Apopulation of bacteria and fungi normally exist on the canine ocular surface. Gram-positive aerobes are most common, with Staphylococcus spp, Bacillus spp, Corynebacterium spp, and Streptococcus spp predominating.1–7 Gram-negative and anaerobic bacteria can also be recovered from the conjunctival sac of dogs.1,4 Fungi are found on the cornea of 14% to 22% dogs, with Alternaria spp, Cladosporium spp, Pénicillium spp, and Aspergillus spp most frequently isolated.7,8 These microbes have the potential to become pathogens if the appropriate circumstances exist, such as development of a corneal ulcer or access to the intraocular space.
Cataract surgery is commonly performed on dogs by veterinary ophthalmologists, and septic endophthalmitis is a potentially devastating complication, which occurs most often in the early postoperative period.9 The periocular flora is the most common source of bacterial contamination, and potential routes of infection include incisions (subsequent to dehiscence), externalized vitreous strands, full-thickness corneal sutures, or introductions to the anterior chamber at the time of surgery.10,11 Contamination of the intraocular space during surgery is frequent; it occurs in up to 24% of canine eyes.10,12 In an effort to minimize bacterial contamination during surgery, the ocular surface and periocular skin are routinely prepared for surgery with topical 0.5% povidone iodine solution.13,14 Additionally, surgeons use various regimens of antimicrobials administered before, during, and after surgery to reduce the risk of infection.a It is standard protocol at the Western College of Veterinary Medicine to use a topically administered fluoroquinolone before and then 4 times daily for 3 weeks after cataract surgery. Ofloxacin is chosen for perioperative prophylactic administration because it has high corneal penetration and typically exceeds the MIC needed to kill most of the common ocular contaminants.15 The 3-week period is chosen because this approximates the amount of time required for healing of the corneal incision and for sutures used to close the incision to dissolve.
It is believed that chronic use of topically applied antimicrobials results in a shift in the normal microbial flora and may facilitate the emergence of resistant bacterial strains. In human medicine, excessive use of fluoroquinolones has resulted in a marked increase in the resistance of many gram-positive bacteria, including coagulase-negative Staphylococcus spp, which account for most cases of septic endophthalmitis after cataract surgery.16,17 In addition to development of resistance to 1 type of antimicrobial by mutation, lateral transfer of resistance genes may lead to resistance to multiple types of antimicrobials. Examples of this are MRSA and methicillin-resistant Staphylococcus epidermidis, which now have high degrees of resistance to fluoroquinolones.16–21
To the authors' knowledge, effects of prolonged topical administration of antimicrobials to the ocular surface of dogs on the bacterial population have not been described in the literature. It is important for veterinary ophthalmologists to know whether antimicrobials used prophylactically to prevent postoperative infection are effective and whether they contribute to the development of antimicrobial resistance. The purpose of the study reported here was to evaluate changes in bacteria and antimicrobial resistance of those bacteria cultured from the conjunctiva of dogs after cataract surgery. We hypothesized that long-term use of topically applied fluoroquinolones after cataract surgery would be associated with changes to the types of bacteria cultured and an increase in antimicrobial resistance of those bacteria.
Materials and Methods
Animals
Sixteen client-owned dogs admitted from February 2010 through November 2013 to the Western College of Veterinary Medicine for bilateral phacoemulsification and intraocular lens implantation were included in the study. Only dogs residing within the province of Saskatchewan that could be returned to our institution for all reevaluations were included. Exclusion criteria included keratoconjunctivitis sicca, blepharitis, or use of topically or systemically administered antimicrobials during the 2 months preceding the study. All dogs had been treated with topically applied diclofenac sodium 0.1% every 12 hours for at least 3 weeks before surgery. Informed consent was obtained from all owners. The study was performed in accordance with the Canadian Council on Animal Care guidelines for experimental animal use, and the University of Saskatchewan Animal Care Committee approved the research protocol.
Clinical examination
All dogs underwent a complete ophthalmic examination performed by a board-certified veterinary ophthalmologist 24 hours before and 12 hours and 1, 3, and 6 weeks after surgery. This included neuro-ophthalmic examination, Schirmer tear testing, rebound tonometry, fluorescein staining, biomicroscopic examination, and indirect ophthalmoscopy after application of tropicamide 0.5%. Electroretinography was completed 24 hours before surgery to ensure appropriate retinal function. Ocular B-mode ultrasonography was performed to identify preoperative posterior segment abnormalities.
Preoperative and postoperative treatments
Topically applied medications initiated before surgery were ofloxacin 0.3%,b diclofenac sodium 0.1%, prednisolone acetate 1%, and atropine sulfate 1%. Each was administered twice the night before surgery (approx 3-hour interval between administrations) and twice the morning of surgery (approx 1-hour interval between administrations). After surgery, topical treatment consisted of ofloxacin 0.3% (every 6 hours for 3 weeks) and diclofenac sodium 0.1% and prednisolone acetate 1% (every 6 hours for 3 weeks, every 8 hours for 3 weeks, and then every 12 hours for 3 weeks). If postoperative ocular hypertension was detected, treatment was modified to include dorzolamide hydrochloride 2%-timolol maleate 0.5% (every 8 hours), travaprost 0.004% (every 12 hours), or both.
Cataract surgery
Dogs were anesthetized, and a nondepolarizing neuromuscular blockade was used. The ocular surface was prepared in accordance with sterile technique by use of 1:50 povidone iodine solutionc to irrigate the cornea and conjunctival surface and wash the periocular skin. Dogs received 1 dose of cefazolin (22 mg/kg, IV) at the start of surgery. Board-certified veterinary ophthalmologists performed a 1-handed endocapsular phacoemulsification followed by automated irrigation and aspiration. The anterior chamber was maintained by use of sodium hyaluronate 1.8% or hydroxypropylmethylcellulose 2% (or both). A 12- to 14-mm, 41-diopter, foldable, soft acrylic 1-piece intraocular lens was implanted. The 3.5-mm corneal incision was closed in a routine manner with 9–0 polyglactin-910 suture.d Surgery was always performed first on the left eye followed by the right eye. Each dog wore an Elizabethan collar for 3 weeks after surgery to prevent self-trauma.
Culture of conjunctival swab samples
Aerobic and anaerobic cultures were performed on samples collected from the conjunctival fornices of each eye 24 hours before surgery (before ofloxacin treatment was initiated; week 0) and 1, 3, and 6 weeks after surgery. Swab samples were collected before administration of topical ophthalmic preparations to avoid inhibitory effects on organism growth.22 Briefly, the eyelids were held open to expose the inferior and superior conjunctival sac. Swab samples for aerobice and anaerobicf culture were collected by rolling sterile swabs within the upper and lower conjunctival fornices; investigators were careful to avoid contact with eyelashes or skin of the eyelids. Swabs used for aerobic culture were moistened with the supplied transport medium prior to collection of a sample. Swab samples were inoculated into transport medium and immediately submitted to the microbiology laboratory at the Western College of Veterinary Medicine for culture.
Bacterial culture procedures
Samples were streaked onto plates within 12 hours after collection. Swab samples were streaked directly onto plates containing blood agar (2 plates), MacConkey agar (1 plate), and brain-heart infusion with 7.5% NaCl solution. One blood agar plate was incubated under 5% CO2, the MacConkey agar plate was incubated aerobically, and the second blood agar plate was incubated under anaerobic conditions; these 3 plates were incubated at 37°C. The brain-heart infusion with 7.5% NaCl solution was incubated aerobically at 37°C. The aerobic plates were examined for growth daily for 3 consecutive days and again on day 7. The anaerobic blood agar plate was examined for growth at 48 hours and incubated for 3 additional days. To increase the chances of isolating MRSA, subculturing was performed. At 24 hours of incubation, a sample from the plate containing brain-heart infusion with 7.5% NaCl solution was swabbed onto a plate containing a commercial MRSA chromogenic agar,g which was then incubated at 35°C. Swabs were rotated on the agar surface over a quarter of each plate (streak 1), and then a series of 3 perpendicular streaks were made with bacterial loops over each remaining quarter (streaks 2, 3, and 4).
Semiquantitative counting was performed and scored as follows: low = 1 to 9 colonies in streak 1; moderate = ≥ 10 colonies in streak 2; and heavy = ≥ 10 colonies in streaks 3 or 4. Isolates were identified by use of standard biochemical tests and stored at −80°C for susceptibility testing. Commonly recognized pathogens were identified to the species level; other organisms were identified to the genus level.
Antimicrobial susceptibility testing
Isolates were tested for susceptibility to 21 antimicrobials belonging to 12 drug classes: penicillin, oxacillin, ampicillin, and amoxicillin-clavulanic acid (β-lactams); gentamicin and neomycin (aminoglycosides); enrofloxacin, levofloxacin, ofloxacin, and moxifloxacin (fluoroquinolones); vancomycin (glyco-peptides); erythromycin (macrolides); clindamycin and lincomycin (lincosamides); linezolid (oxazolidin-ones); tetracycline (tetracyclines); quinupristin-dalfopristin (streptogramines); chloramphenicol (phenicols); trimethoprim-sulfamethoxazole (folate inhibitors); and fusidic acid and mupirocin (miscellaneous). In vitro antimicrobial susceptibility for ofloxacin was determined by use of a commercially available test,h and the remainder of the antimicrobial susceptibility tests were performed by use of Kirby-Bauer disk diffusion. Breakpoints were interpreted in accordance with CLSI guidelines23 when veterinary standards existed and other CLSI guidelines24 when veterinary standards did not exist. When no standards for breakpoints existed, the data were excluded. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 strains were used as control samples.
Statistical analysis
Statistical analysis was completed by use of commercially available software.i The frequency for positive culture results and the proportion of cultures with > 1 organism were calculated for each time point. First, a generalized linear mixed-effect model was fitted, with each of the 16 dogs as a cluster and eye (right vs left) nested within each dog and culture type (aerobic vs anaerobic) nested within each eye. There was no significant effect of right eye versus left eye or anaerobic versus aerobic culture; thus, proportions were calculated by use of the total number of swab samples for each respective time point (n = 64). Bivariate analysis was performed by use of a generalized linear mixed models procedure to evaluate outcome of a positive culture result for the covariates of sex, concurrent diabetes mellitus, age, large- or small-breed dog, and year in which the samples were obtained. A generalized linear mixed-effect model was used to compare frequency of positive culture results and positive culture results with > 1 organism at each time point. Frequency of the category of bacterial load (no, low, moderate, and heavy) was calculated for each time point. The temporal pattern of bacterial load was analyzed with logistic regression analysis to compare outcomes for no and low bacterial load with moderate and heavy bacterial load. When the same organism was obtained on both aerobic and anaerobic cultures for the same eye, the organism was counted only once in calculation of bacterial type at each time point. Ofloxacin MICs were rounded to the nearest dilution, and data were dichotomized by use of ≥ 4 μg/mL, which corresponded to the resistance breakpoint for Staphylococcus pseudintermedius. The frequency of isolates with MICs ≥ 4 μg/mL at each time point after surgery was compared with the frequency of such isolates at week 0 by use of the Fisher exact test; a Bonferroni correction was used to account for multiple comparisons (0.05/3 = 0.017). Values of P < 0.05 were considered significant.
Results
Multiple dog breeds were represented in the study; 6 dogs were considered to be large-breed dogs (Labrador Retriever [n = 4], Samoyed cross [1], and Border Collie [1]), and 10 were considered to be small-breed dogs (Norwich Terrier [1], Pug [1], Boston Terrier [1], Toy Poodle [1], Havanese [1], Havanese cross [1], Lhasa Apso [1], Lhasa Apso cross [1], and Bichon Frise cross [2]). Mean ± SD age was 7.1 ± 3.4 years. Eleven dogs were neutered females, and 5 were neutered males. Six of the 16 dogs were diabetic.
Surgical complications were rare and occurred in 2 of 16 dogs during the study period. One dog (a 5-year-old neutered male Pug) developed a unilateral retinal detachment at 4 weeks after surgery. An 8-year-old neutered male Boston Terrier had incisional dehiscence in the left cornea 1 week after surgery, which was repaired with primary sutures; the corneal incision healed without further complications after the repair. That eye had positive culture results before surgery (Aeromonas spp) and at week 1 (Bacillus spp); both of the organisms were susceptible to ofloxacin.
A total of 256 swab samples were collected, and 78 bacteria were cultured (Table 1). Of the 78 organisms, S pseudintermedius was the most frequently cultured organism (21 [26.9%]), followed by coagulase-negative Staphylococcus spp (19 [24.4%]), Corynebacterium spp (12 [15.4%]), Streptococcus spp (10 [12.8%]), miscellaneous gram-positive organisms (10 [12.8%]), and gram-negative organisms (6 [7.7%]). Aerobic culture yielded 49 organisms (49/128 aerobic swab samples = 38.3% positivity rate), whereas anaerobic culture yielded 29 organisms (29/128 anaerobic swab samples = 22.7% positivity rate).
Frequency of 78 bacteria cultured from swab samples collected from the conjunctival fornices of 16 dogs that received topical treatment with ofloxacin for 3 weeks after cataract surgery.
| Bacteria | No. of bacteria | % |
|---|---|---|
| Most frequently cultured organisms | 62 | 79.5 |
| Staphylococcus pseudintermedius | 21 | 26.9 |
| Coagulase-negative Staphylococcus spp | 19 | 24.4 |
| Corynebacterium spp | 12 | 15.4 |
| Streptococcus spp | 10 | 12.8 |
| Miscellaneous gram-positive organisms | 10 | 12.8* |
| Streptococcus canis | 3 | 3.8 |
| Enterococcus spp | 2 | 2.6 |
| Bacillus spp | 2 | 2.6 |
| Clostridium perfringens | 1 | 1.3 |
| Gram-positive rods | 1 | 1.3 |
| Gram-positive cocci | 1 | 1.3 |
| Miscellaneous gram-negative organisms | 6 | 7.7* |
| Mannheimia spp | 2 | 2.6 |
| Pseudomonas spp | 1 | 1.3 |
| Aeromonas spp | 1 | 1.3 |
| Neisseria spp | 1 | 1.3 |
| Moraxella spp | 1 | 1.3 |
Within a category, percentages may not sum to the total because of rounding.
For each of the 64 swab samples/wk, the frequency of obtaining a positive culture result was 13 (20%), 11 (17%), 20 (31%), and 23 (36%) for weeks 0, 1, 3 and 6, respectively. There was a significant increase in the proportion of positive culture results obtained at week 6, compared with the proportions at weeks 0 (P = 0.034) and 1 (P = 0.011). The number of cultures from which > 1 organism was cultured was 1 of 13, 1 of 11, 6 of 20, and 2 of 23 for weeks 0, 1, 3, and 6, respectively. There was no significant difference in the frequency for culture of multiple organisms from an individual eye. There was a significant increase in the proportion of cultures with a moderate to heavy bacterial load over time (Figure 1). Compared with the bacterial load at week 0, the bacterial load was more likely to be moderate or heavy at weeks 3 (P = 0.007) and 6 (P < 0.001). Similarly, the bacterial load was significantly more likely to be moderate or heavy at weeks 3 (P = 0.001) and 6 (P < 0.001), compared with the bacterial load at week 1.
Bacterial load for 78 organisms cultured from swab samples collected at various times from the conjunctival fornices of 16 dogs that received topical treatment with ofloxacin for 3 weeks after cataract surgery. The sample collected 24 hours before cataract surgery was designated as week 0. Swabs were rotated on the agar surface over a quarter of each plate (streak 1), and then a series of 3 perpendicular streaks were made with bacterial loops over each remaining quarter of the plate (streaks 2, 3, and 4). The percentage of cultures with no or low (1 to 9 colonies in streak 1) bacterial load (gray bars) or with moderate (≥ 10 colonies in streak 2) or heavy (≥ 10 colonies in streaks 3 or 4) bacterial load (black bars) was compared among time points. *Value differs significantly (P = 0.007) from the corresponding value at week 0. †Value differs significantly (P = 0.001) from the corresponding value at week 1. ‡Value differs significantly (P < 0.001) from the corresponding values at weeks 0 and 1.
Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.207
Staphylococcus pseudintermedius was the most frequently cultured organism at weeks 0 (5/12), 1 (4/12), and 6 (8/19); however, this organism had the lowest frequency at week 3 (1/20; Table 2). This pattern was in contrast to that of coagulase-negative Staphylococcus spp, which were the most frequently cultured organisms at week 3 (10/20), but were cultured less frequently at weeks 0 (1/12), 1 (2/12), and 6 (3/19).
Frequency of bacteria cultured from swab samples collected at various times from the conjunctival fornices of 16 dogs that received topical treatment with ofloxacin for 3 weeks after cataract surgery.
| Bacteria | Week 0 | Week 1 | Week 3 | Week 6 |
|---|---|---|---|---|
| S pseudintermedius | 5 | 4 | 1 | 8 |
| Coagulase-negative Staphylococcus spp | 1 | 2 | 10 | 3 |
| Corynebacterium spp | 0 | 2 | 2 | 4 |
| Streptococcus spp | 2 | 0 | 3 | 2 |
| Miscellaneous gram-positive organisms | 1 | 3 | 2 | 2 |
| Miscellaneous gram-negative organisms | 3 | 1 | 2 | 0 |
| Total | 12 | 12 | 20 | 19 |
Week 0 = Sample obtained 24 hours before cataract surgery.
Penicillin was the antimicrobial with the highest frequency of resistant organisms (28/53 [52.8%]) and was followed by ofloxacin (22/57 [38.6%]; data not shown). Comparisons of resistance to each antimicrobial and for each organism were not possible because of low and variable numbers of organisms over time. Analysis of the combined MIC data for ofloxacin for all organisms revealed a significant increase in the proportion of ofloxacin-resistant bacteria at week 3, compared with the proportion at week 0 (Table 3). Because coagulase-negative Staphylococcus spp and S pseudintermedius were the predominant organisms in the study, antimicrobial resistance for these organisms was subjectively evaluated, and the peak of resistance to all the fluoroquinolones tested was evident at week 3 (Figure 2).
The MIC breakpoints for ofloxacin for all bacteria cultured from swab samples collected from the conjunctival fornices of 16 dogs that received topical treatment with ofloxacin for 3 weeks after cataract surgery.
| MIC (|ig/mL| | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Time (wk) | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | Resistant* |
| 0 | 3 | 6 | 1 | 1 | 0 | 0 | 0 | 1 | 1/12 |
| 1 | 1 | 1 | 3 | 3 | 1 | 0 | 0 | 2 | 3/11 |
| 3 | 1 | 2 | 2 | 2 | 3 | 0 | 0 | 9 | 12/19† |
| 6 | 5 | 3 | 0 | 3 | 0 | 2 | 0 | 2 | 4/15 |
Organisms with an MIC of ≥ 4 μg/mL, which corresponded to the susceptibility breakpoint for S pseudintermedius, were considered resistant.
Represents the number of organisms with antimicrobial resistance/total number of organisms.
Proportion of resistant organisms differs significantly (P < 0.003) from the proportion of resistant organisms at week 0.
Proportion of Staphylococcus pseudintermedius and coagulase-negative Staphylococcus spp that were resistant to various fluoroquinolones. There were 6 (5 S pseudintermedius and I coagulase-negative Staphylococcus sp), 6 (4 S pseudintermedius and 2 coagulase-negative Staphylococcus spp), II (I S pseudintermedius and I0 coagulase-negative Staphylococcus spp), and II (8 S pseudintermedius and 3 coagulase-negative Staphylococcus spp) organisms for weeks 0, I (gray bars), 3 (white bars), and 6 (black bars), respectively. Notice that there was no antimicrobial resistance at week 0. Resistance to all the fluoroquinolone antimicrobials was highest at week 3; at that time, coagulase-negative Staphylococcus spp accounted for most of the bacterial isolates.
Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.207
Discussion
The most interesting findings of the study reported here were that there was a significant change in the bacterial population in the conjunctiva of dogs after cataract surgery. This included an alteration of the predominant bacteria from S pseudintermedius to coagulase-negative Staphylococcus spp, an increase in bacterial numbers, and an increase in resistance to ofloxacin.
The bacteria most commonly cultured in the present study were gram-positive aerobes, which is similar to results of other studies.1–5 Overall, S pseudintermedius and coagulase-negative Staphylococcus spp were the most frequently cultured bacteria, accounting for 40 of 78 (51.3%) organisms. This compares favorably with results of previous studies. Staphylococcus spp are the predominant organisms in the conjunctival sac of clinically normal dogs and dogs with extraocular disease, with frequencies ranging from 39.3% to 58.8% of isolates.25–27
Composition of the bacterial population in the conjunctival fornices was altered during the period of topical application of ofloxacin. Staphylococcus pseudintermedius was the organism most commonly present in the conjunctiva prior to initiation of topical ofloxacin (5/12) but was reduced at 3 weeks (1/20). Conversely, culture of coagulase-negative Staphylococcus spp was uncommon before topical application of ofloxacin, but they were the most frequently cultured organism at 3 weeks (10/20). This is in contrast to results for horses in which coagulase-negative Staphylococcus spp were commonly cultured before and during treatment with antimicrobials or antimicrobial-corticosteroid combination ophthalmic preparations.28 Changes in the types of bacteria present appeared to be short term for most organisms. Three weeks after discontinuation of ofloxacin, the frequency for most types of bacteria returned to values similar to those prior to onset of ofloxacin administration.
The frequency of positive culture results increased over time, and bacterial load within eyes was greater at weeks 3 and 6 than at weeks 0 and 1. These changes indicated an increase in the amount of bacteria present in the conjunctival fornices after cataract surgery and that the effects continued even after discontinuation of topical application of ofloxacin.
A possible explanation for these changes in the bacterial population during the postoperative period was the development of antimicrobial resistance and selection for different bacteria. At weeks 3 and 6 when coagulase-negative Staphylococcus spp were the most common organisms, most of these organisms were resistant to ofloxacin. Resistance to fluoroquinolones is increasing, particularly among grampositive organisms, and may develop with exposure to low drug concentrations, repeated bacterial exposure to low amounts of antimicrobials, or use of intermittent or tapering dosing patterns over long periods.29–36
Dogs in the present study simultaneously received topically administered corticosteroids to control postoperative uveitis. These drugs may reduce capability of the local immune system and therefore contribute to changes in bacterial flora.37 Topical application of corticosteroids alone or in combination with ciprofloxacin applied every minute for 3 hours does not significantly affect bacterial colony counts in the conjunctiva of healthy humans.38 No significant differences in bacterial population changes were detected between clinically normal horses treated with antimicrobials or antimicrobial-corticosteroid combination ophthalmic preparations over a 2-week period.28 However, the authors are aware of no controlled studies on the effects of short-term or chronic use of topically applied corticosteroids on bacterial flora in dogs, and the effects of such administration on the bacterial flora of the dogs in the present study are not known.
Changes in the conjunctival flora after cataract surgery are not likely to be attributable solely to topical application of an antimicrobial. A prospective controlled study28 in which investigators evaluated the effects of topical application of antimicrobial and antimicrobial-corticosteroid ophthalmic preparations to eyes of horses on ocular surface flora revealed a reduction in positive results for bacterial cultures, quantity of gram-positive bacteria, and number of bacterial species cultured in all groups, including untreated control eyes and eyes treated with topically applied artificial tears, after treatment for 1 week. Repopulation toward pretreatment numbers of gram-positive bacteria was detected after treatment for 2 weeks, and similar to results for eyes of dogs in the study reported here, repopulation of the normal flora of the eyes of those horses was evident after discontinuation of interventions.28 This illustrates that multiple host and environment factors influence microbial populations. In addition to topical application of an antimicrobial, there were multiple interventions to the eyes of the dogs in the present study, including placement of corneal sutures, use of an Elizabethan collar after surgery, and topical application of other medications containing preservatives. All of these variables may have impacted the conjunctival flora population.
The authors are not aware of any epidemiological studies on the development of septic endophthalmitis in veterinary medicine. Investigators for a study9 of canine eyes enucleated or eviscerated after phacoemulsification reported endophthalmitis as a cause of failure in 17 of 21 eyes enucleated during the first 3 months after surgery. This was associated most often with suppurative keratitis, followed by incisional dehiscence and corneal perforation away from the incision site. Bacteria were identified in only 2 dogs.9 There were no dogs with septic endophthalmitis in the study reported here. Incisional dehiscence was detected in 1 dog at 1 week after surgery. This dog continued to receive topically applied ofloxacin and did not develop septic endophthalmitis. The prevalence of septic endophthalmitis reported in the veterinary literature is low; the combined results for retrospective studies39–41 of complications following cataract surgery of dogs indicate it as a complication in 4 of 705 (0.57%) eyes, and there is a lack of information as to the causative microbe. Similarly, the incidence of postoperative septic endophthalmitis in humans is low, with a value of 0.09% in 1 retrospective study.42
In humans, pathogens responsible for endophthalmitis appear to originate from microflora of the eyelids and conjunctiva.11,43 Coagulase-negative Staphylococcus spp account for most cases of endophthalmitis after cataract or vitrectomy surgery, and there is increasing fluoroquinolone resistance among coagulase-negative Staphylococcus spp cultured from patients with endophthalmitis.44–49 It is particularly concerning that long-term topical use of ofloxacin as a prophylactic treatment in dogs may contribute to an increase in the population of periocular coagulase-negative Staphylococcus spp after 3 weeks, concurrent with an increase in fluoroquinolone resistance of those organisms at that time.
Similar to other observational studies, results for the study reported here should be interpreted with caution. Antimicrobial resistance found in vitro does not always correlate with resistance in clinical situations. A combination of the pharmacokinetics and pharmacodynamics of drug, infection site, and MIC is needed to properly predict in vivo efficacy of antimicrobials against target pathogens. It should be mentioned that susceptibility breakpoints have not been established for topically applied antimicrobials. Thus, susceptibility data for ocular pathogens are customarily interpreted by use of breakpoints established by the CLSI for systemically administered antimicrobials. These are based on drug concentrations expected to be attained in serum, plasma, or CSF and are likely to differ from those achievable in an eye after topical administration. These CLSI breakpoints are used for testing susceptibility of ocular isolates with the assumption that concentrations for topically applied antimicrobials are at least equal to or greater than concentrations for systemic administration.50,51 Additionally, veterinary-specific susceptibility breakpoints have not been established for many drugs, including ofloxacin; thus, when veterinary-specific information was lacking, breakpoints for humans were used to define resistance. Caution must be used when extrapolating breakpoints among species.
Not all isolates were speciated, and coagulase-negative Staphylococcus spp were categorized together for simplicity. However, this was an oversimplification, and DNA sequencing to further identify coagulase-negative Staphylococcus spp would have been valuable because this group may contain numerous and diverse species. Other limitations of the present study included a small sample size, reliance on owner compliance, and lack of a control population. Because this study involved the use of client-owned animals, we elected not to include a group of dogs undergoing the same interventions but not receiving topically applied antimicrobial prophylaxis. However, a population of dogs undergoing cataract surgery and receiving a non-fluoroquinolone antimicrobial could have acted as a control group. Therefore, we cannot absolutely conclude that long-term use of topically applied antimicrobials, specifically fluoroquinolones, were responsible for the changes in ocular flora that were detected.
Findings for the present study indicated that under the conditions of the clinical protocol used, there was a change in the conjunctival bacterial populations, an increase in bacterial numbers, and an increase in bacterial resistance to ofloxacin after cataract surgery. Additionally, although these changes were most apparent at 3 weeks after surgery, increased frequency of positive culture results and increased bacterial load were still evident at 6 weeks (ie, 3 weeks after the discontinuation of the antimicrobial). Multiple interventions as well as host and environment factors may affect conjunctival bacterial populations. Limitations regarding nonestablished veterinary-specific susceptibility breakpoints as well as susceptibility breakpoints for topically applied antimicrobials moderate interpretation of the clinical relevance of the resistance data. However, the results suggested there was a potential for selection of resistance in the normal host flora, which indicates a need for ongoing consideration of the prophylactic use of ophthalmic antimicrobials with regard to the development of antimicrobial resistance.
Acknowledgments
Supported by the Companion Animal Health Fund of the University of Saskatchewan.
Presented as an abstract at the 45th Annual Meeting of the American College of Veterinary Ophthalmologists, Fort Worth, Tex, October 2014.
The authors thank Dr. Joseph Rubin for technical assistance.
ABBREVIATIONS
| ATCC | American Type Culture Collection |
| CLSI | Clinical and Laboratory Standards Institute |
| MIC | Minimum inhibitory concentration |
| MRSA | Methicillin-resistant Staphylococcus aureus |
Footnotes
Herring IP, Dorbandt DM. Canine cataract surgery: a survey of surgical techniques and management protocols employed by ACVO diplomates, in Proceedings. 43rd Annu Meet Am Coll Vet Ophthalmol 2012;42.
PMS-ofloxacin, Pharmascience Inc, Montreal, QC, Canada.
Povidone-iodine 10%, Rougier, Toronto, ON, Canada.
Vicryl, Ethicon Inc, Johnson & Johnson Medical Products, Markham, ON, Canada.
BBL CultureSwab collection and transport system, Becton Dickinson & Co, Sparks, Md.
BD BBL Vacutainer anaerobic specimen collector, Becton, Dickinson & Co, Sparks, Md.
BBL CHROMagar MRSA, Becton Dickinson & Co, Sparks, Md.
Etest, AB BIODISK, Solna, Sweden.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
References
1. Thangamuthu R, Varshney JP, Rathore BS. Conjunctival flora of clinically healthy and diseased eyes of dogs. Haryana Vet 2002; 41: 38–40.
2. Teixeira AL, Maia FBN, Alvarenga LS, et al. Aerobic conjunctival flora of healthy dogs in Sao Paulo. Arq Bras Med Vet Zootec 2006; 58: 1024–1029.
3. Urban M, Wyman M, Rheins M, et al. Conjunctival flora of clinically normal dogs. J Am Vet Med Assoc 1972; 161: 201–206.
4. McDonald PJ, Watson AD. Microbial flora of normal canine conjunctivae. J Small Anim Pract 1976; 17: 809–812.
5. Varges R, Penna B, Martins G, et al. Antimicrobial susceptibility of staphylococci isolated from naturally occurring canine external ocular diseases. Vet Ophthalmol 2009; 12: 216–220.
6. Furiani N, Scarampella F, Martino PA, et al. Evaluation of the bacterial microflora of the conjunctival sac of healthy dogs and dogs with atopic dermatitis. Vet Dermatol 2011; 22: 490–496.
7. Samuelson DA, Andresen TL, Gwin RM. Conjunctival fungal flora in horses, cattle, dogs, and cats. J Am Vet Med Assoc 1984; 184: 1240–1242.
8. Verneuil M, Durand B, Marcon C, et al. Conjunctival and cutaneous fungal flora in clinically normal dogs in Southern France. J Mycol Med 2014; 24: 25–28.
9. Moore DL, McLellan GJ, Dubielzig RR. A study of the morphology of canine eyes enucleated or eviscerated due to complications following phacoemulsification. Vet Ophthalmol 2003; 6: 219–226.
10. Ledbetter EC, Millichamp NJ, Dziezyc J. Microbial contamination of the anterior chamber during cataract phacoemulsification and intraocular lens implantation in dogs. Vet Ophthalmol 2004; 7: 327–334.
11. Speaker MG, Milch FA, Shah MK, et al. Role of external bacterial flora in the pathogenesis of acute postoperative endophthalmitis. Ophthalmology 1991; 98: 639–649.
12. Taylor MM, Kern TJ, Riis RC, et al. Intraocular bacterial contamination during cataract surgery in dogs. J Am Vet Med Assoc 1995;206: 1716–1720.
13. Roberts SM, Severin GA, Lavach JD. Antibacterial activity of dilute povidone-iodine solutions used for ocular surface disinfection in dogs. Am J Vet Res 1986; 47: 1207–1210.
14. Ciulla TA, Starr MB, Masket S. Bacterial endophthalmitis prophylaxis for cataract surgery. Ophthalmology 2002; 109: 13–26.
15. Yu-Speight AW, Kern TJ, Erb HN. Ciprofloxacin and ofloxacin aqueous humor concentrations after topical administration in dogs undergoing cataract surgery. Vet Ophthalmol 2005; 8: 181–187.
16. Mamalis N. The increasing problem of antibiotic resistance. J Cataract Refract Surg 2007; 33: 1831–1832.
17. Karacal H, Kymes SM, Apte RS. Retrospective analysis of etiopathogenesis of all cases of endophthalmitis in a large tertiary referral center. Int Ophthalmol 2007; 27: 251–259.
18. Asbell PA, Sahm DF, Shaw M, et al. Increasing prevalence of methicillin resistance in serious ocular infections caused by Staphylococcus aureus in the United States: 2000–2005. J Cataract Refract Surg 2008;34: 814–818.
19. Sotozono C, Fukuda M, Ohishi M, et al. Vancomycin ophthalmic ointment 1% for methicillin-resistant Staphylococcus aureus or methicillin-resistant Staphylococcus epidermidis infections: a case series. BMJ Open 2013; 3: e001206.
20. Dave SB, Toma HS, Kim SJ. Ophthalmic antibiotic use and multidrug-resistant Staphylococcus epidermidis. A controlled, longitudinal study. Ophthalmology 2011; 118: 2035–2040.
21. Olson R, Donnenfeld E, Bucci FA Jr, et al. Methicillin resistance of Staphylococcus species among health care and non-health care workers undergoing cataract surgery. Clin Ophthalmol 2010; 4: 1505–1514.
22. Kleinfeld J, Ellis PP. Effects of topical anesthetics on growth of microorganisms. Arch Ophthalmol 1966; 76: 712–715.
23. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility disk and dilution susceptibility tests for bacteria isolated from animals. CLSI document VET-01S. 3rd ed. Wayne, Pa: Clinical and Laboratory Standards Institute, 2015.
24. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement. CLSI document M100-S24. Wayne, Pa: Clinical and Laboratory Standards Institute, 2014.
25. Tolar EL, Hendrix DV, Rohrbach BW, et al. Evaluation of clinical characteristics and bacterial isolates in dogs with bacterial keratitis: 97 cases (1993–2003). J Am Vet Med Assoc 2006; 228: 80–85.
26. Wang L, Pan Q, Zhang L, et al. Investigation of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China. Vet Ophthalmol 2008; 11: 145–149.
27. Gerding PA Jr, McLaughlin SA, Troop MW. Pathogenic bacteria and fungi associated with external ocular disease in dogs: 131 cases (1981–1986). J Am Vet Med Assoc 1988; 193: 242–244.
28. Gemensky-Metzler AJ, Wilke DA, Kowalski JJ, et al. Changes in bacterial and fungal ocular flora of clinically normal horses following experimental application of topical antimicrobial or antimicrobial-corticosteroid ophthalmic preparations. Am J Vet Res 2005; 66: 800–811.
29. McDonald M, Blondeau JM. Emerging antibiotic resistance in ocular infections and the role of fluoroquinolones. J Cataract Refract Surg 2010; 36: 1588–1598.
30. Blondeau JM. Fluoroquinolones: mechanism of action, classification, and development of resistance. Surv Ophthalmol 2004; 49(suppl 2): S73–S78.
31. Fintelmann RE, Hoskins MD, Leitman M, et al. Topical fluoroquinolone use as a risk factor for in vitro fluoroquinolone resistance in ocular cultures. Arch Ophthalmol 2011; 129: 399–402.
32. Kim SJ, Toma HS. Antimicrobial resistance and ophthalmic antibiotics: 1-year results of a longitudinal controlled study of patients undergoing intravitreal injections. Arch Ophthalmol 2011; 129: 1180–1188.
33. Milder E, Vander J, Shah C, et al. Changes in antibiotic resistance patterns of conjunctival flora due to repeated use of topical antibiotics after intravitreal injection. Ophthalmology 2012; 119: 1420–1424.
34. Dorrepaal SJ, Gale J, El-Defrqey S, et al. Resistance of ocular flora to gatifloxacin in patients undergoing intravitreal injections. Can J Ophthalmol 2014; 49: 66–71.
35. Blondeau JM, Hansen G, Metzler K, et al. The role of PK/PD parameters to avoid selection and increase of resistance: mutant prevention concentration. J Chemother 2004; 16(suppl 3): S1–S19.
36. Hwang DG. Fluoroquinolone resistance in ophthalmology and the potential role for newer ophthalmic fluoroquinolones. Surv Ophthalmol 2004; 49(suppl 2): S79–S83.
37. Pappa KS. Corticosteroid drugs. In: Mauger TF, Elson LC, eds. Havener's ocular pharmacology. 6th ed. Mosby Year Book Inc: St Louis, 1994;364–428.
38. Ermis SS, Aktepe OC, Inan UU, et al. Effect of topical dexamethasone and ciprofloxacin on bacterial flora of healthy conjunctiva. Eye (Lond) 2004; 18: 249–252.
39. Klein HE, Krohne SG, Moore GE, et al. Postoperative complications and visual outcomes of phacoemulsification in 103 dogs (179 eyes): 2006–2008. Vet Ophthalmol 2011;14: 114–120.
40. Azoulay T, Dulaurent T, Isard PF, et al. Immediately sequential bacterial cataract surgery in dogs: a retrospective analysis of 128 cases (256 eyes) [in French]. J Fr Ophtalmol 2013; 36: 645–651.
41. Sigle KJ, Nasisse MP. Long-term complications after phacoemulsification for cataract removal in dogs: 172 cases (1995–2002). J Am Vet Med Assoc 2006; 228: 74–79.
42. Ravindran RD, Venkatesh R, Chang DF, et al. Incidence of post-cataract endophthalmitis at Aravind Eye Hospital: outcomes of more than 42,000 consecutive cases using standard sterilization and prophylaxis protocols. J Cataract Refract Surg 2009; 35: 629–636.
43. Bannerman TL, Rhoden DL, McAllister SK, et al. The source of coagulase-negative staphylococci in endophthalmitis vitrectomy study: a comparison of eyelid and intraocular isolates using pulsed-field gel electrophoresis; the Endophthalmitis Vitrectomy Study Group. Arch Ophthalmol 1997; 115: 357–361.
44. Oguz H, Satici A, Guzey M, et al. Microbiological analysis of aqueous humor in phacoemulsification. Jpn J Ophthalmol 1999; 43: 162–165.
45. Driebe WT Jr, Mandelbaum S, Forster RK, et al. Pseudophakic endophthalmitis. Diagnosis and management. Ophthalmology 1986; 93: 442–448.
46. Weber DJ, Hoffman KL, Thoft RA, et al. Endophthalmitis following intraocular lens implantation. Report of 30 cases and review of the literature. Rev Infect Dis 1986; 8: 12–20.
47. Han DP, Wisniewski SR, Wilson LA, et al. Spectrum and susceptibilities of microbiologic isolates in the endophthalmitis vitrectomy study (Erratum published in Am J Ophthalmol 1996; 122:920). Am J Ophthalmol 1996;122: 1–17.
48. Miller DM, Vedula AS, Flynn HW Jr, et al. Endophthalmitis caused by Staphylococcus epidermidis: in vitro antibiotic susceptibilities and clinical outcomes. Ophthalmic Surg Lasers Imaging 2007; 38: 446–451.
49. Miller D, Flynn PM, Scott IU, et al. In vitro fluoroquinolone resistance in staphylococcal endophthalmitis isolates. Arch Ophthalmol 2006; 124: 479–483.
50. Kaliamurthy J, Jesudasan CAN, Geraldine P, et al. Comparison of in vitro susceptibilities of ocular bacterial isolates to gatifloxacin and other topical antibiotics. Ophthalmic Res 2005; 37: 117–122.
51. Parmar P, Salman A, Kalavathy CM, et al. Comparison of topical gatifloxacin 0.3% and ciprofloxacin 0.3% for the treatment of bacterial keratitis. Am J Ophthalmol 2006; 141: 282–286.
