The smallest number of isolates were found to be resistant to ofloxacin and the largest number to erythromycin (Fig. 2). Comparisons of fluoroquinolone sensitivity for the most common gram-positive and gram-negative organisms recovered revealed a similar profile for ofloxacin, ciprofloxacin, and norfloxacin, although several (e.g., S. pneumoniae, E. faecalis, and viridans-group streptococci) were less resistant to ofloxacin (Fig. 3). Interstrain differences in sensitivity between ofloxacin and gentamicin were similar to the pattern found for the fluoroquinolones (Fig. 4).

The selection of antimicrobial therapy for ocular infections must account for the presence of resistant strains.⁵⁷ Among vitreous isolates, over half of enterococci are resistant to gentamicin or cefazolin, and up to a third or more of staphylococci are methicillin-resistant, leading to the recommendation for use as part of the empiric vancomycin treatment of postoperative endophthalmitis.⁵⁸ The absence of vancomycin resistance in a multicenter study of endophthalmitis therapy⁵⁹ is noteworthy, but surveillance data from systemic infections indicating increasing vancomycin resistance is of concern. Because vancomycin is often a treatment option of last resort, its use as routine prophylaxis during ophthalmic surgery should be avoided whenever possible.⁶⁰

It is impossible to devise a strategy for using antibiotics in clinical ophthalmology that would completely obviate the development of antibiotic resistance, for to do so would violate a basic tenet of evolutionary theory: The exposure of a species (bacteria) to a destructive environmental element (antibiotic) results in the natural selection of those traits (antibiotic resistance) that will allow the species to survive. There are, however, some approaches that collectively could help reduce the severity of this problem.

REDUCED ANTIBIOTIC USE

Antibiotics should be judiciously prescribed to patients for the shortest amount of time possible.²⁹ Antibiotic use policies must be established at local, national, and international levels. Educational and monitoring programs should be developed.^{2,29,34,39,62} Essential elements of any policy would include the withholding of empiric antibiotic therapy for patients with infections likely to be self-limiting and/or of viral etiology,²⁹ adopting an abbreviated (e.g., 72 hours), high dose (e.g., drops administered at least four times daily) prophylactic regimen for many ophthalmic surgical procedures,^34,63–66 and eliminating the practice of using antibiotics as growth enhancers for livestock.^2,39,67

HIGH DOSING

Data from several studies support the notion that an antibiotic should be given at the highest dose, consistent with an acceptable product-safety profile. Peak serum antibiotic concentrations less than 10 times the MIC of the organism predispose to the emergence of resistant bacterial subpopulations.^68–70 It is unclear, however, whether it is the peak concentration that is the primary pharmacokinetic determinant of antibiotic resistance or the area under the curve (AUC). The AUC is important in monitoring the therapy of pneumonia in which resistant strains are more likely to appear when the area under the inhibitory curve (AUIC) is low. A sufficiently high AUIC would ensure that the achieved drug level exceeds the target organism's MIC for at least 80% of the treatment interval.⁷¹ The opportunity for resistance to develop is lowered if the antibiotic is used at a high dose during the early treatment phase.⁷²

A tapering antibiotic regimen is prone to promote the development of resistance by repeatedly exposing bacteria to prolonged levels of antibiotic below the MIC. Thus an important principle of antibiotic prescribing is that antibiotic administration should be dosed never less than the frequency adequate to exceed the MIC of relevant pathogens (e.g., four times daily). At the conclusion of therapy, the antibiotic should be discontinued abruptly rather than tapered.

USE OF NARROW-SPECTRUM AGENTS

A narrow-spectrum antibiotic should always be prescribed if possible,²⁹ because broad-spectrum agents needlessly expose nonpathogenic organisms to the selective pressures that inevitably result in the development of resistance. To illustrate the importance of this phenomenon, suppose an individual has been previously exposed to a broad-spectrum antibiotic under conditions that allow for the acquisition of resistant genes by the normal flora. Subsequent exposure of the individual to a pathogen could conceivably lead to the transfer of resistance genes via plasmids or transposons from the commensal to the pathogenic species and promote the development of an infection intractable to antibiotic therapy.

COMBINATIONS AND CYCLING

The use of antibiotic combinations is a common practice in treating serious ocular infections such as microbial keratitis and endophthalmitis. Besides providing broad-spectrum coverage, combination therapy reduces the emergence of resistant strains. Antibiotic cycling is another strategy for reducing the development of resistance.^73,74 Switching from one antibiotic to another every several weeks can be done during long-term prophylaxis.⁷⁵ The use of biocides that nonspecifically target living cells is a useful option for short-term prophylaxis, such as before ophthalmic surgery.⁷⁶ Appropriate use of disinfectants in the operating room and eye clinic can also slow the spread of resistant microorganisms^77,78 and is an essential adjunct to antimicrobial use in surgical prophylaxis.

IMMUNIZATION

Ultimately, preventing ocular infections through mucosal vaccines may prove feasible. The role of immunization in reducing the global burden of infectious diseases of the eye remains a future challenge.

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