P N E U M O C O C C A L I N F E C T I O N S O F T H E E Y E

MASAO O K U M O T O , M.A., AND GILBKRT S M O L I N , M.D.

San Francisco, California

Although the treatment of pneumococcal spectrum. Although pneumococci organisms infections has progressed substantially over the years, the pneumococcus remains a fre­quent and troublesome agent of ocular in­fectious disease. In this role it ranks second in importance to Staphylococcus aureus in the San Francisco area. Among the Proctor Foundation's laboratory records of 10,242 patients of all ages who were studied bac-teriologically between October 1966 and February 1973 (including many preopera-tive patients and many with clinically quiet eyes), we found 196 from whom pneumo­cocci were isolated on culture or identified in stained ocular scrapings or exudates.

In the ensuing report, we summarize vari­ous clinical data on these 196 cases and dis­cuss the best way to recover pneumococci from the eye. The antibiotic sensitivities of 116 pneumococcal isolates, and the typing of 20 of these isolates, are also reported.

The pneumococcus, currently designated Streptococcus pneumoniae,1 is seldom tested for antibiotic sensitivity, probably because pneumococci are generally regarded as high­ly susceptible to most antibiotics and sul-fonamides and as failing to develop anti­biotic resistance. In the last decade, however, occasional strains have shown significant re­sistance to penicillin,2'3 tetracycline,4 '5 and erythromycin.6 '7

For the treatment of known pneumococci infections, many ophthalmologists prescribe Neosporin ointment or drops routinely, per­haps because of the great popularity they enjoy by virtue of their broad antimicrobial

From the Francis I. Proctor Foundation for Re­search in Ophthalmology and the Department of Ophthalmology, University of California, San Fran­cisco. This study was supported in part by program grant EY-00310 from the National Institutes of Health.

Reprint requests to Masao Okumoto, 31S-S, Uni­versity of California, San Francisco, CA 94143.

are known to be resistant to polymyxin B, they are commonly thought to be sensitive to the other components of Neosporin (oint­ment contains neomycin and bacitracin; drops, neomycin and gramicidin). It is a re­grettable fact, however, that patients with pneumococcal infections respond poorly to Neosporin therapy. For this and several other reasons—the widespread use of Neo­sporin in ophthalmic practice, the natural variation in resistance among pneumococcal strains, and recent reports of increasing pneumococcal resistance to several antibi­otics—we undertook to test the antibiotic sensitivities of a substantial number of pneumococcal isolates from the human eye.

During the last 27 months of this study (December 1970 to February 1973), we col­lected 116 isolates of pneumococci from a variety of ocular sources. Dr. Franklin E. Cignetti made 63 of the isolations during a bacteriologic study of an Indian population surveyed and treated for trachoma in Ari­zona. These 63 cases were not included in the series of pneumococcus-infected patients subjected to clinical evaluation, however; we used them for the antibiotic sensitivity study only. By means of the disk agar-diffusion method, we tested all 116 strains against 13 antimicrobial agents, and by means of the tube-dilution method, we tested 20 strains, selected at random, against neomycin.

In 1943, von Sallmann8 reported that pneumococcal types 3, 7, and 10 were the types found most commonly on the external eye. The typing of pneumococci is rarely done now, however, even by state public health authorities. As a favor to us, Dr. C. H. Mordhorst of the State Serum Insti­tute in Copenhagen, Denmark, arranged for his colleague, Dr. Erna Lund, to type 20 of our isolates, selected at random.

346

VOL. 77, NO. 3 PNEUMOCOCCAL INFECTIONS OF THE EYE 347

MATERIALS AND METHODS

The antimicrobial disks (Difco Labora­tories) used to test sensitivity contained the following amounts of the following anti­microbial agents: bacitracin, 2 uni ts ; chlor-amphenicol, 5 \Lg; erythromycin, 2 txg; Frira-dantin, SO \i.g; Gantrisin, 50 [xg; gentamicin, 10 |xg; methicillin, 5 [Ag; neomycin, 5, 10, and 30 |xg; Novobiocin, 5 \xg; penicillin G, 2 units; polymyxin B, SO uni ts ; streptomy­cin, 2 [Jig; and tetracycline, 5 [Jig.

The following materials were also used: neomycin sulfate (Upjohn) ; optochin dif­ferentiation disks (Difco) ; blood agar plates: blood agar base ( B B L ) and 7^2% sheep blood; brain-heart infusion broth ( B B L ) ; and mannitol salt-agar plates (Difco) .

The pneumococci were isolated from cul­tures of ocular material collected in the Uni­versity of California San Francisco Eye Clinic, in the Proctor Foundation private practice unit, and in Arizona during a field survey of trachoma. For the routine collec­tion of material for study in the Proctor Foundation microbiology laboratory, we moisten a sterile cotton-tipped applicator in nutrient broth and swab it over the conjunc­tiva and eyelid margins without any previous anesthesia. In some instances material for culture is also taken from the cornea. The swabbed material is streaked immediately over a blood-agar plate and a mannitol salt-agar plate. (S. aureus or 6*. epidermidis are recovered from more than 9 0 % of the cul­tures studied in the Proctor Foundation laboratory, and the mannitol salt-agar plate differentiates them.)

In this study the patients in the Proctor Foundation private office unit received 0.5% proparacaine hydrochloride before the scrap­ings were taken, and the inoculated plates were transported at once to the Proctor lab­oratory located in the same building. The culture plates and slides of material col­lected in the Eye Clinic were placed initially in a 37°C incubator until they could be de­livered by messenger to the Proctor labora­

tory two blocks away. Cultures of the Ari­zona material were incubated for one or two days and transported to San Francisco by air, and from the airport to the Proctor laboratory as personal baggage. Cultures that had not been incubated for a full 48 hours were placed in the 37°C incubator immedi­ately on arrival and were incubated for at least 48 hours before the final reading. W e identified the pneumococci on the basis of colony morphology, alpha hemolysis, a 6-mm or greater inhibition of growth to an opto­chin disk, and the microscopic appearance of the diplococci on Gram- or Giemsa-stained slides.

W e subcultured all isolates on blood agar and lyophilized each isolate, using a Virtis lyophilizer and sterile skim milk as the sus­pending agent. W e then performed the anti­biotic sensitivity tests and the optochin tests on the fresh isolates after the first or second transfer. The concentration of the 13 anti­microbial agents in the disks was the lowest concentration commercially available except in the neomycin disks. Neomycin was used in three concentrations—low, intermediate, and high. The two extra disks of neomycin made a total of 15 antimicrobial disks for each isolate.

In determining the disk antibiotic sensi­tivities, we suspended a few colonies of the pneumococci from a 24-hour subculture in 0.5 ml of nutrient broth, using a Vortex mixer. W e then dipped a sterile cotton-tipped applicator in this suspension and swabbed it over a blood-agar plate, streaking the en­tire surface of the plate back and forth in one direction, and then at right angles to the first direction. Two plates were inocu­lated with each isolate. W e placed the 15 antimicrobial disks on the plates (seven on one plate and eight on the other) , using a Difco multiple disk dispenser and pressing the disks firmly on the agar to ensure good contact with the medium. W e then placed the plates in a 37°C incubator and read the zones of inhibition at 24 hours (or occasionally, if the growth was slight, at 48 hours ) , mea-

348 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH, 1974

suring and recording the diameters of the zones.

Using Lorian's method,9 we made tube-dilution antibiotic sensitivity determinations for neomycin on 20 randomly selected strains. One gram of neomycin sulfate was reconstituted with sterile saline and diluted to provide 200 [J.g/ml. For each isolate, five tubes of neomycin sulfate solution contain­ing 2 ml in twofold decreasing concentrations were prepared, starting with 200 pig/ml, so that on addition of the pneumococcal sus­pension the final concentrations were thus 100, 50, 25, 12.5, and 6.25 ^.g/ml. A sixth tube with 2 ml of saline served as the con­trol.

For each isolate to be tested we prepared an 18-hour culture of the pneumococcus grown in brain-heart infusion broth at 37°C, diluted each culture 1:1,000 with the broth, and pipetted 2 ml of the diluted suspension into each of the six tubes. All tubes were incubated at 37°C and the results read the following day. The lowest concentration that inhibited growth was designated as the "min­imum inhibitory concentration" (M.I.C.). The suspensions in the clear tubes were sub-cultured on blood agar, and the lowest con­centrate showing no growth on subculture was designated as the "minimum bactericidal concentration" (M.B.C.).

TABLE 1

CLINICAL CONDITIONS FROM WHICH PNEUMOCOCCI WERE RECOVERED ( 1 9 6 CASES)

Clinical Conditions No. of Cases % of Cases

Conjunctivitis Infected tear sac Corneal ulcer Irritated eyes Preoperative eyes Trachoma Miscellaneous keratitis Endophthalmitis Keratitis sicca Erythema multiforme Blepharitis Miscellaneous

89 39 14 12 11 9 5 3 3 2 2 7

45.5 19.9 7.1 6.1 5.6 4.6 2.5 1.5 1.5 1.0 1.0 3.5

RESULTS

Clinical data—The 196 patients from whom pneumococci were recovered in the Proctor microbiology laboratory between October 1966 and February 1973 were pa­tients with a wide variety of clinical condi­tions (Table 1). As one would expect, a large percentage (almost half) were cases of conjunctivitis. Infected tear sacs were the second most common disorder (20%) and corneal ulcers the third (7%) . Information on the patient's age and whether one or both eyes were affected was given in 161 of the 196 histories and was omitted from the re­maining 35. The age distribution showed a preponderance of cases occurring in the very young, 43% of the 161 patients (from among 10,242 patients of all ages) being less than 10 years old (Table 2) ; 96 of the 161 cases were unilateral, 65 bilateral.

Laboratory identification—The scrapings for culture and direct examination were taken primarily from the eyelid margins and conjunctivas. In 95 cases the pneumococcus was isolated on culture or identified by di­rect examination in scrapings from both the eyelid margins and the conjunctivas. We found the organism in material from the con­junctiva alone in 45 cases, in material from the eyelid margins alone in 25 cases, and in material from the cornea in 21 cases. In the corneal cases we also recovered the organism from other areas of the eye. In one case of bilateral endophthalmitis the organism was recovered in cultures of aqueous humor from one eye and was seen by direct examination of stained slides of aqueous humor from both eyes (Table 3).

Our laboratory findings indicate the im­portance of using both cultures and the di­rect examination of scrapings for identifica­tion purposes. In 56 cases the cultures were positive for pneumococci when the scrapings were negative, and in eight cases the scrap­ings were positive and the cultures negative. In 81 cases the cultures were positive but scrapings were not done, and in seven cases

VOL. 77, NO. 3 P N E U M O C O C C A L I N F E C T I O N S O F T H E E Y E 349

the scrapings were positive but cultures were not done. In the corneal ulcer cases, pneumo-cocci were identified in 12 cases by direct examination of stained scrapings only, and in nine cases by cultures only (Table 4) .

Antibiotic sensitivity pattern—The anti­biotic disk sensitivity tests showed that pneumococci isolated from the eye were sus­ceptible to a variety of antimicrobial agents. In Table 5 the average sizes, and ranges of size, of the zones of inhibition are listed in the order of decreasing zone size. Although zone size is influenced by several factors other than the effect of an agent on the or­ganism in question, it still serves as a useful guide.

The most effective drugs were penicillin G, erythromycin, and Furadantin. Although methicillin, tetracycline, and bacitracin showed moderately sized zones of inhibition, a few pneumococcal strains were resistant to tetracycline. The average zone sizes of chlor-amphenicol, Novobiocin, and Gantrisin indi­cated that they were also useful drugs, but the remaining antibiotics—gentamicin, strep­tomycin, polymyxin B, and neomycin—ap­peared to be poor drugs for use against pneumococcal infections.

There were only small differences between the reactions of the Arizona isolates and the San Francisco isolates. In general, the San Francisco isolates showed smaller average zone sizes, and this may have reflected the

TABLE 2

AGE DISTRIBUTION OF 161 PATIENTS FROM WHOM PNEUMOCOCCI WERE RECOVERED

Age, yr

0- 9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99

No. of Patients

70 20 21

7 13 9 9 7 4 1

% of Pa

43 12 13 4 8 6 6 4 2 1

TABLE 3

RECOVERY OF PNEUMOCOCCI IN CULTURES OR IDENTIFIED IN SCRAPINGS BY

DIRECT EXAMINATION

Cultures Scrapings Positive Positive

Conjunctiva positive (eyelid negative)

Eyelid margin positive (con­junctiva negative)

Conjunctiva and eyelid margin positive

Cornea positive Aqueous humor positive Not specified

36

21

89 9 1

23

9

4

5 12 2

27

TABLE 4

POSITIVE CULTURES VS. POSITIVE IDENTIFICATIONS BY DIRECT SLIDE EXAMINATION

Cultures and Slides No. of Cases

Culture and slide positive 44 Culture positive; slide negative 56 Slide positive; culture negative 8 Culture positive; slide not done 81 Slide positive; culture not done 7

Total 196

patients' greater exposure to antibiotic ther­apy in this area. The single exception was the reaction to tetracycline, which was slight­ly greater in the San Francisco group than in the Arizona group. This in turn may have reflected the tetracycline exposure of some of the Arizona patients who had participated in a doxycycline treatment program for tra­choma.

In the tube-dilution tests of pneumococcal sensitivity to neomycin, the 20 randomly se­lected isolates showed an overall neomycin resistance (Table 6).

Pneumococcal typing—The results of the typing of the 20 randomly selected strains that was performed at the State Serum In­stitute in Copenhagen, Denmark, by Dr. Erna Lund are tabulated in Table 7. The Danish10 and American systems of nomen-

350 A M E R I C A N J O U R N A L O F O P H T H A L M O L O G Y MARCH, 1974

TABLE 5

ANTIBIOTIC DISK SENSITIVITIES OF 116 PNEUMOCOCCAL ISOLATES: AVERAGE DIAMETER OF ZONES OF INHIBITION AND RANGE OF ZONE SIZES

Isolates from Arizona (63 cases) Isolates from San Francisco (53 cases)

Agent

Penicillin G Erythromycin Furadantin Methicillin Tetracycline Bacitracin Chloramphenicol Novobiocin Gantrisin Gentamicin Neomycin, 30 MK Streptomycin Polymyxin B Neomycin, 10 jig Neomycin, S jig

Average Zone Size, mm

40 28 27 27 21 19 19 18 13 11

7 6* 6 6 6

Range of Zone Sizes, mm

26-46 10-46 18-36 16-36 6-30 8-30

12-26 12-32 6-30 6-22 6-26 6-12 6- 8 6-10 6- 6

Average Zone Size, mm

36 27 26 25 23 18 19 16 14 12

7 6 6 6 6

Range of Zone Sizes, mm

20-48 16-42 16-42

8-40 6-34 7-34

12-34 12-24 6-26 6-20 6-12 6-10 6-10 6 - 6 6- 6

* Since the diameter of the disks is 6 mm, a measurement of 6 mm was interpreted as "no inhibition."

clature are given for each strain. Seven of the isolates were "rough" strains—i.e., strains that were noncapsulated, avirulent, and could not be typed.

DISCUSSION

Pneumococci are regarded as delicate, fastidious organisms that are highly suscep­tible to many chemotherapeutic agents. It has been said, moreover, that they never de­velop resistance. In our series, all strains were highly sensitive to penicillin G and erythromycin, and occasional strains were resistant to tetracycline. It is to these three antibiotics that pneumococcal resistance has recently been reported elsewhere.2"7

The sizes of the zones of inhibition rep­resented the sensitivity of the organism to the lowest commercially available concentra­tions of the various antibiotics tested, diam­eters exceeding 20 mm probably represent­ing significant sensitivity. Any inhibition in­duced by the lowest concentration of a drug probably indicates some degree of sensitiv­ity ; but if the drug happens to diffuse poorly

TABLE 6

NEOMYCIN SENSITIVITY OF 20 PNEUMOCOCCAL ISOLATES : TUBE-DILUTION TEST

Isolate

No.

19 21 22 40 46 501

50J

54 58 62 64 65 67 70 71 75 79 84 85

107

Average

Source

SF SF SF Ariz. SF SF SF Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. SF SF SF

Minimum Inhibitory

Concentration, j i g /ml

100 25 25 50 25

100 50 25 25

100 12.5 12.5 6.25

12.5 25 12.5 12.5

6.25 25 25

33.75

Minimum Bactericidal

Concentration, jug/ml

100 25 50 50 25

100 100 50 25

100 25 12.5 6.25

12.5 50 12.5 25

6.25 50 25

42.5

VOL. 77, NO. 3 PNEUMOCOCCAL INFECTIONS OF THE EYE 351

through agar, the organism's sensitivity may be masked. Since we were particularly in­terested in the performance of neomycin, we also tested the intermediate and high concen­trations of this one antibiotic.

The results of testing these three concen­trations by the disk agar-diffusion test, and the results of the quantitative tube-dilution test, were consistently negative, only one pneumococcal strain responding to the high­est disk concentration of 30 jxg, and all strains requiring an average M.I.C. of 33 lAg/ml in the tube-dilution test. Bacitracin is a moderate inhibitor of pneumococci, but polymyxin B and neomycin are almost uni­formly noninhibitory. Although Neosporin ointment, which consists of these three anti­biotics, has a broad antimicrobial spectrum and is certainly a valuable antibiotic for a number of ocular infections, it is clearly a poor choice for pneumococcal infec­tions. We did not test gramicidin, which is the third antibacterial agent in Neosporin

TABLE 7

TYPING OF 20 PNEUMOCOCCAL ISOLATES

I

No.

29 30 33 35 36 37 40 41 42 43 44 45 46 47 48 49 50 51 76

107

solate

Source

SF SF Ariz. Ariz. Ariz. Ariz. Ariz. Ariz. SF SF SF SF SF SF SF SF SF SF Ariz. SF

Type of Pnei:

Danish Nomenclature

28 F 11 A 35 A 33 F

R* 35 F 14

R* 21

R* R* R*

6 B R*

13 19 F 14 35 F

R* 6 B

imococcus

U.S. Nomenclature

28 11 35 70

35 14

21

26

13 19 14 35

26

* R indicates rough, nontypable.

drops, but assumed that its inhibiting effect would be similar to that of bacitracin for which it is a substitute.

The fact that pneumococci in corneal ul­cers could be identified by direct examina­tion of stained slides more often than they could be recovered in cultures might have been due to one or both of the following fac­tors: (1) The presence of antimicrobial drugs might have inhibited the cultural growth of the pneumococci in spite of the fact that nonviable pneumococci still appeared as poorly stained diplococci in smears. (2) The supply of carbon dioxide might have been inadequate to ensure growth in culture; Austrian and Collins11 made the interesting observation that about 8% of pneumococcal strains fail to grow unless 5 to 10% carbon dioxide is present. This is particularly interesting in view of the inter­fering effect of carbon dioxide that Rags-dale and San ford12 noted when they were do­ing the optochin test.

Ocular infections did not seem to be caused by any particular pneumococcal types. On the contrary, a highly diverse scattering of types was found, as well as a number of rough strains. According to a personal com­munication from Dr. Lund (Copenhagen), these rough strains occur under natural con­ditions and do not represent a mutation in­duced by prolonged passage on artificial media. In 25 eye specimens, Lund13 found five pneumococcal types, all of which she also found in our series. But in contrast to von Sallman's report in the early 1940s that types 3, 7, and 10 were found characteristi­cally on the external eye, in our series no type or types could be considered typical of ocular isolates. It was interesting that rough, avirulent strains were recovered from the ex­ternal eye and that they were found among both the San Francisco and the Arizona isolates.

SUMMARY

Although pneumococci have recently been reported to be resistant to penicillin, eryth-

352 AMERICAN JOURNAL OF OPHTHALMOLOGY MARCH, 1974

romycin, and tetracycline, in this study ocu­lar isolates were highly sensitive to penicil­lin and erythromycin and only slightly re­sistant to tetracycline.

Pneumococci were resistant to neomycin and polymyxin B, i.e., two of the three constituents of Neosporin, which must be considered a poor choice for the control of pneumococcal infections.

Rough, avirulent strains of pneumococci were recovered from the eye. There were indications that carbon dioxide-dependent strains of pneumococci might occur in the eye. Pneumococci are apparently much more likely to be recovered from ocular scrapings by culture than by direct microscopic ex­amination ; but the use of both methods yields the best results.

ACKNOWLEDGMENT

We thank Frank Cignetti, M.D., University of Pittsburgh, for contributing many of the strains of pneumococci used in this study, and Drs. Erna Lund and Carl H. Mordhorst of the State Serum Institute, Copenhagen, Denmark, for typing of 20 of our strains.

REFERENCES

1. International Committee on Nomenclature of Bacteria: Subcommittee on Streptococci and Pneu­

mococci. Minutes of the Meeting (Moscow), July 21, 1966. Int. J. Systematic Bact. 17:281, 1967.

2. Hansman, D., Stuart, J., Devitt, L., and Doug­las, R.: Increased resistance to penicillin of pneu­mococci isolated from man. N. Engl. J. Med. 284:17S, 1971.

3. Gunnison, B., Fraher, M. A., Pelcher, E., and Jawetz, E.: Penicillin-resistant variants of pneu­mococci. Appl. Microbiol. 16:311, 1968.

4. Schaedler, R. W., Choppin, P. W., and Zabriskie, J. B.: Pneumonia caused by tetracycline-resistant pneumococci. N. Engl. J. Med. 270:127, 1964.

5. Turner, G. C.: Tetracycline-resistant pneumo­cocci in a general hospital. Lancet 2:1292, 1963.

6. Kislak, J. W.: Type 6 pneumococcus resistant to erythromycin and lincomycin. N. Engl. J. Med. 276:852, 1967.

7. Dixon, J. M. S.: Pneumococcus resistant to erythromycin and lincomycin. Lancet 1 :S73, 1967.

8. Von Sallman, L.: Penicillin and sulfadiazine in the treatment of experimental intraocular infection with pneumococcus. Arch. Ophthalmol. 30 :426, 1943.

9. Lorian, V.: Antibiotics and Chemotherapeutic Agents in Clinical and Laboratory Practice. Spring­field, Charles C Thomas, 1966, p. 80.

10. Lund, E.: Laboratory diagnosis of pneumo­coccus infections. Bull. WHO 23 :5, 1960.

11. Austrian, R., and Collins, P.: Importance of carbon dioxide in the isolation of pneumococci. J. Bacteriol. 92:1281, 1966.

12. Ragsdale, R. A., and Sanford, J. P.: Inter­fering effect of incubation in carbon dioxide on the identification of pneumococci by optochin discs. Appl. Microbiol. 22:854, 1971.

13. Lund, E.: Distribution of pneumococcus types at different times in different areas. Bayer Sym­posium III. Berlin, Springer-Verlag, 1971, p. 49.

O P H T H A L M I C MINIATURE

In the process of reconstructing this event in his mind, Franz grimaced and groaned. All the festive lights of Friedrichstrasse had been stamped out by his boot. H e would have to take the glasses to be repaired: only one lens was still in place and that was cracked. H e palpated rather than re-examined the cripple.

Vladimir Nabokov King, Queen, Knave

McGraw-Hill, 1968