ABOUT THE AUTHOR:
Tracy A. Smith of SimbaKui Silver and Golden Persians teaches High School Chemistry and Biology. She has her BS degree in Biology and Chemistry from Washington College where she graduated Magna Cum Laude in 2000 and received Honors on her thesis research.
She also has her Master's Degree in Education from Wesley College.
Tracy has been breeding silver and golden Persians since 1997 under the Simbakui cattery name.
As a result of their short and concave facial features, the lacrimal sac and nasolacrimal duct of most Persian cats is blocked, causing an excessive coagulation of debris and an overflow of tears from the eyes. Consequently, Persian cats suffer from chronic eye infections. The objective of this experiment was to identify the bacteria contributing to the chronic conjunctivitis, and then in turn, determine the best antibacterial agent to treat the infection. The ocular discharge from both eyes of ten different Persian cats was swabbed and cultured; in all cats, only one visually distinct type of colony was isolated. Further analysis of the isolated colonies revealed the same single species of bacteria was present in both eyes of all ten Persian cats. Upon gram staining this isolated bacteria, it was discovered to be of the genus Staphylococcus. Further physiological and biochemical tests revealed a species that, according to Bergey’s Manual, resembled Staphylococcus felis. Using the Kirby-Bauer method of antimicrobic sensitivity testing, the drugs novobiocin, erythromycin, ceftriaxone, sulfamethoxazole, ciprofloxacin, chloramphenicol, gentamycin, streptomycin, and bacitracin were found to be effective against the isolated S. felis. Analysis of the results revealed that the antibiotics gentamycin, erythromycin, and bacitracin/novobiocin/polymyxin B all had a substantial effect on the number of S. felis present in the Persian cats’ eyes and the amount of brown tearing. After treatment, no bacteria could be cultured from all ten Persian cats’ eyes. In addition, the brown tearing completely disappeared in almost all of the cats in this study after being treated with one of the three chosen sensitive antibiotics. This suggests the amount of S. felis present is closely related to the amount and severity of the brown tearing.
As a result of their short and concave underlying facial bone structure, the lacrimal sac and the nasolacrimal duct of most Persian cats is blocked at the lacrimal puncta, causing an excessive coagulation of debris and an overflow of tears from the lacrimal glands: Excessive tearing is a common characteristic of Persian cats and is caused by abnormal drainage of tears and may result in epiphora (Faculty of Cornell Feline Health Center 1997). The two lacrimal puncta are the small openings to the canaliculi (ducts) leading to the lacrimal sac. The nasolacrimal duct drains the sac into the nose. The ducts of the lacrimal system are already very small in felines and the facial conformation of extremely short-nosed, large-eyed cats, namely Persians, is the single most common cause of occlusion (blockage) of the lacrimal system and resulting abnormal drainage of tears. Consequently, because of the epiphora and the blocked lacrimal system, Persian cats suffer from chronic eye infections. Bacteria build up in the obstructed lacrimal passages and thrive on the debris deposited from the coagulated tears, inducing chronic conjunctivitis, with characteristic symptoms such as a brown, mucus-like discharge from the eye, blinking, and an exposed third eyelid. In severe cases, the conjunctiva is swollen red (Faculty of Cornell Feline Health Center 1997). It is important to note, however, that conjunctivitis is not painful (Carlson and Giffin 1995).
A new species of staphylococci was recently discovered in 1989 by Shizunobu Igimi et al. He identified a group of coagulase negative staphylococci from infections in cats, but not in infections of dogs. The lesions included external otitis, cystitis, abscesses, wounds, and other skin infections (Igimi et. al. 1989). Igimi et. al. isolated a total of 39 strains of S. felis from cats suffering somatic infections between 1982-1988. The 39 strains were basically homogeneous. All exhibited facultative growth, no coagulation, no oxidase production, nitrate reduction, arginine dihydrolase production, urease production, no novobiocin or bacitracin resistance, and acid production from sucrose, mannitol, mannose, trehalose, lactose, galactose, ribose, and glycerol (Igimi et. al. 1989). Igimi et. al. described S. felis as a gram positive cocci of 0.8 -1.2 micrometers in diameter that occur singly, in pairs, and predominantly in irregular clusters (1989). They are nonmotile, nonsporeforming, and most strains are unpigmented. In addition, S. felis can grow in the presence of 15% NaCl after 72 hours and most strains have very weak hemolytic activity. Igimi et. al. discovered all strains in their study to be highly susceptible to penicillin G and ampicillin, and moderately susceptible to chloramphenicol and erythromycin (1989).
S. felis can be easily distinguished from other coagulase negative staphylococci on the basis of biochemical reactions. However, one species of staphylococci, S. simultans, closely mimics S. felis. Bacitricin susceptibility is the primary method for distinguishing S. felis from S. simultans (Igimi et. al. 1994). S. felis was the only type of staphylococci isolated from conjunctivitis in felines to date; furthermore, in the Igimi et. al. study, it accounted for 45% of all staphylococci isolates from the cat (1994).
Intraocular antibacterial therapy is a necessity for most Persian cats. Deep-seated infections caused by the occlusion of the lacrimal system due to facial conformation of the cat is usually very difficult to treat. Repeated cleansing of the eye with a dilute solution of boric acid and specific topical or oral antibiotics tailored to sensitivities are the primary approaches to this problem.
The antibiotic treatment of choice is usually tetracycline, a bacteriostatic agent that inhibits certain steps in the pathway from ribosomal transcription to protein synthesis, since it is secreted in tears and binds that part of the tears that stains the fur around the eye the characteristic brown color (Carlson and Giffin 1995). If improvement is due only to the binding action of the drug, then the face around the eyes remains wet with tears but not discolored. If the brown staining returns after treatment, then long-term administration is recommended. However, since this drug is only bacteriostatic, that is, it only inhibits growth and replication, it does not kill the bacteria infecting the eye. Examples of bacteriostatic agents include chloramphenicol, macrolides (erythromycin), and sulfonamides. Chloramphenicol, the macrolides, and the aminoglycosides (streptomycin, gentamycin, neomycin) function in the same manner as tetracycline, by inhibiting protein synthesis (Allen 1998). Whereas the tetracyclines, chloramphenicol, and the macrolides are bacteriostatic, the aminoglycosides are bactericidal. Using a bactericidal agent rather then a bacteriostatic agent is beneficial since it actually kills the harmful bacteria, causing the infection to almost completely subside. Other examples of bactericidal drugs include the penicillins, bacitracin, novobiocin, polymyxins, and trimethoprim-sulfonamide combinations. The penicillins and bacitracin function to inhibit bacterial cell wall synthesis. The efficacy of these agents require active bacterial replication and therefore concurrent use of a bacteriostatic agent will impair the effectiveness of these drugs. Novobiocin and polymyxin B are disrupters of bacterial cell membranes and are generally bactericidal but are not dependent on the rate of bacterial growth (Allen 1998).
This experiment was intended to isolate and identify bacteria from ocular discharge experienced by Persian cats, and, subsequently, to find an effective treatment. The cats were treated with an antibiotic tailored to the cultures and sensitivities of the isolated bacteria and the treatment was evaluated in terms of amount of brown tearing experienced before and after treatment. In addition, standard plate counts before and after antibiotic therapy were also conducted.
Materials and Methods:
Isolation: Ten samples of ocular discharge were collected bilaterally from ten different, healthy Persian cats exhibiting moderate to severe brown tearing and conjunctivitis. The ocular discharge was plated via the quadrant streak method onto nutrient agar and the cultured bacteria was isolated.
Counting: A standard plate count from each Persian cat was conducted on nutrient agar using undiluted and diluted tears (1:100) in distilled water. The number of bacteria per milliliter was determined for each Persian cat.
Quantifying: The brown tearing was quantified in all ten Persian cats by measuring the amount of tearing (determined in mm by the length of the horizontal band of brown tearing under the eyes) and each was classified as severe (>6mm) or moderate (2-5mm). Cats with less than 2 mm of brown tearing before treatment were not available in this experiment.
Identification: Only one visually distinct colony-type appeared on the nutrient agar plates used for isolation suggesting a monoculture. This isolated bacteria was gram stained and the morphological structure was determined. Biochemical tests were performed on the isolated bacteria according to Benson (1998) to determine physiological characteristics. These tests included mixed acid, adonitol, lactose, arabinose, sorbitol, glucose with gas production, and dulcitol fermentations, acetoin, urease, catalase, hydrogen sulfide, cadaverine, putrescine, and indole productions, nitrate reduction, and citrate utilization. An Enterotube II (Becton-Dickinson) was utilized for some of the tests for results only, not for enterobacteriaceae identification. The isolated bacteria was also tested for motility using semisolid media and aerobic/anaerobic growth using Fluid Thioglycollate Medium. The isolated bacteria was identified using Bergey’s Manual.
Antimicrobic Sensitivity Testing: The identified bacteria was tested for antimicrobic sensitivity using the Kirby-Bauer Method according to Benson (1998). Antibiotic disks used in the sensitivity testing (manufactured by BBL) included novobiocin (30 micrograms), erythromycin (15 micrograms), ceftriaxone (30 micrograms), sulfamethoxazole (25 micrograms), ciprofloxacin (5 micrograms), chloramphenicol (30 micrograms), ampicillin (10 micrograms), penicillin (10 micrograms), and bacitracin (10 micrograms). Antibiotics manufactured by Difco that were also used in the sensitivity testing were gentamycin (10 micrograms), tetracycline (30 micrograms), and streptomycin (10 micrograms). The bacteria was classified as sensitive, intermediate, or resistant for each antibiotic disk according to Benson (1998).
Treatment: The availability of ophthalmic preparations containing these antibiotics found to be most effective was determined in the Handbook of Veterinary Drugs. The drugs considered the safest and that were approved for ophthalmic use in cats were listed as gentamycin, erythromycin, and bacitracin/polymyxin B combination. These three drugs were used to treat three or four cats each. After 10 days of treatment, a standard plate count was conducted for the nine cats and the amount of bacteria present per milliliter was determined. The amount of brown tearing after treatment was quantified by measuring the proportion of the eye covered by brown mucus (in mm) after 24 hours as previously described.
Analysis: The number of bacteria present and the amount of brown tearing was determined before and after treatment. The effectiveness of the intraocular antibacterial therapy was concluded.
One visually distinct colony was cultured from each of the ten Persian cats’ ocular discharge via the quadrant streak method onto nutrient agar. Biochemical and physiological characteristics of this organism are listed in Table 1. The isolated bacteria cells stained gram-positive and were predominantly found in grape-like clusters of cocci around 0.8 - 1.2 micrometers in diameter. This bacteria was found to be facultatively anaerobic as growth occurred in both aerobic and anaerobic portions of the thioglycolate medium. The organisms were nonmotile in semisolid media and nonsporeforming. Colonies were unpigmented, circular, slightly convex, and opaque with a white tint with smooth and shiny surfaces. These organisms grew in the presence of 15% NaCl after 68 hours at 37 degrees Celsius. The isolated bacteria exhibited no coagulation when added to rabbit plasma. The organisms also failed to hemolyze Sheep-Blood Agar indicating they do not produce Beta-hemolysin. They were able to ferment glucose and lactose with the production of gas. The isolated bacteria was able to produce urease, utilize citrate as its sole carbon source, and reduce nitrate to use its oxygen as a final hydrogen acceptor in anaerobic respiration . They were negative for mixed acid fermentation, acetoin production, and hydrogen sulfide production. No acid was produced from sorbitol, adonitol, or dulcitol.
The isolated bacteria was found to be highly susceptible to novobiocin, erythromycin, ceftriaxone, sulfamethoxazole, ciprofloxacin, chloramphenicol, gentamycin, streptomycin, and bacitracin (Table 2). They were found to be resistant to tetracycline, ampicillin, and penicillin.
Analysis of the results revealed that the gentamycin, erythromycin, and bacitracin/polymyxin B/neomycin combination all had a substantial effect on the amount of brown tearing (Figure 1) as well as the number of bacteria present in all ten Persian cats’ eyes (Figure 2). Before treatment, the Persian cats experienced a maximum of 1.71 x 10 7 and a minimum of 1.00 x 10 7 bacteria per mL ocular discharge. The maximum portion of the eye covered in brown tears before therapy was 9 mm and the minimum was 3 mm, with an average of 5.15 mm. After treatment, no bacteria could be cultured from any of the ten Persian cats (Figure 2) and minimal tearing occurred (Figure 1).
The FIGURES and GRAPHS referenced below are not available although they were part of the original thesis:
Table 1: Physiological and Biochemical characteristics of the isolated bacteria on nutrient agar pate.
Table 2: Antibiotic Sensitivity for the isolated bacteria on the nutrient agar plate.
Figure 1: Amount of brown tearing before and after treatment with antibiotic ophthalmic preparations. Cats 1-3 were treated with gentamycin, cats 4-7 were treated with erythromycin, and cats 7-10 were treated with bacitracin/polymyxin B/neomycin combination.
Figure 2: Number of bacteria present in the eyes of the ten Persian cats. One microliter of tearing was pipetted from the eyes of each Persian cat and added to 99 microliters of distilled water. This 1:100 dilution plated countable results (<300 colonies per plate). After treatment, nothing could be cultured from the eyes of all ten Persian cats.
The present study suggests the presence of Staphylococcus felis in the ocular microflora of the Persian cats. The sampled isolate in this experiment was characterized by 24 biochemical and physiological reactions (Table 1) listed in Bergey’s Manual (1998) and 12 antibiotic susceptibility tests (Table 2). It was identified as Staphylococcus felis using the Tables of characteristics listed in Bergey’s Manual. This isolate displayed typical characteristics of this species. It has not been confirmed if S. felis is a normal inhabitant of the conjunctival sac in felines, but it has been isolated from the eyes of domestic cats not experiencing conjunctivitis (Lilenbaum 1999). This suggests that S. felis is part of the normal mircobiota of felines. However, in Persian cats which experience occlusion of the lacrimal duct system, these results suggest that S. felis reach very high concentrations and build up in the blocked passages, causing disease.
This hypothesis is supported by the fact there appears to be a substantial relationship between the number of bacteria present in the Persian cats’ eyes and the amount of brown tearing as well as the fact that after treatment with any of the three chosen sensitive antibiotics (gentamycin, erythromycin, or bacitracin/neomycin/polymyxin B), the amount of brown tearing experienced by all ten Persian cats was substantially reduced to a minimum of 0 mm and a maximum of .9 mm (Figure 1). It was determined that both bacteriostatic drugs were just as effective as bactericidal drugs in most cases (Figure 1).
Interestingly, S. felis was found to be resistant to three antibiotics which had previously been proven to be effective against this organism in a previous experiment in our lab using cats from the same household: All strains [of S. felis] are highly susceptible to penicillin G and tetracycline (Igimi et. al. 1989). Although the tetracycline and penicillin proved to be effective against the S. felis in a previous experiment, this study revealed a resistance to these two antibiotics. This suggests the bacteria may have developed a resistance to these drugs since previous studies. This finding is consistent with the fact that all Persian cats used in this experiment have been treated with tetracycline and penicillin in the past, approximately one year prior to the study. A more comprehensive study should be performed to conclude the most effective antibiotic treatment against S. felis. In addition, further experimentation is needed to determine if the S. felis is in fact the cause of the brown tearing experienced by Persian cats. This would include using a larger sample of Persian cats from different households/cohorts, using a control group, as well as using more sensitive methods for culturing disease causing agents, both viral and bacterial.
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