- Email: [email protected]
Lincomycin Levels in Human Ocular Fluids and Serum Following Subconjunctival Injection
LINCOMYCIN L E V E L S IN H U M A N O C U L A R FLUIDS A N D SERUM F O L L O W I N G S U B C O N J U N C T I V A L INJECTION GERARD
L.
BOYLE,
F . I . M . L . T . , MICHAEL L .
LICHTIG,
M.D,
A N D
H. LEOPOLD, M . D .
IRVING
New York, New York
Lincomycin HCl monohydrate (Lincocin) is derived from the actinomycete, Streptomyces lincolnensis var. lincolnensis. Its mechanism of antibacterial activity is by virtue of its inhibition of bacterial protein synthesis. In vitro, the antibiotic is active primarily against Gram-positive organisms with the exception of enterococci. McPherson and associates have shown that bacteriostatic levels of certain antibiotics appear in the aqueous humor as rapidly as 30 seconds following subconjunctival injection. Becker reported that significant levels of lincomycin are achieved in human serum and in aqueous humor afer intramuscular injection to patients with noninflamed eyes. Since there are certain potential advantages to the use of lincomycin in certain patients (penicillin sensitivity), the purpose of this study was to evaluate ocular penetration of lincomycin in humans following subconjunctival injection. 1
2
3
4
injections of 0.25 ml of lincomycin (300 mg/ ml) at intervals varying between two minutes to 17 hours and 40 minutes before surgery. The injections were given either before or after retrobulbar and eyelid blocks, but in no case was subconjunctival anesthetic administered. Prior to corneoscleral section, paracentesis of the anterior chamber was performed under sterile conditions using a 25-gauge needle on a disposable 2.5 ml syringe. Approximately 0.1 to 0.2 ml of aqueous humor was thus obtained for determination of aqueous humor lincomycin levels. Simultaneously, 10 ml of venous blood was drawn for serum assay of lincomycin. Following delivery of the lens, lens tissue from 20 of the patients studied was also obtained. Samples of aqueous humor, serum, and lens tissue were stored at —20° C until lincomycin assays were performed. 5
Levels of lincomycin were determined by the tube dilution technique, with a total volume of 1 ml in each tube. The lincomycin standard containing 1 ¡xg/ml in broth was prepared just prior to testing from a standard solution (1000 |xg/ml) dissolved in distilled water. Two parallel series of tubes were set up. One contained appropriate volumes of the standard (1 ¡xg/ml) lincomycin and the other appropriate volumes of the specimen to be assayed either undiluted or diluted. (The dilution factor depended upon the concentration of antibiotic in the test sample.) Diluent, and test organism (staphylococcus aureus six-hour culture diluted 10~ , giving a Lumetron optical density reading of 0.025 with a 580 mu, filter) was added to each tube so as to attain a volume of 1 ml for each tube. Control tubes were also prepared to verify sterility of the samples and the diluent, and viability of the test organism. The tubes were incubated at 37° C for 18 hours, and the end point of complete inhibition for each 6
MATERIALS AND METHODS
The 27 subjects used in the study were patients admitted to the hospital for elective ocular surgery, usually cataract extraction. One patient had bilateral operations and was studied twice. None of the subjects had ocular inflammatory disease. The subjects ranged in age from 25 to 85 years ; 20 were older than 60 years. Six of the 27 patients in the study were diabetic and five reported allergies to penicillin, aspirin, or barbiturates. The 27 patients scheduled for elective cataract extraction were given subconjunctival From the Department of Ophthalmology, Mount Sinai School of Medicine of the City University of New York. This study was supported in part by a N.I.H. Pharmacology grant and a grant from the Upjohn Company. Reprint requests to Irving H. Leopold, M . D , 11 East 100th Street and Fifth Avenue, New York, New York 10029.
1303
3
AMERICAN JOURNAL OF OPHTHALMOLOGY
1304
series, as indicated by the first tube showing no visible growth, was recorded. The Staph, aureus test organism was found to be consistently inhibited by 0.3 ¡/.g/ml of the lincomycin standard solution. Each lens tissue sample was weighed and then homogenized in 1 ml of sterile broth and further diluted on the basis of weight of the sample to give 100 mg/ml of tissue. After centrifugation of the homogenate, the supernatent was assayed undiluted. Concentrations of antibiotic in serum, lens tissue, and aqueous humor were calculated by dividing the minimum inhibitory concentration ( M I C ) of the standard lincomycin by the volume of the sample required to inhibit growth of the standard inoculum and multiplied by the sample dilution used. Results are expressed as ng/ml. Con-
JUNE, 1971
centration of antibiotic per ml in sample: MIC/ml antibiotic standard Sample volume X Dilution of sample RESULTS
The data reveal that within a relatively short time after subconjunctival injection, lincomycin reaches significant concentrations in aqueous humor (Fig. 1 ) . Levels of lincomycin were observed as early as two minutes following subconjunctival injection in the uninflamed eye. These levels persisted for up to four and one-half hours with high peaks at one to two hours. In each case, antibiotic concentration in 100 mg of lens tissue was less than 0.3 [Ag/ml (0.3 ¡xg/ml was the
•(135)
so ^ 40 g
• -
Aqueous
° -
Serum
35
30
Iu
25
•5
20
g
S o o
15 10
8 • Op>-i—r
T—i—i—i—i—r
—r
i—i—i
102030405060 2 3 4 5 6 7 —Minutes
1—
o oo i — i — i1 —- i — i — i — i — r ° ^ - 0 |
8 9 10
II
12 13 14 15 16 17 18
Hours After Subconjunctival
Injection
I
Fig. 1 (Boyle, Lichtig, and Leopold), Lincomycin levels in serum and aqueous humor following subconjunctival injection.
VOL. 71, NO. 6
SUBCONJUNCTIVAL LINCOMYCIN
minimal assayable concentration with the method and standard organism employed). The serum showed a relatively significant level 10 minutes after the subconjunctival injection and this level was still detectable up to 12 hours later. It remained relatively constant during that period except for a peak level at one and one-half hours. The data also indicates that maximal levels of lincomycin are obtained in the aqueous humor and serum within two hours after subconjunctival injection, and relatively significant levels are still present at 12 hours. Adverse reactions seemed limited to a rather marked C h e m o s i s which appeared within a few minutes of subconjunctival injection in all patients studied. In most of these patients, subconjunctival hemorrhages also appeared, which were apparently unrelated to the mechanical trauma of the injection. The Chemosis was noted to subside considerably after several hours' time, in those patients so studied (i.e., those with the longer intervals between subconjunctival injection and anterior chamber paracentesis). Patients complained of little or no pain when the antibiotic was administered, even when anesthesia was limited to topical tetracaine solution. No adverse systemic effects were noted in any of the subjects as revealed through 12channel blood analysis, blood counts, urinalysis, and complete physical examinations. All patients have been followed for a minimum of three months postoperatively. DISCUSSION
The necessary treatment of intraocular infections due to Gram-positive organisms by subconjunctival injection of chemotherapeutic agents depends upon the susceptibility of the pathogen to the antibiotic, the reaction of the patient, permeability associated with inflammation, and the maintenance of effective therapeutic levels. Langham reported that the maximum concentration of an antibiotic in the aqueous humor of a normal eye for a given concentration in the blood stream is dependent not only on the rate of entry but also on its de-
1305
gree of absorption on the proteins of the plasma. Leopold has shown from clinical data that intraocular infections, once established, are difficult to control and that cultures of the intraocular fluids in these cases are often sterile and therefore are not often helpful in detecting the cause of the infection or in selecting the antibiotic. He concluded that once the infection has developed, subconjunctival injections of penicillin (1,000,000 units), along with streptomycin (50,000 units), will not control intraocular infection in all cases, nor will systemic or intravitreal administration of chloramphenicol or systemic, intravitreal, or subconjunctival administration of terramycin. 8
In treatment of intraocular infections, an antibiotic may be used prior to surgical procedures, whether it is to be extraction of a foreign body or a cataract, or repair of a ruptured globe. In this way one can establish a concentration of the antibiotic in the blood stream and in the ocular fluids prior to opening the eye. In vitro studies (summarized in Table 1) reported by McCall, Steigbigel, and Finland, McGehee, Smith, Wilcox, and Finland, and, Barber and Waterworth indicate that low concentrations of lincomycin inhibit several common pathogens. Although a direct correlation cannot be assumed between in vitro and in vivo susceptibility of bacteria, it seems likely that in àn inflamed eye, aqueous humor levels of lincomycin can be attained which may be active against susceptible Gram-positive organisms. 9
10
11
Following subconjunctival or intramuscular injection, lincomycin has been demonstrated to produce adequate aqueous humor levels in man in the noninflamed eye similar to that shown for other leading antibiotics, and it could prove useful in ocular infections due to Gram-positive pathogens susceptible to lincomycin.
7
SUMMARY
After 0.25 ml of a solution containing 300 mg/ml of lincomycin was administered subconjunctivally to each of 27 hospitalized pa-
1306
AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1971
TABLE 1 SUSCEPTIBILITY OF VARIOUS PATHOGENS TO LINCOMYCIN
Minimum Inhibition Concentration Pathogen
McCall and Associates'
(jig/ml)
McGehee and Associates
10
Barber and Waterworth Range
11
Staph, aureus Strep. (Group A ) D. pneumoniae Strep, viridans Enterococcus H. Influenzae N. gonorrhoeae X. meningitidis
Range
Median
Range
Median
0..78 - >100 0.,04 0. 19 0.,0050. 39 0 .02 0.,78 25.,00 - >100 6..25 - 100 6..25 25,,0
0,.78 0,.04 0 .04 0 .09 50,.0 12.,5 12 .5
0,.8 3. 1 0,.040. 2 0 .020. 2 0 .020. 4 25,.0 - >100 6,,3050 0 .0225 100 >100
1,.60 0. 04 0.,20 0,.04 >100 25.,0 12 .5 100
tients, aqueous humor, serum, and lens tissue samples were obtained at varying postinjection intervals. Significant concentrations of lincomycin were demonstrated
in aqueous
humor samples as early as two minutes after injection. Peak levels of the antibiotic in aqueous humor occur between one and three hours after injection, followed by a gradual decline. Minimal levels of the antibiotic persisted in the serum and aqueous humor for 12 hours. All the lens tissue samples showed a concentration of less than 0.3 u.g/ml. ACKNOWLEDGMENT
We thank Dr. S. S. Schneierson, Director of Microbiology, for his helpful comments and criticism. We also thank Dr. J. D. Panzer, of the Upjohn Company, for his advice. We are also indebted to Drs. J. Bloomfield, S. Sherman, S. Wunsh, and E. Siegel, who helped to obtain some of the specimens, and to the Upjohn Company for supplying the lincomycin (Lincocin) used in this study. REFERENCES
1. Mason, D. J, Dietz, A., and DeBoer, G.: Lincomycin, a new antibiotic. I. Discovery and biological properties. Antimicrob. Chemotherap. 554, 1962.
-
0 .5 - 2..00 0..06- 0,.12 0 .06- 0 .50 4,.00-16 .0 4,.00-16 .0 32 .0 >32 .0
2. Josten, J. J, and Allen, P. M. : The mode of action of lincomycin. Biochem. Biophys. Res. Comm. 14:241, 1964. 3. McPherson, S. D , Presley, G. D , and Crawford, J. R. : Aqueous humor assays of subconjunctival antibiotics. Am. J. Ophth. 66:430, 1968. 4. Becker, E. F. : The intraocular penetration of lincomycin. Am. J. Ophth. 67:963, 1969. 5. Riley, F. C , Boyle, G. L , and Leopold, I. H. : Intraocular penetration of cephaloridine in humans. Am. J. Ophth. 66:1042, 1968. 6. Schneierson, S. S, and Torharsky, B. : A method for the determination of aureomycin in the blood. J. Bacteriol. 57:483, 1949. 7. Langham, M. : Factors affecting the penetration of antibiotics into aqueous humor. Brit. J. Ophth. 35:614, 1951. 8. Leopold, I. H. : Surgery of ocular trauma : Therapy of secondary intraocular infection. Arch. Ophth. 48:738, 1952. 9. McCall, C. E , Steigbigel, N , and Finland, M. : Lincomycin: activity in vitro and absorption and excretion in normal young men. Am. J. Med. Sei. 254:144, 1967. 10. McGehee, R. F , Smith, C. B , Wilcox, C , and Finland, M. : Comparative studies of antibacterial activity in vitro and absorption and excretion of lincomycin and clinimycin. Am. J. Med. Sei. 265 : 279, 1968. 11. Barber, M , and Waterworth, P. M. : Antibacterial activity of lincomycin and pristamycin : a comparison with erythromycin. Brit. Med. J. 2:603, 1964.
L.
BOYLE,
F . I . M . L . T . , MICHAEL L .
LICHTIG,
M.D,
A N D
H. LEOPOLD, M . D .
IRVING
New York, New York
Lincomycin HCl monohydrate (Lincocin) is derived from the actinomycete, Streptomyces lincolnensis var. lincolnensis. Its mechanism of antibacterial activity is by virtue of its inhibition of bacterial protein synthesis. In vitro, the antibiotic is active primarily against Gram-positive organisms with the exception of enterococci. McPherson and associates have shown that bacteriostatic levels of certain antibiotics appear in the aqueous humor as rapidly as 30 seconds following subconjunctival injection. Becker reported that significant levels of lincomycin are achieved in human serum and in aqueous humor afer intramuscular injection to patients with noninflamed eyes. Since there are certain potential advantages to the use of lincomycin in certain patients (penicillin sensitivity), the purpose of this study was to evaluate ocular penetration of lincomycin in humans following subconjunctival injection. 1
2
3
4
injections of 0.25 ml of lincomycin (300 mg/ ml) at intervals varying between two minutes to 17 hours and 40 minutes before surgery. The injections were given either before or after retrobulbar and eyelid blocks, but in no case was subconjunctival anesthetic administered. Prior to corneoscleral section, paracentesis of the anterior chamber was performed under sterile conditions using a 25-gauge needle on a disposable 2.5 ml syringe. Approximately 0.1 to 0.2 ml of aqueous humor was thus obtained for determination of aqueous humor lincomycin levels. Simultaneously, 10 ml of venous blood was drawn for serum assay of lincomycin. Following delivery of the lens, lens tissue from 20 of the patients studied was also obtained. Samples of aqueous humor, serum, and lens tissue were stored at —20° C until lincomycin assays were performed. 5
Levels of lincomycin were determined by the tube dilution technique, with a total volume of 1 ml in each tube. The lincomycin standard containing 1 ¡xg/ml in broth was prepared just prior to testing from a standard solution (1000 |xg/ml) dissolved in distilled water. Two parallel series of tubes were set up. One contained appropriate volumes of the standard (1 ¡xg/ml) lincomycin and the other appropriate volumes of the specimen to be assayed either undiluted or diluted. (The dilution factor depended upon the concentration of antibiotic in the test sample.) Diluent, and test organism (staphylococcus aureus six-hour culture diluted 10~ , giving a Lumetron optical density reading of 0.025 with a 580 mu, filter) was added to each tube so as to attain a volume of 1 ml for each tube. Control tubes were also prepared to verify sterility of the samples and the diluent, and viability of the test organism. The tubes were incubated at 37° C for 18 hours, and the end point of complete inhibition for each 6
MATERIALS AND METHODS
The 27 subjects used in the study were patients admitted to the hospital for elective ocular surgery, usually cataract extraction. One patient had bilateral operations and was studied twice. None of the subjects had ocular inflammatory disease. The subjects ranged in age from 25 to 85 years ; 20 were older than 60 years. Six of the 27 patients in the study were diabetic and five reported allergies to penicillin, aspirin, or barbiturates. The 27 patients scheduled for elective cataract extraction were given subconjunctival From the Department of Ophthalmology, Mount Sinai School of Medicine of the City University of New York. This study was supported in part by a N.I.H. Pharmacology grant and a grant from the Upjohn Company. Reprint requests to Irving H. Leopold, M . D , 11 East 100th Street and Fifth Avenue, New York, New York 10029.
1303
3
AMERICAN JOURNAL OF OPHTHALMOLOGY
1304
series, as indicated by the first tube showing no visible growth, was recorded. The Staph, aureus test organism was found to be consistently inhibited by 0.3 ¡/.g/ml of the lincomycin standard solution. Each lens tissue sample was weighed and then homogenized in 1 ml of sterile broth and further diluted on the basis of weight of the sample to give 100 mg/ml of tissue. After centrifugation of the homogenate, the supernatent was assayed undiluted. Concentrations of antibiotic in serum, lens tissue, and aqueous humor were calculated by dividing the minimum inhibitory concentration ( M I C ) of the standard lincomycin by the volume of the sample required to inhibit growth of the standard inoculum and multiplied by the sample dilution used. Results are expressed as ng/ml. Con-
JUNE, 1971
centration of antibiotic per ml in sample: MIC/ml antibiotic standard Sample volume X Dilution of sample RESULTS
The data reveal that within a relatively short time after subconjunctival injection, lincomycin reaches significant concentrations in aqueous humor (Fig. 1 ) . Levels of lincomycin were observed as early as two minutes following subconjunctival injection in the uninflamed eye. These levels persisted for up to four and one-half hours with high peaks at one to two hours. In each case, antibiotic concentration in 100 mg of lens tissue was less than 0.3 [Ag/ml (0.3 ¡xg/ml was the
•(135)
so ^ 40 g
• -
Aqueous
° -
Serum
35
30
Iu
25
•5
20
g
S o o
15 10
8 • Op>-i—r
T—i—i—i—i—r
—r
i—i—i
102030405060 2 3 4 5 6 7 —Minutes
1—
o oo i — i — i1 —- i — i — i — i — r ° ^ - 0 |
8 9 10
II
12 13 14 15 16 17 18
Hours After Subconjunctival
Injection
I
Fig. 1 (Boyle, Lichtig, and Leopold), Lincomycin levels in serum and aqueous humor following subconjunctival injection.
VOL. 71, NO. 6
SUBCONJUNCTIVAL LINCOMYCIN
minimal assayable concentration with the method and standard organism employed). The serum showed a relatively significant level 10 minutes after the subconjunctival injection and this level was still detectable up to 12 hours later. It remained relatively constant during that period except for a peak level at one and one-half hours. The data also indicates that maximal levels of lincomycin are obtained in the aqueous humor and serum within two hours after subconjunctival injection, and relatively significant levels are still present at 12 hours. Adverse reactions seemed limited to a rather marked C h e m o s i s which appeared within a few minutes of subconjunctival injection in all patients studied. In most of these patients, subconjunctival hemorrhages also appeared, which were apparently unrelated to the mechanical trauma of the injection. The Chemosis was noted to subside considerably after several hours' time, in those patients so studied (i.e., those with the longer intervals between subconjunctival injection and anterior chamber paracentesis). Patients complained of little or no pain when the antibiotic was administered, even when anesthesia was limited to topical tetracaine solution. No adverse systemic effects were noted in any of the subjects as revealed through 12channel blood analysis, blood counts, urinalysis, and complete physical examinations. All patients have been followed for a minimum of three months postoperatively. DISCUSSION
The necessary treatment of intraocular infections due to Gram-positive organisms by subconjunctival injection of chemotherapeutic agents depends upon the susceptibility of the pathogen to the antibiotic, the reaction of the patient, permeability associated with inflammation, and the maintenance of effective therapeutic levels. Langham reported that the maximum concentration of an antibiotic in the aqueous humor of a normal eye for a given concentration in the blood stream is dependent not only on the rate of entry but also on its de-
1305
gree of absorption on the proteins of the plasma. Leopold has shown from clinical data that intraocular infections, once established, are difficult to control and that cultures of the intraocular fluids in these cases are often sterile and therefore are not often helpful in detecting the cause of the infection or in selecting the antibiotic. He concluded that once the infection has developed, subconjunctival injections of penicillin (1,000,000 units), along with streptomycin (50,000 units), will not control intraocular infection in all cases, nor will systemic or intravitreal administration of chloramphenicol or systemic, intravitreal, or subconjunctival administration of terramycin. 8
In treatment of intraocular infections, an antibiotic may be used prior to surgical procedures, whether it is to be extraction of a foreign body or a cataract, or repair of a ruptured globe. In this way one can establish a concentration of the antibiotic in the blood stream and in the ocular fluids prior to opening the eye. In vitro studies (summarized in Table 1) reported by McCall, Steigbigel, and Finland, McGehee, Smith, Wilcox, and Finland, and, Barber and Waterworth indicate that low concentrations of lincomycin inhibit several common pathogens. Although a direct correlation cannot be assumed between in vitro and in vivo susceptibility of bacteria, it seems likely that in àn inflamed eye, aqueous humor levels of lincomycin can be attained which may be active against susceptible Gram-positive organisms. 9
10
11
Following subconjunctival or intramuscular injection, lincomycin has been demonstrated to produce adequate aqueous humor levels in man in the noninflamed eye similar to that shown for other leading antibiotics, and it could prove useful in ocular infections due to Gram-positive pathogens susceptible to lincomycin.
7
SUMMARY
After 0.25 ml of a solution containing 300 mg/ml of lincomycin was administered subconjunctivally to each of 27 hospitalized pa-
1306
AMERICAN JOURNAL OF OPHTHALMOLOGY
JUNE, 1971
TABLE 1 SUSCEPTIBILITY OF VARIOUS PATHOGENS TO LINCOMYCIN
Minimum Inhibition Concentration Pathogen
McCall and Associates'
(jig/ml)
McGehee and Associates
10
Barber and Waterworth Range
11
Staph, aureus Strep. (Group A ) D. pneumoniae Strep, viridans Enterococcus H. Influenzae N. gonorrhoeae X. meningitidis
Range
Median
Range
Median
0..78 - >100 0.,04 0. 19 0.,0050. 39 0 .02 0.,78 25.,00 - >100 6..25 - 100 6..25 25,,0
0,.78 0,.04 0 .04 0 .09 50,.0 12.,5 12 .5
0,.8 3. 1 0,.040. 2 0 .020. 2 0 .020. 4 25,.0 - >100 6,,3050 0 .0225 100 >100
1,.60 0. 04 0.,20 0,.04 >100 25.,0 12 .5 100
tients, aqueous humor, serum, and lens tissue samples were obtained at varying postinjection intervals. Significant concentrations of lincomycin were demonstrated
in aqueous
humor samples as early as two minutes after injection. Peak levels of the antibiotic in aqueous humor occur between one and three hours after injection, followed by a gradual decline. Minimal levels of the antibiotic persisted in the serum and aqueous humor for 12 hours. All the lens tissue samples showed a concentration of less than 0.3 u.g/ml. ACKNOWLEDGMENT
We thank Dr. S. S. Schneierson, Director of Microbiology, for his helpful comments and criticism. We also thank Dr. J. D. Panzer, of the Upjohn Company, for his advice. We are also indebted to Drs. J. Bloomfield, S. Sherman, S. Wunsh, and E. Siegel, who helped to obtain some of the specimens, and to the Upjohn Company for supplying the lincomycin (Lincocin) used in this study. REFERENCES
1. Mason, D. J, Dietz, A., and DeBoer, G.: Lincomycin, a new antibiotic. I. Discovery and biological properties. Antimicrob. Chemotherap. 554, 1962.
-
0 .5 - 2..00 0..06- 0,.12 0 .06- 0 .50 4,.00-16 .0 4,.00-16 .0 32 .0 >32 .0
2. Josten, J. J, and Allen, P. M. : The mode of action of lincomycin. Biochem. Biophys. Res. Comm. 14:241, 1964. 3. McPherson, S. D , Presley, G. D , and Crawford, J. R. : Aqueous humor assays of subconjunctival antibiotics. Am. J. Ophth. 66:430, 1968. 4. Becker, E. F. : The intraocular penetration of lincomycin. Am. J. Ophth. 67:963, 1969. 5. Riley, F. C , Boyle, G. L , and Leopold, I. H. : Intraocular penetration of cephaloridine in humans. Am. J. Ophth. 66:1042, 1968. 6. Schneierson, S. S, and Torharsky, B. : A method for the determination of aureomycin in the blood. J. Bacteriol. 57:483, 1949. 7. Langham, M. : Factors affecting the penetration of antibiotics into aqueous humor. Brit. J. Ophth. 35:614, 1951. 8. Leopold, I. H. : Surgery of ocular trauma : Therapy of secondary intraocular infection. Arch. Ophth. 48:738, 1952. 9. McCall, C. E , Steigbigel, N , and Finland, M. : Lincomycin: activity in vitro and absorption and excretion in normal young men. Am. J. Med. Sei. 254:144, 1967. 10. McGehee, R. F , Smith, C. B , Wilcox, C , and Finland, M. : Comparative studies of antibacterial activity in vitro and absorption and excretion of lincomycin and clinimycin. Am. J. Med. Sei. 265 : 279, 1968. 11. Barber, M , and Waterworth, P. M. : Antibacterial activity of lincomycin and pristamycin : a comparison with erythromycin. Brit. Med. J. 2:603, 1964.