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Anaerobic Corneal Ulcers
Anaerobic Corneal Ulcers L. DOUGLAS PERRY, MD, JOHN H. BRINSER, BA, HARRY KOLODNER, MD
Abstract: In a series of 162 bacterial corneal ulcers, 27 were culture positive for anaerobic organisms. Applying strict microbiologic criteria, 11 ulcers were determined to be due to anaerobic infection. No morphologic characteristic was identified to distinguish anaerobic from other types of corneal ulcers. All of the anaerobic ulcer patients manifested one or more predisposing factors. A total of 13 anaerobes were cultured from the 11 confirmed anaerobic ulcers. Five previously unreported anaerobic strains were identified as causes of bacterial keratitis. Over one third of the anaerobic organisms occurred in mixed cultures with other organisms. Most of the anaerobic isolates were susceptible to all antibiotics routinely used for their treatment. The use of topical chloramphenicol is recommended for treatment of confirmed anaerobic ulcers, and topical cefazolin or one of the other cephalosporins effective against anaerobes is suggested to be included in the treatment of all ulcers requiring broad spectrum antibiotic coverage. [Key words: anaerobic corneal ulcers, anaerobic ocular infections, bacterial keratitis, cornea, ocular microbiology.] Ophthalmology 89:636-642, 1982
Successful therapy of a bacterial corneal ulcer. requires rapid identification of the causative organism and initiation of appropriate antibiotic therapy. 1 In many instances, however, the corneal culture is negative in an apparently infected ulcer. Possible explanations for such a negative culture include: (1) antibiotic pretreatment, (2) inadequate culture methods, and (3) true culture negativity. While the role of aerobic organisms in causing corneal inflammation has long been appreciated, it was not until recently that Ostler and Okumot0 2 and Jones and Robinson 3 confirmed nons pore-forming anaerobes as a cause of bacterial corneal ulcers. Believing that many of our own culture negative corneal ulcers were really due to anaerobic organisms that we could not culture with standard techniques, an improved methodology for anaerobic organisms was instituted in our From the Department of Ophthalmology, University of South Florida, Tampa, Florida. Presented at the Eighty-sixth Annual Meeting of the American Academy of Ophthalmology, Atlanta, Georgia, November 1-6, 1981. Supported in part by a grant from the Central Florida Lions Eye Bank. Reprint requests to L. Douglas Perry, MD, Department of Ophthalmology, University of South Florida, Box 21, 12901 N. 30th Street, Tampa, FL 33612.
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ocular microbiology laboratory in 1977. Twenty-seven corneal ulcers from which anaerobic organisms were grown are reported on herein.
MATERIALS AND METHODS SPECIMEN COLLECTION AND TRANSPORT A TION
Conjunctival cultures are taken prior to corneal culturing using a sterile thioglycollate-moistened cotton applicator rolled along the length of the inferior conjunctival cul-de-sac. Plating on the left side of the culture plates is immediately done. Once inoculation on solid media has been accomplished, the moistened cotton applicator tip is broken off in a tube of thioglycollate broth maintained at room temperature. A similarly moistened cotton applicator is then rolled along the inferior lid margin and applied to the right side of the previously inoculated plate. The moistened cotton applicator is then put in thioglycollate broth. In most instances, conjunctival and lid cultures of the patient's opposite eye are also performed. Several drops of topical proparacaine hydrochloride are then instilled in the involved eye over a period of several minutes. The cornea is scraped at the slit lamp with a Kimura platinum spatula. Usually the peripheral edge 0161-6420/82/0600/636/$00.85
© American Academy of Ophthalmology
PERRY, et al • ANAEROBIC ULCERS
of the ulcer is chosen for the site of culturing rather than a central necrotic area. In addition to multiple culture plate inoculations, several slides are also prepared for subsequent staining. Care is maintained throughout the corneal culturing procedure to avoid contact between the lids and platinum spatula. Following inoculation of solid media and preparation of slides the patient is reclined in the examining chair and broth media are inoculated. A sterile cotton applicator is moistened with the appropriate broth and "twirled" in the ulcer. The applicator is then broken off at its tip into the broth media. The time required for the entire culturing procedure is usually less than 30 minutes. Most specimens are taken at our medical center and immediately transported to the ocular microbiology laboratory. Upon arrival in the laboratory, designated solid and broth media from all sources are immediately placed in an anaerobic jar. If the specimens are taken at a site distant from the laboratory, an anaerobic jar is brought to the culture location and the appropriate plates and broths are placed in the activated jar. In no instance does the period of time between performing the conjunctival and lid cultures and placement in an anaerobic environment exceed 45 minutes. For corneal cultures, there is a maximum of 30 minutes between plating and placement in the anaerobic environment. MICROBIOLOGY
Anaerobic media used for cultures of the conjunctiva and lid include an anaerobic blood agar plate and liquidthioglycollate broth enriched with vitamin K and hemin. Both media are incubated anaerobically. For corneal cultures, an additional cooked meat broth is inoculated and incubated anaerobically. Separate thioglycollate broth cultures from the conjunctiva, lid, and cornea are also taken and incubated aerobically. In order to establish proper anaerobic conditions in the incubation jar (Oxoid), an evacuation-replacement technique is used. After evacuating the jar with a vacuum, a gas mixture of carbon dioxide (10%), hydrogen (10%), and nitrogen (80%) is infused. This is repeated a minimum of five times. Achievement of an anaerobic environment is indicated when an oxidation-reduction potential indicator strip turns colorless. We also frequently employ an alternate method of creating an anaerobic environment with a disposable hydrogen and carbon dioxide generating kit. The anaerobic jar is incubated for 48 hours at 35 C before being opened. Negative cultures are reexamined every 72 hours for two weeks before being discarded. Identification of obligate anaerobes is accomplished by Gram's stain reaction and morphology, colonial morphology, lipase and lecithinase reaction, nitrate reduction, and their action on litmus milk.4 Biochemical characterization is accomplished using a commercially available identification system API-20A (Analytab Products, Inc.).
SENSITIVITY TESTING
Antibiotic susceptibility testing is performed using a modified disc diffusion technique. 5 The antibiotics and their disc potencies include: ampicillin (10 J-tg), carbenicillin (100 J-tg), cephalothin (30 J-tg), chloramphenicol (30 J-tg), clindamycin (2 J-tg), doxycycline (30 J-tg), erythromycin (15 J-tg), pencillin (10 units), tetracycline (30 J-tlt) and Cefamandole (30 J-tg). Anaerobic blood agar plates are swabbed with Bauer-Kirby standardized suspension of the organism and allowed to dry for 15 minutes. The antibiotic discs are pressed firmly onto the surface of the agar plate, three discs per plate. After 48 hours incubation at 35 C in the anaerobic jar, the zone diameter around each disc is measured with a caliper and results recorded. CRITERIA
Recovery of a bacterial organism from a corneal culture does not necessarily imply that the isolated organism is responsible for the disease process. To determine an organism's pathogenicity for the corneal ulcer from which it was cultured, certain criteria have been established. Those cases that are considered "confirmed" due to anaerobic organisms fall into one of three groups: (1) all three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is positive; (2) two out of three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is positive; or (3) all three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is negative. The criteria for a "probable" anaerobic corneal infection include those cases in which all corneal cultures are positive and the organism is seen on Gram's stain, but light growth of the anaerobe is also present on the conjunctival or lid cultures. The criteria for a "possible" anaerobic corneal ulcer include those cases where some or all of the corneal culture media are positive, but the Gram's stain is negative and the number of anaerobic colonies from the conjunctiva or lid exceeds that from the cornea.
RESULTS Between January 1, 1977 and June 30, 1981 a total of 326 patients with apparent corneal infections were cultured in our laboratory. Bacteria were recovered from 162 ulcer patients. Anaerobic organisms were grown from 27 (17%) of the bacterial ulcers. Applying the criteria for pathogenicity described, 11 ulcers (7%) had a confirmed anaerobic etiology. Two additional ulcers (1 %) were considered probably to be infected by anaerobes, while the remaining 14 ulcers (9%) were regarded as only possibly infected by these organisms. In many instances in the latter group, aerobic organisms were also cultured, casting significant doubt on the role of the cultured anaerobe in causing keratitis. Seven of 11 confirmed anaerobic ulcers were Gram's 637
OPHTHALMOLOGY • JUNE 1982 • VOLUME 89 • NUMBER 6
stain positive and in nine , all three anaerobic corneal media were positive for growth of the infecting organism. No morphologic characteristics were noted that could be used to distinguish anaerobic from aerobic bacterial keratitis. However, all 27 patients did manifest one or more predisposing factors that may have contributed to their ulcer development (Table 1.) There were 29 anaerobes cultured from 27 patients (Table 2). Two anaerobes were recovered from each of two patients in the confirmed group . Peptococcus anaerobius and Bifidobacterium eriksonii were cultured from one anaerobic ulcer, while from another Propionibacterium acnes and Clostridium perjringens were grown. Only one anaerobe was grown from the remaining 25 ulcers. However, there were ten ulcers (37%) from which anaerobes were grown that were also culture positive for aerobic bacterial or fungal organisms (Table 3). From one ofthe two ulcers positive for two anaerobic organisms, the aerobe Pseudomonas alcaligenes was also cultured. Mixed anaerobic and anaerobic-aerobic organisms were therefore recovered from 11 of 27 (41%) ulcers. Thioglycollate broth incubated in air is the most frequently used medium for recovery of anaerobic organisms. A comparison of its ability to recover anaerobes from ocular sources with that of multiple solid and broth media incubated under anaerobic conditions is therefore indicated. In our series thioglycollate broth was always positive when a single organism was cultured (16 ulcers). From the 11 ulcers positive
Table 1. Predisposing Factors in Anaerobic-Positive Corneal Ulcers Confirmed 11 Ulcers
Probable 2 Ulcers
Possible 14 Ulcers
6 (55%) 5 (45%)
1 (50%)
12 (86%) 5 (36%)
4 (36%)
2 (100%)
2 (14%)
Previous ocular disease or surgery Trauma Soft contact lens wearing Topical corticosteroids preceding bacterial keratitis
2 (18%)
4 (29%)
Table 2. Anaerobic Organisms Cultured from 27 Corneal Ulcers Organism Propionibacterium acnes Peptococcus asaccharolyticus Clostridium perfringens Bacteroides melaninogenicus Bifidobacterium eriksonii Pc. prevotii Pc . anaerobius Total
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Confirmed Probable Possible Total 6 2 1 1 1 1 1 13
12 1
2
14
19 3 1 3 1 1 1 29
Table 3. Mixed Anaerobic/Aerobic Corneal Infections Confirmed
Organism
Probable
Staphylococcus aureus (3) Enterobacter aerogenes (1) Pseudomonas aeruginosa (1) Fusarium solani (1)
Propionibacterium acnes
with Bacteroides melaninogenicus with Peptococcus ' anaerobius and with Bifidobacterium ' eriksonii Total
Possible
Staphylococcus Corynebacterium epidermidis (1) pseudodiphthepseUdomonas} riticum (1) alcaligenes
Streptococcus sanguis /I (1)
1
Pseudomonas alcaligenes
7
2
• Recovered from same ulcer.
for mixed organisms (13 anaerobes , 9 aerobes, 1 fungus) only two anaerobes were recovered using thioglycollate broth incubated aerobically . Overall, 18 of 29 anaerobic isolates (62%) and 10 of 15 (67%) from confirmed and probable anaerobic ulcers were identified using thioglycollate broth alone. From one of two mixed anaerobic ulcers C. perjringens, a rapidly growing aerotolerant organism, was grown in thioglycollate broth. From another patient with fungal keratitis P. acnes was recovered. Eleven other isolates of anaerobic organisms from mixed cultures were not identified using thioglycollate broth alone due to overgrowth of other organisms in the broth. Antibiotic susceptibility tests using ten antibiotics known to be active against most anaerobes were performed on 20 isolates (Table 4). All of the anaerobes except for two strains of Bacteroides melaninogenicus were susceptible to all antibiotics tested. One of the two isolates was not susceptible to penicillin and ampicillin while the other showed resistance to tetracycline and erythromycin as well as penicillin and ampicillin. Table 4. Antibiotic Susceptibility of Anaerobic Organisms Derived from Corneal Ulcers
Antibiotic Chloramphenicol Clindamycin Cephaloridine Tetracycline Ampicillin Penicillin Erythromycin Carbenicillin Doxycycline Cefamandole
Number of Isolates Tested
Number of Isolates Susceptible
Percent Susceptible
20 20 20 14 19 19 16 6 6 2
20 20 20 13 17 17 15 6 6 2
100 100 100 93 89 89 94 100 100 100
PERRY, et al • ANAEROBIC ULCERS
Our routine therapy for apparent bacterial keratitis consists of topical hourly gentamicin (9 mg/mt) and either bacitracin (10,000 units/ml) or cefazolin (50 mg/ml). Gram-stained corneal scrapings when definitely positive are used to make appropriate antibiotic selection. In most instances subconjunctival antibiotics (gentamicin and methicillin or cefazolin) are given only on the first day of therapy. Intravenous antibiotics are used rarely. Eight ofthe 13 patients regarded as having confirmed or probable anaerobic ulcers were switched to more conventional therapy for anaerobic organisms (chloramphenicol or carbenicillin) once the anaerobic nature of the infection was appreciated (Table 5). Final visual acuities of seven of eight appropriately treated ulcers are known and range from 20/20 to light perception. Four anaerobic ulcer patients were treated initially with gentamicin only or in combination with bacitracin. Both of these drugs are regarded as being of no value in treatment of anaerobic infection. Nevertheless, all four ulcers improved on this therapy, and the treatment was continued even after the anaerobic nature of the ulcer was known. One patient with recurrent erosion syndrome responded to patching only and received no antibiotics. His minimal keratitis cleared completely in two to three weeks without visual loss.
DISCUSSION The majority of organisms present in the skin, mouth, tracheobronchial tree, gastrointestinal tract, and genitourinary system are anaerobes. More than three fourths of the organisms in the mouth are anaerobic. 6.7 In the colon anaerobic bacteria outnumber aerobes by 1000 to 1 and in the skin, mouth, and vagina by 10 to 1.8 Over one third of the fecal mass is composed of bacterial cells, mostly anaerobes. It is not surprising, then, that anaerobes might be implicated in a host of infectious disease processes. However, general acceptance by the clinician of the role of anaerobes in human disease has awaited the more re-
cent advances in anaerobic microbiology. Now even the smallest hospital clinical laboratory can isolate and identify anaerobic organisms .9 In the past a bacteriologically negative culture from an abcess would have caused the clinician to make a diagnosis of "sterile pus." It is now generally recognized that many such cases represent anaerobic infection .IO Acceptance of the role of anaerobes in producing systemic infections has also been delayed by 'the clinician's belief in the concept of "one infection-one organism. " Physicians have tended to ignore anaerobes recovered from material also positive for aerobes and have frequently considered them contaminants. However, the polymicrobial nature of systemic anaerobic infection is now well recognized. II Generally more than one anaerobe and one or more aerobes can be recovered from clinical material resulting from anaerobic infection.12 In fact, there may be a synergistic relationship between anaerobes and aerobes 13 with the aerobe ' s replication producing a reduced oxidationreduction potential, a factor favoring anaerobic proliferation. 14 While there have been scattered reports of corneal ulcers due to C. perfringens, 15.16 an anaerobic sporeforming bacillus, a causal role for nonspore-forming anaerobes in infectious keratitis was not established until 1976. In that year Ostler and Okumot0 2 described a 10-year-old girl who developed deep keratitis following a penetrating holly leaf injury due to a member of the genus Peptostreptococcus. In 1976 Wilson also noted his previous isolation of Peptococcus morbi/orum from a corneal ulcer.17 Jones and Robinson 3 subsequently reported on their experience with anaerobes as etiologic agents in a variety of ocular infections, including five cases of confirmed anaerobic corneal ulcers. All five of their patients had had preexisting ocular disease, and three had received topical corticosteroids. None of their patients had antecedent trauma. Liesegang and Forster18 in 1980 reported on 238 positive bacterial corneal cultures from which two anaerobes (both Propionibacterium species) were identified.
Table 5. Anaerobic Keratitis Treated with Conventional Antibiotics Organism
Ulcer Location
Antibiotic Given
P. acnes P. acnes P. acnes P. acnes and C. perfringens Pc. asaccharolyticus Pc. prevotii
Paracentral Central (descemetocele) Central Inferocentral Inferonasal Inferior
Bifidobacterium eriksonii and P. anaerobius Bacteroides melaninogenicus
Chloramphenicol Chloramphenicol, Chloramphenicol, Chloramphenicol, Chloramphenicol Chloramphenicol , bacitracin
Central Central
Chloramphenicol Chloramphenicol
carbenicillin cefazolin carbenicillin gentamicin ,
Final Visual Acuity Allen cards 10 feet LP* 20/20 20/40 20/200 20/30 20/200 Unknown
• LP = light perception.
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Based upon our microbiologic criteria, 11 of 162 (7%) bacterial ulcers were caused by anaerobic organisms. Although this is not a large number, a significant percentage of corneal cultures would have been false negative if anaerobic cultures had not been performed. We feel the microbiologic criteria described will allow differentiation of ulcers due to anaerobic infection from ulcers due to other causes but from which anaerobes are also grown as a result of their colonization of periocular tissues. The finding of 14 positive anaerobic corneal cultures from patients felt to have keratitis on some other basis is c'onsistent with the observation of several investigators that anaerobes are present in the normal flora of periocular tissue 19- 22 and in increased frequency in the inflamed conjunctiva. 22 ,23 Our incidence of anaerobic recovery from periocular tissues of 14 of 162 ulcers (9%) is less than the 13 to 52% reported by others and may reflect insensitivity of our method and loss of organisms by contact with oxygen prior to establishment of an anaerobic environment. We have not used prereduced media, and a delay of 30 to 45 minutes between inoculation of the cultures and putting the plates in activated anaerobic jars frequently occurs. Also, the anaerobic cultures are examined initially at 48 hours rather than waiting the recommended five to seven days. This early exposure to oxygen may destroy incipient growth of the more fastidious anaerobes including P. acnes. 24 Greater attention to anaerobic methodology in the future may result in an increased incidence of both true and false positive anaerobic corneal ulcers. The anaerobic culture technique described results in significantly greater recovery of anaerobic organisms than would have occurred using a single thioglycollate broth maintained in air. While thioglycollate broth incubated aerobically was positive in all 16 ulcers from which only one anaerobe was recovered, only two of 13 anaerobes cultured from the 11 mixed ulcers were isolated using it. This relative inability to recover anaerobes from mixed cultures was due to overgrowth in thioglycollate broth of the more fastidious anaerobic organisms by rapidly growing aerobes and anaerobes. Use of this method of culturing anaerobes would therefore reduce by one third the incidence of culture positivity. A variety of factors are known to predispose patients to the development of systemic anaerobic infections. 9 These include: (1) tissue necrosis from trauma, surgery, aerobic, and facultative bacterial infection; (2) anoxia of tissues from vascular injury, compression, shock, or tissue edema; (3) human and animal bites; (4) malignancies of the colon, uterus, and lung, (5) previous antibiotic therapy ; and (6) certain systemic illnesses such as diabetes, collagen diseases, and those requiring systemic corticosteroid therapy. As in systemic anaerobic infections, a variety offactors predisposing to the development of confirmed and probable anaerobic ulcers were noted. These predisposing factors included previous ocular disease or surgery, 640
trauma, soft contact lens wearing, and use of topical corticosteroids. There were seven different anaerobic organisms isolated from the cornea in our series. Except for P. acnes and C. perfringens, the others have not been described as causes of anaerobic keratitis. These previously unreported organisms are: Bacteroides melaninogenicus, Bijidobacterium eriksonii, Peptococcus asaccharolyticus, Peptococcus prevotti, and Peptococcus anaerobius. A variety of antibiotics are effective against anaerobes. If it were not for Bacteroides fragilis, a common cause of systemic infection, penicillin would be the drug of choice for all anaerobic infections. However, B. fragilis is resistant to levels of penicillin safely achieved by systemic administration. Therefore, chloramphenicol is the drug of choice for most anaerobes,25 but its potential systemic toxicity limits its usefulness. While the cephalosporins are not usually considered drugs of choice in anaerobic infections due to resistance of B. fragilis, they are quite effective against the majority of anaerobic strains isolated. 26 Carbenicillin27 and ampicillin28 are also active against most anaerobes, but the isoxazolyl penicillins such as methicillin and oxacillin have little activity. Clindamycin is very effective against anaerobes 29 but due to its potential to produce life-threatening pseudomembranous colitis, its systemic use is reserved for more serious infections. The aminoglycosides are considered ineffective against most anaerobes at the serum levels achievable without nephrotoxicity. 30 All of our ocular isolates except for two strains of B. melaninogenicus were susceptible to all antibiotics tested. One of these strains was resistant to penicillin and ampicillin, while the other was resistant to penicillin, ampicillin, erythromycin, and tetracycline. Based upon this limited data we feel antibiotic susceptibility testing is helpful but not critical and that therapy should be instituted on the basis of probable susceptibility of the isolated organism. In choosing topical antibiotics for initial ulcer therapy, we rely heavily on broad spectrum antibiotic coverage. For initial therapy we prefer cefazolin in combination with gentamicin because of cefazolin's known efficacy against most anaerobic as well as grampositive aerobic organisms. Once an anaerobe is identified and its pathogenicity for the ulcer confirmed, topical chloramphenicol is also started. In order to reduce the likelihood of misdiagnosis and inadequate therapy of anaerobic corneal ulcers, we recommend: (1) consider all infected appearing ulcers as potentially due to anaerobes, (2) culture for anaerobes using several media incubated in an anaerobic environment, (3) use a drug, eg, cefazolin, known to be effective against anaerobes in all ulcers requiring broad spectrum antibiotic coverage, and (4) give specific treatment for an anaerobic infection in the presence of a confirmed anaerobic culture even if pathogenic aerobic organisms are also identified from the cornea.
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REFERENCES 1. Jones DB. Initial therapy of suspected microbial corneal ulcers: specific antibiotic therapy based on corneal smears. Surv Ophthalmol 1979; 24:97, 105-16. 2. Ostler HB, Okumoto M. Anaerobic streptococcal corneal ulcer. Am J Ophthalmol 1976; 81 :518-9. 3. Jones DB, Robinson NM. Anaerobic ocular infections . Trans Am Acad Ophthalmol Otolaryngol 1977; 83 :309 - 31 . 4. Allen SO, Siders, JA. Procedures for isolation and characterization of anaerobic bacteria. In: Balows A, Hausler WJ Jr, Truant JP, eds. Manual of Clinical Microbiology, 3rd ed . Washington DC: American Society for Microbiology. 1980; 397- 417. 5. Bodner SJ, Koenig MG, Treanor LL, Goodman JS. Antibiotic susceptibility testing of Bacteroides . Antimicrob Agents Chemother 1972; 2:57-60. 6. Skinner FA, Carr JG, eds. The normal microbial flora of man. The Society for Applied Bacteriology. London : Academic Press 1974; 47-74. 7. Olson RE, Morello JA, Kieft ED. Antibiotic treatment of oral anaerobic infections . J Oral Surg 1975; 33:619 - 21 . 8. WOst J. The diagnosis of anaerobic infections. Experientia 1977; 33:1671-4. 9. Meyer RD, Finegold SM. Anaerobic infections: diagnosis and treatment. South Med J 1976; 69:1178- 95. 10. Nichols RL, Smith Jw. Modern approach to the diagnosis of anaerobic surgical seps is. Surg Cl in North Am 1975 ; 55:21-30. 11. Finegold SM. Anaerobic infections. Surg Clin North Am 1980; 60:49-64. 12. Klastersky J, Coppens L, Mombelli G. Anaerobic infection in cancer patients: comparative evaluation of clindamycin and cefoxitin. Antimicrob Agents Chemother 1979; 16:366-71 13. Dankert J, Holloway Y, Bouwma J. Bacterial synergy in mixed aerobic/anaerobic infections . Lancet 1980; 1(8170):714. 14. Levison ME. The importance of anaerobic bacteria in infectious diseases . Med Clin North Am 1973; 57:1015-27. 15. Majekodunmi S, Odugbemi T. Clostridium we/chii corneal ulcer: a case report. Can J Ophthalmol 1975; 10:290-2.
16. Stern GA, Hodes BL, Stock EL. Clostridium perfringens corneal ulcer. Arch Ophthalmol 1979; 97:661-3. 17. Wilson LA. Bacterial corneal ulcers. In: Duane TD, ed. Clinical Ophthalmology. Hagerstown : Harper & Row, 1980; Vol. 4, Chapter 18. 18. Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol 1980; 90:38-47. 19. Matuura H. Anaerobes in the bacterial flora of the conjunctival sac . Jpn J Ophthalmol1971 ; 15:116- 24 . 20. Perkins RE, Kundsin RB, Pratt MV, et al. Bacteriology of normal and infected conjun ctiva. J Clin Microbiol1975; 1:147-9. 21. McNatt J, Allen SO, Wilson LA, Dowell VR Jr. Anaerobic flora of the normal human conjunctival sac. Arch Ophthalmol 1978; 96:1448-50. 22. Brook I, Pettit TH, Martin WJ, Finegold SM. Anaerobic and aerobic bacteriology of acute conjun ctivitis. Ann Ophthalmol 1979; 11 :389-93. 23. Brook I. Anaerobic and aerobic bacterial flora of acute conjunctivitis in children. Arch Ophthalmol 1980; 98:833-5. 24. Wren MWD. Prolonged primary incubation in the isolation of anaerobic bacteria from clinical specimens. J Med Microbiol 1980; 13:257-63 . 25. Nichols RL, Schumer W, Nyhus LM, et al. Anaerobic infections . Am Fam PhYSician 1976; 14(4):100-10. 26. Ernst EC, Berger S, Barza M, et al. Activity of cefamandole and other cephalosporins against aerobic and anaerobic bacteria. Antimicrob Agents Chemother 1976; 9:8 52-5. 27. Fiedelman W, Webb CD. Clinical evaluation of carbenici llin in the treatment of infection due to anaerobic bacteria. Curr Ther Res 1975; 18:441-51. 28. Rahman M. Sensitivities of clinically significant organisms to four cephalosporins, ampicillin and mecillinam. Curr Med Res Opin 1980; 7:9 6-100. 29. Levison ME, Santoro J, Bran JL, et al. In vitro activity and clinical efficacy of clindamycin in the treatment of infections due to anaerobic bacteria. J Infect Dis 1977; 135(suppl): 549-53. 30. Martin WJ , Gardner M, Washington JA II. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical spec imens . Antimicrob Agents Chemother 1972; 1:1 48-58.
Discussion by Thomas J. Liesegang, MD Ophthalmologists have lagged behind other specialists in establishing the role of anaerobes in clinical disease . Anaerobes are important and perhaps are the predominant pathogen in many serious superficial and deep abscesses of the body , although they are infrequently found in most acute infections. Until 1977,1 only scattered reports on anaerobes appeared in the ophthalmic literature. The report by Perry et al offers further stimulus for re-examination of our laboratory techniques to ensure recovery of responsible organisms in microbial keratitis . In most large series of patients with ulcerative keratitis, about 45% of the cases are classified as From Mayo Clinic, Rochester, Minnesota.
culture negative. Specific attention to anaerobic culturing techniques could help identify the pathogen in some of these cases. The clinical setting for anaerobic infection elsewhere in the body has been established.2 The tissue is usually severely compromised, Gram' s stain shows extensive variability in morphologic characteristics and coloration, and a synergism seems to exist between anaerobes and aerobes. 3 Within the ocular tissues , a similar polymicrobial nature of anaerobes is beginning to emerge. As in most corneal infections, however, predisposing factors are present frequently, and no distinguishing morphologic features are evident before laboratory testing.
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Universally accepted criteria for establishing the presence of a true pathogen in microbial keratitis are still lacking. In this paper, very strict criteria were used with regard to isolation medium, results of Gram's stain, and associated cultures of the eyelid and conjunctiva. Previous reports of anaerobic isolation have usually been based on a single isolation medium, with or without confirmation by Gram's stain. Most microbiologists would agree that the authors have probably chosen the three ideal media to support primary anaerobic isolation. For simplicity, most ocular microbiology laboratories use only thioglycolate broth supplemented with hemin, vitamin K, and serum. The present study, as well as a study several years ago by Rosenblatt et al,4 revealed the shortcomings of using thioglycolate broth alone. These deficiencies are disturbing because they necessitate addition of other culture media to an already long list. The current study indicates that we cannot predict those corneal infections in which anaerobes should be suspected clinically. Using thioglycolate broth alone does not allow us to monitor contamination, quantitate the isolate, or prevent strict anaerobes from being overgrown by aerobes and facultative anaerobes. The use of a solid medium overcomes some of these problems but necessitates immediate placement in an anaerobic setting, probably best accessible in the area of inoculation. Basically, three devices are available for producing an anaerobic environment. The anaerobic glove box and prereduced anaerobic sterilized media are quite expensive and complex and require considerable space and training of technicians. Fortunately, the simple anaerobic jars, equipped with either a Gas-Pak or an evacuation system, have proved to be sensitive to most clinical pathogens (Rosenblatt JE, personal communication, 1981). This sensitivity may be due to the phenomenon of coincubation, wherein other anaerobes produce volatile growth-enhancing products in close proximity. Now that anaerobes are respected as pathogens, antibiotic susceptibility testing is recommended in serious infections because specific therapy occasionally is crucial. Previous rules about the predictability of antibiotic activity against anaerobes may no longer be applicable because of genetic mutation. Researchers have developed different antibiotics for management of these infections, and recommendations may change during the next several years on the basis of periodic surveys.
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Yet to be developed, however, is a reliable antibiotic susceptibility testing system. The authors used an agar diskdiffusion technique, but this procedure is frequently faulty because of the variable growth rate of the anaerobic bacteria. More useful perhaps are the broth disk-elution technique of Kurzynski et aJ5 and the agar-dilution test of Sutter et al. 6 The handling of anaerobic cultures involves more than the simple addition of a different medium. Because of the expense, certain collection and transport rules must be enforced or most laboratories will simply refuse to process the cultures. Contamination with normal flora must be avoided. Any air in swabs, in transport, or during processing can interfere with the recovery rate. Reliance on a single liquid medium for isolation may be unreasonable, as these authors have pointed out. The laboratory should take special precautions with regard to the addition of certain supplements (eg, vitamin K, hemin, and serum) and should carefully monitor the setup of the anaerobic jar with all of its catalysts and indicators. The main thrust of this paper is accurate: unless the clinician and the microbiologist perform the additional steps necessary to detect these anaerobes, these pathogens will continue to be overlooked. REFERENCES 1. Jones DB, Robinson NM. Anaerobic ocular infections. Trans Am Acad Ophthalmol Otolaryngol 1977; 83:309-31. 2. Finegold SM. Anaerobic infections. Surg Clin North Am
1980; 60:49-64. 3. Dankert J, Holloway Y, Bouwma J. Bacterial synergy in mixed aerobic/anaerobic infections. Lancet 1980; 1(8170):714. 4. Rosenblatt JE, Fallon A, Finegold SM. Comparison of methods for isolation of anaerobic bacteria from clinical specimens. Appl Microbiol 1973; 25:77-85 5. Kurzynski TA, Yrios JW, Helstad AG, Field CR. Aerobically incubated thioglycolate broth disk method for antibiotic susceptibility testing of anaerobes. Antimicrob Agents Chemother
1976; 10:727-32. 6. Sutter VL, Barry AL, Wilkins TO, Zabransky RJ. Collaborative evaluation of a proposed reference dilution method of susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 1979; 16:495- 502.
Abstract: In a series of 162 bacterial corneal ulcers, 27 were culture positive for anaerobic organisms. Applying strict microbiologic criteria, 11 ulcers were determined to be due to anaerobic infection. No morphologic characteristic was identified to distinguish anaerobic from other types of corneal ulcers. All of the anaerobic ulcer patients manifested one or more predisposing factors. A total of 13 anaerobes were cultured from the 11 confirmed anaerobic ulcers. Five previously unreported anaerobic strains were identified as causes of bacterial keratitis. Over one third of the anaerobic organisms occurred in mixed cultures with other organisms. Most of the anaerobic isolates were susceptible to all antibiotics routinely used for their treatment. The use of topical chloramphenicol is recommended for treatment of confirmed anaerobic ulcers, and topical cefazolin or one of the other cephalosporins effective against anaerobes is suggested to be included in the treatment of all ulcers requiring broad spectrum antibiotic coverage. [Key words: anaerobic corneal ulcers, anaerobic ocular infections, bacterial keratitis, cornea, ocular microbiology.] Ophthalmology 89:636-642, 1982
Successful therapy of a bacterial corneal ulcer. requires rapid identification of the causative organism and initiation of appropriate antibiotic therapy. 1 In many instances, however, the corneal culture is negative in an apparently infected ulcer. Possible explanations for such a negative culture include: (1) antibiotic pretreatment, (2) inadequate culture methods, and (3) true culture negativity. While the role of aerobic organisms in causing corneal inflammation has long been appreciated, it was not until recently that Ostler and Okumot0 2 and Jones and Robinson 3 confirmed nons pore-forming anaerobes as a cause of bacterial corneal ulcers. Believing that many of our own culture negative corneal ulcers were really due to anaerobic organisms that we could not culture with standard techniques, an improved methodology for anaerobic organisms was instituted in our From the Department of Ophthalmology, University of South Florida, Tampa, Florida. Presented at the Eighty-sixth Annual Meeting of the American Academy of Ophthalmology, Atlanta, Georgia, November 1-6, 1981. Supported in part by a grant from the Central Florida Lions Eye Bank. Reprint requests to L. Douglas Perry, MD, Department of Ophthalmology, University of South Florida, Box 21, 12901 N. 30th Street, Tampa, FL 33612.
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ocular microbiology laboratory in 1977. Twenty-seven corneal ulcers from which anaerobic organisms were grown are reported on herein.
MATERIALS AND METHODS SPECIMEN COLLECTION AND TRANSPORT A TION
Conjunctival cultures are taken prior to corneal culturing using a sterile thioglycollate-moistened cotton applicator rolled along the length of the inferior conjunctival cul-de-sac. Plating on the left side of the culture plates is immediately done. Once inoculation on solid media has been accomplished, the moistened cotton applicator tip is broken off in a tube of thioglycollate broth maintained at room temperature. A similarly moistened cotton applicator is then rolled along the inferior lid margin and applied to the right side of the previously inoculated plate. The moistened cotton applicator is then put in thioglycollate broth. In most instances, conjunctival and lid cultures of the patient's opposite eye are also performed. Several drops of topical proparacaine hydrochloride are then instilled in the involved eye over a period of several minutes. The cornea is scraped at the slit lamp with a Kimura platinum spatula. Usually the peripheral edge 0161-6420/82/0600/636/$00.85
© American Academy of Ophthalmology
PERRY, et al • ANAEROBIC ULCERS
of the ulcer is chosen for the site of culturing rather than a central necrotic area. In addition to multiple culture plate inoculations, several slides are also prepared for subsequent staining. Care is maintained throughout the corneal culturing procedure to avoid contact between the lids and platinum spatula. Following inoculation of solid media and preparation of slides the patient is reclined in the examining chair and broth media are inoculated. A sterile cotton applicator is moistened with the appropriate broth and "twirled" in the ulcer. The applicator is then broken off at its tip into the broth media. The time required for the entire culturing procedure is usually less than 30 minutes. Most specimens are taken at our medical center and immediately transported to the ocular microbiology laboratory. Upon arrival in the laboratory, designated solid and broth media from all sources are immediately placed in an anaerobic jar. If the specimens are taken at a site distant from the laboratory, an anaerobic jar is brought to the culture location and the appropriate plates and broths are placed in the activated jar. In no instance does the period of time between performing the conjunctival and lid cultures and placement in an anaerobic environment exceed 45 minutes. For corneal cultures, there is a maximum of 30 minutes between plating and placement in the anaerobic environment. MICROBIOLOGY
Anaerobic media used for cultures of the conjunctiva and lid include an anaerobic blood agar plate and liquidthioglycollate broth enriched with vitamin K and hemin. Both media are incubated anaerobically. For corneal cultures, an additional cooked meat broth is inoculated and incubated anaerobically. Separate thioglycollate broth cultures from the conjunctiva, lid, and cornea are also taken and incubated aerobically. In order to establish proper anaerobic conditions in the incubation jar (Oxoid), an evacuation-replacement technique is used. After evacuating the jar with a vacuum, a gas mixture of carbon dioxide (10%), hydrogen (10%), and nitrogen (80%) is infused. This is repeated a minimum of five times. Achievement of an anaerobic environment is indicated when an oxidation-reduction potential indicator strip turns colorless. We also frequently employ an alternate method of creating an anaerobic environment with a disposable hydrogen and carbon dioxide generating kit. The anaerobic jar is incubated for 48 hours at 35 C before being opened. Negative cultures are reexamined every 72 hours for two weeks before being discarded. Identification of obligate anaerobes is accomplished by Gram's stain reaction and morphology, colonial morphology, lipase and lecithinase reaction, nitrate reduction, and their action on litmus milk.4 Biochemical characterization is accomplished using a commercially available identification system API-20A (Analytab Products, Inc.).
SENSITIVITY TESTING
Antibiotic susceptibility testing is performed using a modified disc diffusion technique. 5 The antibiotics and their disc potencies include: ampicillin (10 J-tg), carbenicillin (100 J-tg), cephalothin (30 J-tg), chloramphenicol (30 J-tg), clindamycin (2 J-tg), doxycycline (30 J-tg), erythromycin (15 J-tg), pencillin (10 units), tetracycline (30 J-tlt) and Cefamandole (30 J-tg). Anaerobic blood agar plates are swabbed with Bauer-Kirby standardized suspension of the organism and allowed to dry for 15 minutes. The antibiotic discs are pressed firmly onto the surface of the agar plate, three discs per plate. After 48 hours incubation at 35 C in the anaerobic jar, the zone diameter around each disc is measured with a caliper and results recorded. CRITERIA
Recovery of a bacterial organism from a corneal culture does not necessarily imply that the isolated organism is responsible for the disease process. To determine an organism's pathogenicity for the corneal ulcer from which it was cultured, certain criteria have been established. Those cases that are considered "confirmed" due to anaerobic organisms fall into one of three groups: (1) all three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is positive; (2) two out of three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is positive; or (3) all three corneal culture media are positive, the conjunctival and lid cultures are negative, and the Gram's stain is negative. The criteria for a "probable" anaerobic corneal infection include those cases in which all corneal cultures are positive and the organism is seen on Gram's stain, but light growth of the anaerobe is also present on the conjunctival or lid cultures. The criteria for a "possible" anaerobic corneal ulcer include those cases where some or all of the corneal culture media are positive, but the Gram's stain is negative and the number of anaerobic colonies from the conjunctiva or lid exceeds that from the cornea.
RESULTS Between January 1, 1977 and June 30, 1981 a total of 326 patients with apparent corneal infections were cultured in our laboratory. Bacteria were recovered from 162 ulcer patients. Anaerobic organisms were grown from 27 (17%) of the bacterial ulcers. Applying the criteria for pathogenicity described, 11 ulcers (7%) had a confirmed anaerobic etiology. Two additional ulcers (1 %) were considered probably to be infected by anaerobes, while the remaining 14 ulcers (9%) were regarded as only possibly infected by these organisms. In many instances in the latter group, aerobic organisms were also cultured, casting significant doubt on the role of the cultured anaerobe in causing keratitis. Seven of 11 confirmed anaerobic ulcers were Gram's 637
OPHTHALMOLOGY • JUNE 1982 • VOLUME 89 • NUMBER 6
stain positive and in nine , all three anaerobic corneal media were positive for growth of the infecting organism. No morphologic characteristics were noted that could be used to distinguish anaerobic from aerobic bacterial keratitis. However, all 27 patients did manifest one or more predisposing factors that may have contributed to their ulcer development (Table 1.) There were 29 anaerobes cultured from 27 patients (Table 2). Two anaerobes were recovered from each of two patients in the confirmed group . Peptococcus anaerobius and Bifidobacterium eriksonii were cultured from one anaerobic ulcer, while from another Propionibacterium acnes and Clostridium perjringens were grown. Only one anaerobe was grown from the remaining 25 ulcers. However, there were ten ulcers (37%) from which anaerobes were grown that were also culture positive for aerobic bacterial or fungal organisms (Table 3). From one ofthe two ulcers positive for two anaerobic organisms, the aerobe Pseudomonas alcaligenes was also cultured. Mixed anaerobic and anaerobic-aerobic organisms were therefore recovered from 11 of 27 (41%) ulcers. Thioglycollate broth incubated in air is the most frequently used medium for recovery of anaerobic organisms. A comparison of its ability to recover anaerobes from ocular sources with that of multiple solid and broth media incubated under anaerobic conditions is therefore indicated. In our series thioglycollate broth was always positive when a single organism was cultured (16 ulcers). From the 11 ulcers positive
Table 1. Predisposing Factors in Anaerobic-Positive Corneal Ulcers Confirmed 11 Ulcers
Probable 2 Ulcers
Possible 14 Ulcers
6 (55%) 5 (45%)
1 (50%)
12 (86%) 5 (36%)
4 (36%)
2 (100%)
2 (14%)
Previous ocular disease or surgery Trauma Soft contact lens wearing Topical corticosteroids preceding bacterial keratitis
2 (18%)
4 (29%)
Table 2. Anaerobic Organisms Cultured from 27 Corneal Ulcers Organism Propionibacterium acnes Peptococcus asaccharolyticus Clostridium perfringens Bacteroides melaninogenicus Bifidobacterium eriksonii Pc. prevotii Pc . anaerobius Total
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Confirmed Probable Possible Total 6 2 1 1 1 1 1 13
12 1
2
14
19 3 1 3 1 1 1 29
Table 3. Mixed Anaerobic/Aerobic Corneal Infections Confirmed
Organism
Probable
Staphylococcus aureus (3) Enterobacter aerogenes (1) Pseudomonas aeruginosa (1) Fusarium solani (1)
Propionibacterium acnes
with Bacteroides melaninogenicus with Peptococcus ' anaerobius and with Bifidobacterium ' eriksonii Total
Possible
Staphylococcus Corynebacterium epidermidis (1) pseudodiphthepseUdomonas} riticum (1) alcaligenes
Streptococcus sanguis /I (1)
1
Pseudomonas alcaligenes
7
2
• Recovered from same ulcer.
for mixed organisms (13 anaerobes , 9 aerobes, 1 fungus) only two anaerobes were recovered using thioglycollate broth incubated aerobically . Overall, 18 of 29 anaerobic isolates (62%) and 10 of 15 (67%) from confirmed and probable anaerobic ulcers were identified using thioglycollate broth alone. From one of two mixed anaerobic ulcers C. perjringens, a rapidly growing aerotolerant organism, was grown in thioglycollate broth. From another patient with fungal keratitis P. acnes was recovered. Eleven other isolates of anaerobic organisms from mixed cultures were not identified using thioglycollate broth alone due to overgrowth of other organisms in the broth. Antibiotic susceptibility tests using ten antibiotics known to be active against most anaerobes were performed on 20 isolates (Table 4). All of the anaerobes except for two strains of Bacteroides melaninogenicus were susceptible to all antibiotics tested. One of the two isolates was not susceptible to penicillin and ampicillin while the other showed resistance to tetracycline and erythromycin as well as penicillin and ampicillin. Table 4. Antibiotic Susceptibility of Anaerobic Organisms Derived from Corneal Ulcers
Antibiotic Chloramphenicol Clindamycin Cephaloridine Tetracycline Ampicillin Penicillin Erythromycin Carbenicillin Doxycycline Cefamandole
Number of Isolates Tested
Number of Isolates Susceptible
Percent Susceptible
20 20 20 14 19 19 16 6 6 2
20 20 20 13 17 17 15 6 6 2
100 100 100 93 89 89 94 100 100 100
PERRY, et al • ANAEROBIC ULCERS
Our routine therapy for apparent bacterial keratitis consists of topical hourly gentamicin (9 mg/mt) and either bacitracin (10,000 units/ml) or cefazolin (50 mg/ml). Gram-stained corneal scrapings when definitely positive are used to make appropriate antibiotic selection. In most instances subconjunctival antibiotics (gentamicin and methicillin or cefazolin) are given only on the first day of therapy. Intravenous antibiotics are used rarely. Eight ofthe 13 patients regarded as having confirmed or probable anaerobic ulcers were switched to more conventional therapy for anaerobic organisms (chloramphenicol or carbenicillin) once the anaerobic nature of the infection was appreciated (Table 5). Final visual acuities of seven of eight appropriately treated ulcers are known and range from 20/20 to light perception. Four anaerobic ulcer patients were treated initially with gentamicin only or in combination with bacitracin. Both of these drugs are regarded as being of no value in treatment of anaerobic infection. Nevertheless, all four ulcers improved on this therapy, and the treatment was continued even after the anaerobic nature of the ulcer was known. One patient with recurrent erosion syndrome responded to patching only and received no antibiotics. His minimal keratitis cleared completely in two to three weeks without visual loss.
DISCUSSION The majority of organisms present in the skin, mouth, tracheobronchial tree, gastrointestinal tract, and genitourinary system are anaerobes. More than three fourths of the organisms in the mouth are anaerobic. 6.7 In the colon anaerobic bacteria outnumber aerobes by 1000 to 1 and in the skin, mouth, and vagina by 10 to 1.8 Over one third of the fecal mass is composed of bacterial cells, mostly anaerobes. It is not surprising, then, that anaerobes might be implicated in a host of infectious disease processes. However, general acceptance by the clinician of the role of anaerobes in human disease has awaited the more re-
cent advances in anaerobic microbiology. Now even the smallest hospital clinical laboratory can isolate and identify anaerobic organisms .9 In the past a bacteriologically negative culture from an abcess would have caused the clinician to make a diagnosis of "sterile pus." It is now generally recognized that many such cases represent anaerobic infection .IO Acceptance of the role of anaerobes in producing systemic infections has also been delayed by 'the clinician's belief in the concept of "one infection-one organism. " Physicians have tended to ignore anaerobes recovered from material also positive for aerobes and have frequently considered them contaminants. However, the polymicrobial nature of systemic anaerobic infection is now well recognized. II Generally more than one anaerobe and one or more aerobes can be recovered from clinical material resulting from anaerobic infection.12 In fact, there may be a synergistic relationship between anaerobes and aerobes 13 with the aerobe ' s replication producing a reduced oxidationreduction potential, a factor favoring anaerobic proliferation. 14 While there have been scattered reports of corneal ulcers due to C. perfringens, 15.16 an anaerobic sporeforming bacillus, a causal role for nonspore-forming anaerobes in infectious keratitis was not established until 1976. In that year Ostler and Okumot0 2 described a 10-year-old girl who developed deep keratitis following a penetrating holly leaf injury due to a member of the genus Peptostreptococcus. In 1976 Wilson also noted his previous isolation of Peptococcus morbi/orum from a corneal ulcer.17 Jones and Robinson 3 subsequently reported on their experience with anaerobes as etiologic agents in a variety of ocular infections, including five cases of confirmed anaerobic corneal ulcers. All five of their patients had had preexisting ocular disease, and three had received topical corticosteroids. None of their patients had antecedent trauma. Liesegang and Forster18 in 1980 reported on 238 positive bacterial corneal cultures from which two anaerobes (both Propionibacterium species) were identified.
Table 5. Anaerobic Keratitis Treated with Conventional Antibiotics Organism
Ulcer Location
Antibiotic Given
P. acnes P. acnes P. acnes P. acnes and C. perfringens Pc. asaccharolyticus Pc. prevotii
Paracentral Central (descemetocele) Central Inferocentral Inferonasal Inferior
Bifidobacterium eriksonii and P. anaerobius Bacteroides melaninogenicus
Chloramphenicol Chloramphenicol, Chloramphenicol, Chloramphenicol, Chloramphenicol Chloramphenicol , bacitracin
Central Central
Chloramphenicol Chloramphenicol
carbenicillin cefazolin carbenicillin gentamicin ,
Final Visual Acuity Allen cards 10 feet LP* 20/20 20/40 20/200 20/30 20/200 Unknown
• LP = light perception.
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Based upon our microbiologic criteria, 11 of 162 (7%) bacterial ulcers were caused by anaerobic organisms. Although this is not a large number, a significant percentage of corneal cultures would have been false negative if anaerobic cultures had not been performed. We feel the microbiologic criteria described will allow differentiation of ulcers due to anaerobic infection from ulcers due to other causes but from which anaerobes are also grown as a result of their colonization of periocular tissues. The finding of 14 positive anaerobic corneal cultures from patients felt to have keratitis on some other basis is c'onsistent with the observation of several investigators that anaerobes are present in the normal flora of periocular tissue 19- 22 and in increased frequency in the inflamed conjunctiva. 22 ,23 Our incidence of anaerobic recovery from periocular tissues of 14 of 162 ulcers (9%) is less than the 13 to 52% reported by others and may reflect insensitivity of our method and loss of organisms by contact with oxygen prior to establishment of an anaerobic environment. We have not used prereduced media, and a delay of 30 to 45 minutes between inoculation of the cultures and putting the plates in activated anaerobic jars frequently occurs. Also, the anaerobic cultures are examined initially at 48 hours rather than waiting the recommended five to seven days. This early exposure to oxygen may destroy incipient growth of the more fastidious anaerobes including P. acnes. 24 Greater attention to anaerobic methodology in the future may result in an increased incidence of both true and false positive anaerobic corneal ulcers. The anaerobic culture technique described results in significantly greater recovery of anaerobic organisms than would have occurred using a single thioglycollate broth maintained in air. While thioglycollate broth incubated aerobically was positive in all 16 ulcers from which only one anaerobe was recovered, only two of 13 anaerobes cultured from the 11 mixed ulcers were isolated using it. This relative inability to recover anaerobes from mixed cultures was due to overgrowth in thioglycollate broth of the more fastidious anaerobic organisms by rapidly growing aerobes and anaerobes. Use of this method of culturing anaerobes would therefore reduce by one third the incidence of culture positivity. A variety of factors are known to predispose patients to the development of systemic anaerobic infections. 9 These include: (1) tissue necrosis from trauma, surgery, aerobic, and facultative bacterial infection; (2) anoxia of tissues from vascular injury, compression, shock, or tissue edema; (3) human and animal bites; (4) malignancies of the colon, uterus, and lung, (5) previous antibiotic therapy ; and (6) certain systemic illnesses such as diabetes, collagen diseases, and those requiring systemic corticosteroid therapy. As in systemic anaerobic infections, a variety offactors predisposing to the development of confirmed and probable anaerobic ulcers were noted. These predisposing factors included previous ocular disease or surgery, 640
trauma, soft contact lens wearing, and use of topical corticosteroids. There were seven different anaerobic organisms isolated from the cornea in our series. Except for P. acnes and C. perfringens, the others have not been described as causes of anaerobic keratitis. These previously unreported organisms are: Bacteroides melaninogenicus, Bijidobacterium eriksonii, Peptococcus asaccharolyticus, Peptococcus prevotti, and Peptococcus anaerobius. A variety of antibiotics are effective against anaerobes. If it were not for Bacteroides fragilis, a common cause of systemic infection, penicillin would be the drug of choice for all anaerobic infections. However, B. fragilis is resistant to levels of penicillin safely achieved by systemic administration. Therefore, chloramphenicol is the drug of choice for most anaerobes,25 but its potential systemic toxicity limits its usefulness. While the cephalosporins are not usually considered drugs of choice in anaerobic infections due to resistance of B. fragilis, they are quite effective against the majority of anaerobic strains isolated. 26 Carbenicillin27 and ampicillin28 are also active against most anaerobes, but the isoxazolyl penicillins such as methicillin and oxacillin have little activity. Clindamycin is very effective against anaerobes 29 but due to its potential to produce life-threatening pseudomembranous colitis, its systemic use is reserved for more serious infections. The aminoglycosides are considered ineffective against most anaerobes at the serum levels achievable without nephrotoxicity. 30 All of our ocular isolates except for two strains of B. melaninogenicus were susceptible to all antibiotics tested. One of these strains was resistant to penicillin and ampicillin, while the other was resistant to penicillin, ampicillin, erythromycin, and tetracycline. Based upon this limited data we feel antibiotic susceptibility testing is helpful but not critical and that therapy should be instituted on the basis of probable susceptibility of the isolated organism. In choosing topical antibiotics for initial ulcer therapy, we rely heavily on broad spectrum antibiotic coverage. For initial therapy we prefer cefazolin in combination with gentamicin because of cefazolin's known efficacy against most anaerobic as well as grampositive aerobic organisms. Once an anaerobe is identified and its pathogenicity for the ulcer confirmed, topical chloramphenicol is also started. In order to reduce the likelihood of misdiagnosis and inadequate therapy of anaerobic corneal ulcers, we recommend: (1) consider all infected appearing ulcers as potentially due to anaerobes, (2) culture for anaerobes using several media incubated in an anaerobic environment, (3) use a drug, eg, cefazolin, known to be effective against anaerobes in all ulcers requiring broad spectrum antibiotic coverage, and (4) give specific treatment for an anaerobic infection in the presence of a confirmed anaerobic culture even if pathogenic aerobic organisms are also identified from the cornea.
PERRY, et al • ANAEROBIC ULCERS
REFERENCES 1. Jones DB. Initial therapy of suspected microbial corneal ulcers: specific antibiotic therapy based on corneal smears. Surv Ophthalmol 1979; 24:97, 105-16. 2. Ostler HB, Okumoto M. Anaerobic streptococcal corneal ulcer. Am J Ophthalmol 1976; 81 :518-9. 3. Jones DB, Robinson NM. Anaerobic ocular infections . Trans Am Acad Ophthalmol Otolaryngol 1977; 83 :309 - 31 . 4. Allen SO, Siders, JA. Procedures for isolation and characterization of anaerobic bacteria. In: Balows A, Hausler WJ Jr, Truant JP, eds. Manual of Clinical Microbiology, 3rd ed . Washington DC: American Society for Microbiology. 1980; 397- 417. 5. Bodner SJ, Koenig MG, Treanor LL, Goodman JS. Antibiotic susceptibility testing of Bacteroides . Antimicrob Agents Chemother 1972; 2:57-60. 6. Skinner FA, Carr JG, eds. The normal microbial flora of man. The Society for Applied Bacteriology. London : Academic Press 1974; 47-74. 7. Olson RE, Morello JA, Kieft ED. Antibiotic treatment of oral anaerobic infections . J Oral Surg 1975; 33:619 - 21 . 8. WOst J. The diagnosis of anaerobic infections. Experientia 1977; 33:1671-4. 9. Meyer RD, Finegold SM. Anaerobic infections: diagnosis and treatment. South Med J 1976; 69:1178- 95. 10. Nichols RL, Smith Jw. Modern approach to the diagnosis of anaerobic surgical seps is. Surg Cl in North Am 1975 ; 55:21-30. 11. Finegold SM. Anaerobic infections. Surg Clin North Am 1980; 60:49-64. 12. Klastersky J, Coppens L, Mombelli G. Anaerobic infection in cancer patients: comparative evaluation of clindamycin and cefoxitin. Antimicrob Agents Chemother 1979; 16:366-71 13. Dankert J, Holloway Y, Bouwma J. Bacterial synergy in mixed aerobic/anaerobic infections . Lancet 1980; 1(8170):714. 14. Levison ME. The importance of anaerobic bacteria in infectious diseases . Med Clin North Am 1973; 57:1015-27. 15. Majekodunmi S, Odugbemi T. Clostridium we/chii corneal ulcer: a case report. Can J Ophthalmol 1975; 10:290-2.
16. Stern GA, Hodes BL, Stock EL. Clostridium perfringens corneal ulcer. Arch Ophthalmol 1979; 97:661-3. 17. Wilson LA. Bacterial corneal ulcers. In: Duane TD, ed. Clinical Ophthalmology. Hagerstown : Harper & Row, 1980; Vol. 4, Chapter 18. 18. Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol 1980; 90:38-47. 19. Matuura H. Anaerobes in the bacterial flora of the conjunctival sac . Jpn J Ophthalmol1971 ; 15:116- 24 . 20. Perkins RE, Kundsin RB, Pratt MV, et al. Bacteriology of normal and infected conjun ctiva. J Clin Microbiol1975; 1:147-9. 21. McNatt J, Allen SO, Wilson LA, Dowell VR Jr. Anaerobic flora of the normal human conjunctival sac. Arch Ophthalmol 1978; 96:1448-50. 22. Brook I, Pettit TH, Martin WJ, Finegold SM. Anaerobic and aerobic bacteriology of acute conjun ctivitis. Ann Ophthalmol 1979; 11 :389-93. 23. Brook I. Anaerobic and aerobic bacterial flora of acute conjunctivitis in children. Arch Ophthalmol 1980; 98:833-5. 24. Wren MWD. Prolonged primary incubation in the isolation of anaerobic bacteria from clinical specimens. J Med Microbiol 1980; 13:257-63 . 25. Nichols RL, Schumer W, Nyhus LM, et al. Anaerobic infections . Am Fam PhYSician 1976; 14(4):100-10. 26. Ernst EC, Berger S, Barza M, et al. Activity of cefamandole and other cephalosporins against aerobic and anaerobic bacteria. Antimicrob Agents Chemother 1976; 9:8 52-5. 27. Fiedelman W, Webb CD. Clinical evaluation of carbenici llin in the treatment of infection due to anaerobic bacteria. Curr Ther Res 1975; 18:441-51. 28. Rahman M. Sensitivities of clinically significant organisms to four cephalosporins, ampicillin and mecillinam. Curr Med Res Opin 1980; 7:9 6-100. 29. Levison ME, Santoro J, Bran JL, et al. In vitro activity and clinical efficacy of clindamycin in the treatment of infections due to anaerobic bacteria. J Infect Dis 1977; 135(suppl): 549-53. 30. Martin WJ , Gardner M, Washington JA II. In vitro antimicrobial susceptibility of anaerobic bacteria isolated from clinical spec imens . Antimicrob Agents Chemother 1972; 1:1 48-58.
Discussion by Thomas J. Liesegang, MD Ophthalmologists have lagged behind other specialists in establishing the role of anaerobes in clinical disease . Anaerobes are important and perhaps are the predominant pathogen in many serious superficial and deep abscesses of the body , although they are infrequently found in most acute infections. Until 1977,1 only scattered reports on anaerobes appeared in the ophthalmic literature. The report by Perry et al offers further stimulus for re-examination of our laboratory techniques to ensure recovery of responsible organisms in microbial keratitis . In most large series of patients with ulcerative keratitis, about 45% of the cases are classified as From Mayo Clinic, Rochester, Minnesota.
culture negative. Specific attention to anaerobic culturing techniques could help identify the pathogen in some of these cases. The clinical setting for anaerobic infection elsewhere in the body has been established.2 The tissue is usually severely compromised, Gram' s stain shows extensive variability in morphologic characteristics and coloration, and a synergism seems to exist between anaerobes and aerobes. 3 Within the ocular tissues , a similar polymicrobial nature of anaerobes is beginning to emerge. As in most corneal infections, however, predisposing factors are present frequently, and no distinguishing morphologic features are evident before laboratory testing.
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OPHTHALMOLOGY. JUNE 1982 • VOLUME 89 • NUMBER 6
Universally accepted criteria for establishing the presence of a true pathogen in microbial keratitis are still lacking. In this paper, very strict criteria were used with regard to isolation medium, results of Gram's stain, and associated cultures of the eyelid and conjunctiva. Previous reports of anaerobic isolation have usually been based on a single isolation medium, with or without confirmation by Gram's stain. Most microbiologists would agree that the authors have probably chosen the three ideal media to support primary anaerobic isolation. For simplicity, most ocular microbiology laboratories use only thioglycolate broth supplemented with hemin, vitamin K, and serum. The present study, as well as a study several years ago by Rosenblatt et al,4 revealed the shortcomings of using thioglycolate broth alone. These deficiencies are disturbing because they necessitate addition of other culture media to an already long list. The current study indicates that we cannot predict those corneal infections in which anaerobes should be suspected clinically. Using thioglycolate broth alone does not allow us to monitor contamination, quantitate the isolate, or prevent strict anaerobes from being overgrown by aerobes and facultative anaerobes. The use of a solid medium overcomes some of these problems but necessitates immediate placement in an anaerobic setting, probably best accessible in the area of inoculation. Basically, three devices are available for producing an anaerobic environment. The anaerobic glove box and prereduced anaerobic sterilized media are quite expensive and complex and require considerable space and training of technicians. Fortunately, the simple anaerobic jars, equipped with either a Gas-Pak or an evacuation system, have proved to be sensitive to most clinical pathogens (Rosenblatt JE, personal communication, 1981). This sensitivity may be due to the phenomenon of coincubation, wherein other anaerobes produce volatile growth-enhancing products in close proximity. Now that anaerobes are respected as pathogens, antibiotic susceptibility testing is recommended in serious infections because specific therapy occasionally is crucial. Previous rules about the predictability of antibiotic activity against anaerobes may no longer be applicable because of genetic mutation. Researchers have developed different antibiotics for management of these infections, and recommendations may change during the next several years on the basis of periodic surveys.
642
Yet to be developed, however, is a reliable antibiotic susceptibility testing system. The authors used an agar diskdiffusion technique, but this procedure is frequently faulty because of the variable growth rate of the anaerobic bacteria. More useful perhaps are the broth disk-elution technique of Kurzynski et aJ5 and the agar-dilution test of Sutter et al. 6 The handling of anaerobic cultures involves more than the simple addition of a different medium. Because of the expense, certain collection and transport rules must be enforced or most laboratories will simply refuse to process the cultures. Contamination with normal flora must be avoided. Any air in swabs, in transport, or during processing can interfere with the recovery rate. Reliance on a single liquid medium for isolation may be unreasonable, as these authors have pointed out. The laboratory should take special precautions with regard to the addition of certain supplements (eg, vitamin K, hemin, and serum) and should carefully monitor the setup of the anaerobic jar with all of its catalysts and indicators. The main thrust of this paper is accurate: unless the clinician and the microbiologist perform the additional steps necessary to detect these anaerobes, these pathogens will continue to be overlooked. REFERENCES 1. Jones DB, Robinson NM. Anaerobic ocular infections. Trans Am Acad Ophthalmol Otolaryngol 1977; 83:309-31. 2. Finegold SM. Anaerobic infections. Surg Clin North Am
1980; 60:49-64. 3. Dankert J, Holloway Y, Bouwma J. Bacterial synergy in mixed aerobic/anaerobic infections. Lancet 1980; 1(8170):714. 4. Rosenblatt JE, Fallon A, Finegold SM. Comparison of methods for isolation of anaerobic bacteria from clinical specimens. Appl Microbiol 1973; 25:77-85 5. Kurzynski TA, Yrios JW, Helstad AG, Field CR. Aerobically incubated thioglycolate broth disk method for antibiotic susceptibility testing of anaerobes. Antimicrob Agents Chemother
1976; 10:727-32. 6. Sutter VL, Barry AL, Wilkins TO, Zabransky RJ. Collaborative evaluation of a proposed reference dilution method of susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 1979; 16:495- 502.