Chemotherapy 24: 29-33 (1978)
In vitro Activity of Netilmicin (SCH 20569) Against Bacterial Isolates from 111 Children Gerrard Greenstone, Susan Hammerberg and Melvin l. Marks Department of Pediatrics (Infectious Diseases), McGill University and the McGill Univcrsity-Montreal Children's Hospital Research Institute, Montreal. P.Q.
Key Words. Netilmicin • Antibiotic susceptibility • Schering 20569 • In vitro antibiotics • Aminoglycoside • Bacterial sensitivity Abstract. A new aminoglycoside antibiotic, netilmicin, was tested against 306 clinical isolates from ill children and compared with sisomicin and gentamicin. Activity against Enterobacteriaceae was similar to that of gentamicin but less than that of sisomicin. Two gentamicin-resistant strains of Enterobacteriaceae (Klebsiella, MIC 6.25 »g/ml, Escherichia coli, MIC 12.5 /tg/ml) were susceptible to netilmicin (MIC 3.12 //g/ml). Netilmicin was ineffective against almost all strains of Pseudomonas but active against the majority of strains of Staphylococcus, Neisseria meningitidis and Haemophilus influenzae tested. Disc diffusion sensitivity results correlated in general with the agar dilution test. Netilmicin had little activity against Pseudomonas but may be useful in the treatment of infections due to gentamicin-resistant Enterobacteriaceae.
Infections due to gram-negative bacteria are important causes of illness in young in fants. Presently available drugs are becom ing less effective mainly due to the emerg ence of resistant bacteria. Furthermore, the toxicity of these antimicrobials preclude their use at higher than the presently recom mended dosage schedules. A new aminogly coside antibiotic, netilmicin, appears to have increased activity and low toxicity [2, 6,7].
Materials and Methods The following bacteria obtained from ill chil dren at the Montreal Children’s Hospital were iso lated in pure culture and identified according to standard methods in use in the diagnostic micro biology laboratory: 18 Staphylococcus aureus, 15 Staphylococcus epidermidis, 10 Streptococcus py ogenes Group A, 12 Streptococcus pneumoniae. 31 Haemophilus influenzae, 34 Escherichia coli, 11 Klebsiella sp., 23 Proteus sp. (7 indole-positive, 16 indole-negative), 61 Pseudomonas aeruginosa, 11 Enterobacter sp., 12 Streptococcus faecalis, 34 Salmonella sp., 13 Shigella sp., and 21 Neisseria meningitidis. The comparative activities of netil
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
Introduction
Grecnstone/Hammerberg/Marks
micin, sisomicin and gentamicin were determined by the agar dilution method. The reliability of the disc diffusion method in predicting susceptibility and resistance to netilmicin was also investigated. A standardized disc diffusion method was used [1], The inoculum was applied with a cotton swab and consisted of an overnight broth culture of each bacteria in the following dilutions: 10"* for Staphylococci, 10"4 for Enterobacteriaceae and Pseudomonas, and 10 2 for H. influenzae and Streptococci. All bacteria were grown overnight in Mueller-Hinton broth except for H. influenzae (5°/o Fildes in Mueller-Hinton), and Streptococci (Todd-Hcwitt). Neisseria were swabbed from an agar plate into Mueller-Hinton broth to yield a suspension of approximately 109 CFU/rnl (verified by colony count). A 10“2 dilution of this suspen sion was used as inoculum. Discs containing 10 «g of netilmicin were prepared in our laboratory and also supplied by Schering Corp. Comparative test ing showed these to be interchangeable. Agar dilution testing was performed using the Steers replicator method of inoculation [5], A 10-2 dilution of an overnight growth of each isolate was used as the inoculum (Neisseria were diluted as above). Minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug showing no growth on the surface of the agar. Disc diffusion and agar dilution tests were per formed on Mueller-Hinton agar with a calcium and magnesium content of 50-100 mg/I, and 2035 mg/1 [3], respectively, except for H. influenzae (which was tested on M-H agar with 5°/» Fildes) and Streptococci (M-H agar with 5% sheep blood). The agar for all tests was adjusted to pH 7.4. An tibiotic powders for the agar dilution tests were supplied in pure form by Schering Corp. Control strains run for each experiment included E. coli ATCC No. 25 922, Pseudomonas ATCC No. 27 853 and S. aureus ATCC No. 25 923.
Results A total of 306 bacterial isolates from ill children were tested against gentamicin, si somicin and netilmicin. Figure 1 illustrates the activities of these drugs against 34
Fig. 1. Susceptibility of 34 clinical isolates of E. coli to 3 antibiotics (agar dilution, inoculum replicating method, and 10"2 dilution of overnight broth culture).
strains of E. coli. The majority ( > 95%) of strains were inhibited by ♦ * 1 1 1
27
28
29
mm
Fig. 5. Correlation of agar-dilution and disc (10/(g) sensitivity tests for netilmicin against 187
clinical isolates of Enterobacteriaccae and Pseu domonas.
sensitive Proteus and 2 false-resistant Kleb siella were encountered.
tions of ^ 3.12 t(g/ml of netilmicin, sisomi cin and gentamicin as sensitive and those with an MIC of 6.25 «g/ml as resistant. We encountered only 2 gentamicin-resistant strains of Enterobacteriaceae (E. coli MIC 12.5 ,/(g ml. Klebsiella MIC 6.25 jug/ml) and both of these were sensitive to netilmicin (MIC 3.12 Mg/ml); however, these differ ences in vitro are relatively small and their clinical significance is unknown. Rahal et al. [2] also reported gentamicin-resistant ne tilmicin-sensitive strains of Enterobacter iaceae. This suggests that the drug may hold promise for treatment of infections due to such organisms in both children and adults. Marked in vitro activity against Staphylo cocci, N. meningitidis and H. influenzae
Discussion The activity of netilmicin against bacteria from ill children parallel those reported by Rahal et al. [2] against bacteria isolated from adults. In general, the activity of netil micin is about the same as gentamicin and sisomicin for gentamicin-sensitive Enterobacteriaceae. Pharmacokinetic data in ani mals suggest that achievable serum concen trations of netilmicin would be similar to gentamicin [4]. We have arbitrarily desig nated those strains inhibited by concentra-
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
lOO-i
In vitro Activity of Netilmicin
was noted, however, clinical experience with aminoglycosides is limited for infec tions due to these bacteria. Netilmicin ap pears to offer no advantage over gentamicin against P. aeruginosa [2]. Although disc dif fusion sensitivity testing is generally relia ble, false readings were encountered with only 3/187 isolates tested. Based on our data Enterobacteriaceae and P. aeruginosa netilmicin disc sensitivity testing (10 /tg disc) should be interpreted as follows: disc zone diameter 22 mm = sensitive and 19-21 mm = interme diate. Netilmicin is a new active aminogly coside which may have a useful role in the treatment of infections due to gentamicinresistant Enterobacteriaceae.
33
2
3
4
5
6
Acknowledgement 7 The assistance of Schering Corp. and the coop eration of Dr. C. H. Pai and Mr. S. Sorger are gratefully acknowledged.
collaborative study. Acta path, microbiol. scand., suppl. 217, pp. 68-71 (1971). Rahal, J. J., jr.; Simberkoff, M. S.; Kagan, K., and Moldover, N.: Bacterial efficacy of SCH 20 569 and amikacin against gentamicin-sen sitive and resistant organisms. Antimicrob. Agents Chemother. 9: 595-599 (1976). Reller, L. B.; Schoenknecht, F. D.; Kenny, M. A., and Sherris, J. C.: Antibiotic susceptibility testing of Pseudomonas aeruginosa: Selection of a control strain and criteria for magnesium and calcium content in media. J. infect. Dis. 130: 454-463 (1974). Schering Corporation: Informational material for the investigational drug SCH 20 569 (Schering Corp., Bloomfield, New Jersey 1975). Steers, E.; Foltz, E. L., and Graves, B. S.: Inocula replicating apparatus of routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: 307-311 (1959). Waitz, J. A. and Miller, G. H.: Chemothera peutic evaluation of SCH 20 569, a novel semi synthetic aminoglycoside. Intersci. Conf. Anti microb. Agents Chemother., Washington 1975. Wright, J. J.: A broad-spectrum semisynthetic aminoglycoside antibiotic. Intersci. Conf. Anti microb. Agents Chemother., Washington 1975.
References Dr. Melvin I. Marks, Montreal Children’s Hospital, 2300 Tupper Street, Montreal, PQ, H3H 1P3 (Canada)
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
1 Ericsson, H. M. and Sherris, J. C.: Antibiotic sensitivity testing, report of an international
In vitro Activity of Netilmicin (SCH 20569) Against Bacterial Isolates from 111 Children Gerrard Greenstone, Susan Hammerberg and Melvin l. Marks Department of Pediatrics (Infectious Diseases), McGill University and the McGill Univcrsity-Montreal Children's Hospital Research Institute, Montreal. P.Q.
Key Words. Netilmicin • Antibiotic susceptibility • Schering 20569 • In vitro antibiotics • Aminoglycoside • Bacterial sensitivity Abstract. A new aminoglycoside antibiotic, netilmicin, was tested against 306 clinical isolates from ill children and compared with sisomicin and gentamicin. Activity against Enterobacteriaceae was similar to that of gentamicin but less than that of sisomicin. Two gentamicin-resistant strains of Enterobacteriaceae (Klebsiella, MIC 6.25 »g/ml, Escherichia coli, MIC 12.5 /tg/ml) were susceptible to netilmicin (MIC 3.12 //g/ml). Netilmicin was ineffective against almost all strains of Pseudomonas but active against the majority of strains of Staphylococcus, Neisseria meningitidis and Haemophilus influenzae tested. Disc diffusion sensitivity results correlated in general with the agar dilution test. Netilmicin had little activity against Pseudomonas but may be useful in the treatment of infections due to gentamicin-resistant Enterobacteriaceae.
Infections due to gram-negative bacteria are important causes of illness in young in fants. Presently available drugs are becom ing less effective mainly due to the emerg ence of resistant bacteria. Furthermore, the toxicity of these antimicrobials preclude their use at higher than the presently recom mended dosage schedules. A new aminogly coside antibiotic, netilmicin, appears to have increased activity and low toxicity [2, 6,7].
Materials and Methods The following bacteria obtained from ill chil dren at the Montreal Children’s Hospital were iso lated in pure culture and identified according to standard methods in use in the diagnostic micro biology laboratory: 18 Staphylococcus aureus, 15 Staphylococcus epidermidis, 10 Streptococcus py ogenes Group A, 12 Streptococcus pneumoniae. 31 Haemophilus influenzae, 34 Escherichia coli, 11 Klebsiella sp., 23 Proteus sp. (7 indole-positive, 16 indole-negative), 61 Pseudomonas aeruginosa, 11 Enterobacter sp., 12 Streptococcus faecalis, 34 Salmonella sp., 13 Shigella sp., and 21 Neisseria meningitidis. The comparative activities of netil
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
Introduction
Grecnstone/Hammerberg/Marks
micin, sisomicin and gentamicin were determined by the agar dilution method. The reliability of the disc diffusion method in predicting susceptibility and resistance to netilmicin was also investigated. A standardized disc diffusion method was used [1], The inoculum was applied with a cotton swab and consisted of an overnight broth culture of each bacteria in the following dilutions: 10"* for Staphylococci, 10"4 for Enterobacteriaceae and Pseudomonas, and 10 2 for H. influenzae and Streptococci. All bacteria were grown overnight in Mueller-Hinton broth except for H. influenzae (5°/o Fildes in Mueller-Hinton), and Streptococci (Todd-Hcwitt). Neisseria were swabbed from an agar plate into Mueller-Hinton broth to yield a suspension of approximately 109 CFU/rnl (verified by colony count). A 10“2 dilution of this suspen sion was used as inoculum. Discs containing 10 «g of netilmicin were prepared in our laboratory and also supplied by Schering Corp. Comparative test ing showed these to be interchangeable. Agar dilution testing was performed using the Steers replicator method of inoculation [5], A 10-2 dilution of an overnight growth of each isolate was used as the inoculum (Neisseria were diluted as above). Minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug showing no growth on the surface of the agar. Disc diffusion and agar dilution tests were per formed on Mueller-Hinton agar with a calcium and magnesium content of 50-100 mg/I, and 2035 mg/1 [3], respectively, except for H. influenzae (which was tested on M-H agar with 5°/» Fildes) and Streptococci (M-H agar with 5% sheep blood). The agar for all tests was adjusted to pH 7.4. An tibiotic powders for the agar dilution tests were supplied in pure form by Schering Corp. Control strains run for each experiment included E. coli ATCC No. 25 922, Pseudomonas ATCC No. 27 853 and S. aureus ATCC No. 25 923.
Results A total of 306 bacterial isolates from ill children were tested against gentamicin, si somicin and netilmicin. Figure 1 illustrates the activities of these drugs against 34
Fig. 1. Susceptibility of 34 clinical isolates of E. coli to 3 antibiotics (agar dilution, inoculum replicating method, and 10"2 dilution of overnight broth culture).
strains of E. coli. The majority ( > 95%) of strains were inhibited by ♦ * 1 1 1
27
28
29
mm
Fig. 5. Correlation of agar-dilution and disc (10/(g) sensitivity tests for netilmicin against 187
clinical isolates of Enterobacteriaccae and Pseu domonas.
sensitive Proteus and 2 false-resistant Kleb siella were encountered.
tions of ^ 3.12 t(g/ml of netilmicin, sisomi cin and gentamicin as sensitive and those with an MIC of 6.25 «g/ml as resistant. We encountered only 2 gentamicin-resistant strains of Enterobacteriaceae (E. coli MIC 12.5 ,/(g ml. Klebsiella MIC 6.25 jug/ml) and both of these were sensitive to netilmicin (MIC 3.12 Mg/ml); however, these differ ences in vitro are relatively small and their clinical significance is unknown. Rahal et al. [2] also reported gentamicin-resistant ne tilmicin-sensitive strains of Enterobacter iaceae. This suggests that the drug may hold promise for treatment of infections due to such organisms in both children and adults. Marked in vitro activity against Staphylo cocci, N. meningitidis and H. influenzae
Discussion The activity of netilmicin against bacteria from ill children parallel those reported by Rahal et al. [2] against bacteria isolated from adults. In general, the activity of netil micin is about the same as gentamicin and sisomicin for gentamicin-sensitive Enterobacteriaceae. Pharmacokinetic data in ani mals suggest that achievable serum concen trations of netilmicin would be similar to gentamicin [4]. We have arbitrarily desig nated those strains inhibited by concentra-
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
lOO-i
In vitro Activity of Netilmicin
was noted, however, clinical experience with aminoglycosides is limited for infec tions due to these bacteria. Netilmicin ap pears to offer no advantage over gentamicin against P. aeruginosa [2]. Although disc dif fusion sensitivity testing is generally relia ble, false readings were encountered with only 3/187 isolates tested. Based on our data Enterobacteriaceae and P. aeruginosa netilmicin disc sensitivity testing (10 /tg disc) should be interpreted as follows: disc zone diameter 22 mm = sensitive and 19-21 mm = interme diate. Netilmicin is a new active aminogly coside which may have a useful role in the treatment of infections due to gentamicinresistant Enterobacteriaceae.
33
2
3
4
5
6
Acknowledgement 7 The assistance of Schering Corp. and the coop eration of Dr. C. H. Pai and Mr. S. Sorger are gratefully acknowledged.
collaborative study. Acta path, microbiol. scand., suppl. 217, pp. 68-71 (1971). Rahal, J. J., jr.; Simberkoff, M. S.; Kagan, K., and Moldover, N.: Bacterial efficacy of SCH 20 569 and amikacin against gentamicin-sen sitive and resistant organisms. Antimicrob. Agents Chemother. 9: 595-599 (1976). Reller, L. B.; Schoenknecht, F. D.; Kenny, M. A., and Sherris, J. C.: Antibiotic susceptibility testing of Pseudomonas aeruginosa: Selection of a control strain and criteria for magnesium and calcium content in media. J. infect. Dis. 130: 454-463 (1974). Schering Corporation: Informational material for the investigational drug SCH 20 569 (Schering Corp., Bloomfield, New Jersey 1975). Steers, E.; Foltz, E. L., and Graves, B. S.: Inocula replicating apparatus of routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: 307-311 (1959). Waitz, J. A. and Miller, G. H.: Chemothera peutic evaluation of SCH 20 569, a novel semi synthetic aminoglycoside. Intersci. Conf. Anti microb. Agents Chemother., Washington 1975. Wright, J. J.: A broad-spectrum semisynthetic aminoglycoside antibiotic. Intersci. Conf. Anti microb. Agents Chemother., Washington 1975.
References Dr. Melvin I. Marks, Montreal Children’s Hospital, 2300 Tupper Street, Montreal, PQ, H3H 1P3 (Canada)
Downloaded by: King's College London 137.73.144.138 - 3/6/2018 10:57:24 PM
1 Ericsson, H. M. and Sherris, J. C.: Antibiotic sensitivity testing, report of an international
