ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, Aug. 1978, p. 194-200
0066-4804/78/0014-0194$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 14, No. 2
Printed in U.S.A.
Activity of 5-Episisomicin Compared with That of Other Aminoglycosides KWUNG P. FU AND HAROLD C. NEU* Division of Infectious Diseases, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 6 February 1978
5-Episisomicin, a semisynthetic aminoglycoside, has been shown to inhibit many members of the Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus. At a concentration of 3.1 ,ug/ml, more than 90% of Escherichia coli, Klebsiella pneumoniae, Enterobacter, Citrobacter, indole-positive Proteus, and Providencia were inhibited. 5-Episisomicin had activity against S. aureus, E. coli, Klebsiella, Enterobacter, and Citrobacter similar to gentamicin and netilmicin. It had activity similar to amikacin and netilmicin against many gentamicinresistant bacteria. The activity of 5-episisomicin was greatest at an alkaline pH and in medium of low magnesium content. Synergy of 5-episisomicin and carbenicillin was found to occur most often against Pseudomonas. The increasing incidence of gram-negative infections and particularly of gram-negative bacteremia due to bacteria resistant to the aminoglycosides currently in clinical use prompted us to evaluate the in vitro activity of 5-episisomicin, the 5-epimer of sisomicin (9). We compared the in vitro activity of 5-episisomicin with the in vitro activity of other aminoglycosides against clinical isolates. (This material was presented at the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, N.Y., 12-14 October 1977.)
MATERLALS AND METHODS Antibiotics. Gentamicin, sisomicin, netilmicin, and 5-episisomicin were supplied by Schering Corp. Kanamycin and amikacin were obtained from Bristol Laboratories, and carbenicillin was a gift from Beecham Laboratories. Bacterial isolates. The majority of bacterial isolates were obtained from patients hospitalized at the Columbia-Presbyterian Medical Centerduring the last 2 years. Only single isolates from a patient were used, and isolates felt to be from an outbreak were not used. Gentamicin-resistant isolates, listed in Tables 5 and 6 and used for the analysis of comparative activity of the aminoglycosides, had been collected over the past 7 years. Susceptibility tests. An inoculum of 105 colonyforming units was used except where specified. Minimal inhibitory concentrations (MICs) were determined by twofold dilution of the antibiotics in MuellerHinton agar (Baltimore Biological Laboratory [BBL]) or broth (BBL) by using a multiple-inoculating device as described in detail previously (4). The activity of 5-episisomicin against Bacteroides was deter-
mined by using an anaerobe gas pack jar (BBL) with Mueller-Hinton agar which contained sheep blood and vitamin K. All incubations were for 18 h at 35°C, with the exception of those against Bacteroides, which were for 18 h at 35°C. The MBC was the lowest concentration at which less than five colonies grew. The effect tubes into Columbia blood agar (BBL) and incubating for 18 h at 35°C. the MBC was the lowest concentration at which less than five colonies grew. The effect of growth medium upon the MICs and MBCs was determined with Trypticase soy (BBL), brain heart infusion (BBL), and nutrient broths (BBL). The effect of pH of the medium was determined by use of Mueller-Hinton broth adjusted with sodium hydroxide or hydrochloric acid. The concentration of sodium was less than that which would have affected the results of the assay. The effect of divalent cations on the MIC was determined by addition of magnesium sulfate to nutrient broth. Bacterial killing studies were carried out in Mueller-Hinton broth (BBL) or nutrient broth (BBL) by using organisms incubating in a gyratory shaker at 35°C, with samples of 0.1 ml removed for dilution in broth and plated on Mueller-Hinton agar at varying dilutions. Colony counts were read after 18 h of incubation at 35°C. The activity of 5-episisomicin was determined against isolates with known inactivating enzymes which had been given to us by various investigators in the United States and abroad or which we had earlier characterized through use of the techniques of Davies (1, 6) by using radioactive substrates. Synergy test8. Synergy was defined according to the following criteria: (i) a reduction of the MIC of both antibiotics of fourfold or more and (ii) a decrease of 1 log or more in viable cell count occurring at the end of 4 h with an antibiotic combination that contained the agents at one-fourth the MICs of the individual agents. Partial synergy was defined as a fourfold reduction in MICs of one agent and a twofold or no
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5-EPISISOMICIN
VOL. 14, 1978 reduction in the MIC of the other agent (3). The concentration of carbenicillin ranged from 400 to 1 Ag/ml, and that of 5-episisomicin ranged from 10 to 0.1 pLg/ml. These concentrations would be achieved by administration of 100 mg of carbenicillin per kg and 2 mg of 5-episisomicin per kg.
RESULTS Table 1 gives the overall activity of 5-episisomicin against 506 gram-positive and gram-negative isolates. 5-episisomicin inhibited 97% of Staphylococcus aureus at 0.8 ,ug/ml, but at the concentration of 6.3 ug/ml it inhibited only 22% of Streptococcus fecalis and S. fecium (true enterococci) and inhibited only 9% of S. pyogenes and S. agalactiae. Bacteroides fragilis was not inhibited by 5-episisomicin. By using 6.3 ,ug/ml as a cut-off point, 97% of Escherichia coli, 97% of Enterobacter, 96% of Citrobacter, and 100% of Shigella, Klebsiella, Proteus mirabilis, P. rettgeri, P. vulgaris, P. morganii, and Providencia were inhibited. Indeed, at 6.3 ug/ml 81% of Acinetobacter 87% of Pseudomonas aeruginosa, and 73% of Serratia marcescens were inhibited. It is noteworthy that 5-episisomicin at 1.6 ,ug/ml inhibited 90% of E. coli, 91% of Klebsiellapneumoniae, 58% of P. mirabilis, and 78% of P aeruginosa, the organisms which account for most episodes of gram-negative bacteremia. The effect of growth medium on the activity of 5-episisomicin was tested against organisms recently isolated from the blood or urine of hospitalized patients. Organisms were selected TABLE 1. Activity of 5-episisomicin against gramnegative and gram-positive microorganisms MIC (ug/ml) required to
inhibit:
Organism (no. of isolates) 50%
90%
S. aureus (34) 0.2 Enterococcia (30) 12.5 50 Streptococcib (32) E. coli (34) 0.8 Shigella (23) 3.1 K. pneumoniae (32) 0.4 Enterobacter (37) 0.4 S. marcescens (37) 3.1 Salmonella (22) 0.8 Citrobacter (24) 0.4 P. mirabilis 31) 1.6 P. rettgeri (15) 0.4 P. vulgaris (6) 1.6 P. morganii (26) 0.8 Providencia (29) 0.8 P. aeruginosa (46) 0.8 0.8 Acinetobacter (16) B. fragilis (32) 'S. fecalis and S. fecium. b Group A and B streptococci.
0.8 25
TABLE 2. Effects ofgrowth medium on the 5episisomicin MICs and MBCs Organism
E. coli 3939 Klebsiella 3929 P. rettgeri 3919 P. mirabilis 3378 S. marcescens 3915 P. aerugi-
MIC (MBC) in: Mueller-Hin- Brain heart inNutrient broth ton broth fusion
0.8 (3.1) 0.4 (0.4)
3.1 (3.1) 0.4 (0.4)
6.3 (6.3) 1.6 (3.1)
0.8 (3.1)
0.4 (0.4)
6.3 (6.3)
0.8 (6.3)
3.1 (6.3)
12.5 (12.5)
0.8 (250)
6.3 (250)
12.5 (250)
0.4 (1.6)
0.4 (0.4)
25 (250)
0.2 (1.6)
0.2 (1.6)
0.8 (3.1)
0.8 (250)
6.3 (250)
12.5 (250)
0.2 (0.8)
0.2 (0.2)
0.8 (6.3)
0.4 (3.1)
0.4 (0.4)
6.3 (12.5)
(0.8)
0.2 (0.2)
0.4 (1.6)
0.8 (25)
0.8 (0.8)
0.8 (250)
0.2 (0.8)
0.2 (0.4)
0.4 (0.8)
nosa
3696
P. aeruginosa
3886 P. aeruginosa
3901 P. aeruginosa
3902 P. aeruginosa
3903
P. aerugi-
50.1
nosa
3904 P. aeruginosa
3950
P. aeruginosa
3948
0.6 (3.1) 3.1 (6.3) 6.3 (6.3) P. maltaphilia 3941 a MICs and MBCs are in micrograms per milliliter.
TABLE 3. Effect ofpH upon the activity of 5episisomicina MIC (MBC)b at:
1.6
3.1 1.6 1.6 25 0.8 1.6 6.3 1.6 6.3 3.1 1.6 12.5 12.5 0
195
Organism
pH 7
pH 8
12.5 (25)
3.1 (6.3)
1.6 (3.1)
12.5 (250)
6.3 (250)
3.1 (250)
1.6 (1.6)
0.4 (0.4)
0.2 (0.4)
6.3 (6.3) 12.5 (250)
3.1 (3.1) 6.3 (250)
0.4 (0.8) 3.1 (250)
0.8 (1.6)
0.2 (0.2)
0.2 (0.4)
pH 6
P. mirabilis 3378 S. marcescens 3915 K. pneunoniae 3929 E. coli 3939 P. aeruginosa 3901 P. aeruginosa 3902
Assays
were performed in Mueller-Hinton broth. bMICs and MBCs are in micrograms per milliliter.
a
196
ANTIMICROB. AGENTS CHEMOTHER.
FU AND NEU
so as to be certain that they did not represent the same strain. Three media-were tested: nutrient broth, Mueller-Hinton broth, and brain heart infusion broth. The data are given in Table 2. There was minimal difference in either MICs or MBCs between nutrient broth and MuellerHinton broth, whereas, both MICs and MBCs often were higher in the brain heart infusion medium. The E. coli, Klebsiella, P. mirabilis, and P. rettgeri had MBCs which were miniimally greater than the MICs. Some Serratia (for example, isolate 3915 listed in Table 2) and some Pseudomonas (for example, 3901 and 3950) had an MBC which was 25 ,ug/ml or greater even tht)ugh the MIC was less than 1 ,tg/ml.
Table 3 shows that 5-episisomicin had greatest activity in a basic medium. It was 4- to 16-foldmore active at pH 8 than at pH 6. Nonetheless, a 16-fold greater MBC than MIC was noted with the Serratia and Pseudomonas tested at pH 8. The inhibitory activity of 5-episisomicin against Pseudomonas could be prevented by magnesium. Figure 1 shows the effect of adding varying concentrations of magnesium sulfate to an exponential-phase culture of Pseudomonas to which 5-episisomicin at two times the MIC had been added. In the presence of 0.5 mM MgSO4, there was a reduction in the killing rate, and in the presence of 2.5 mM MgSO4 there was a lag in growth but no reduction in colony-form-
9
S-
*1
N-
Houm FIG. 1. Effect of magnesium upon the bactericidal activity of 5-episisomicin against P. aeruginosa 3948. Magnesium sulfate was added at zero time to a log-phase culture. Symbols: 0, Control; El, control + 5 mM MgS04; *, 5-episisomicin (0.4 pg/ml); I, 5-episisomicin (0.4 pAg/ml) + 0.5 mM MgS04; A, 5-episisomicin (0.4 pg/ml) + 2.5 mM MgS04; A, 5-episisomicin (0.4 pg/ml) + 5 mM MgSO4. The MIC of 5-episisomicin is 0.2 pg/ml. CFU, Colony-forming units.
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5-EPISISOMICIN
VOL. 14, 1978
TABLE 4. Comparative activity of 5 episieomicin with known aminoglycoside antibiotics MIC (gag/ml) requred for: of strair) (no.Organis
50% of strains
90% of strains
Gent Net Tobra Ami 5-Epi Gent Net Tobra S. aureus (34) 0.4 0.4 1.6 3.1 0.8 1.6 1.6 1.6 Enterococci (32) 12.5 6.3 12.5 50 25 25 12.5 25 E. coli (34) 0.8 0.8 0.8 1.6 25 1.6 K. pneumoniae (32) 0.4 0.4 0.8 3.1 25 0.8 Enterobacter (37) 0.8 0.8 1.6 1.6 1.6 0.8 S. marcescens (37) 3.1 50 12.5 25 >50 >50 Citrobacter (24) 0.4 0.4 0.8 1.6 1.6 1.6 Acinetobacter (16) 1.6 1.6 1.6 12.5 >50 >50 P. aeruginosa (39) 3.1 3.1 0.8 3.1 12.5 50 50 12.5 P. rettgeri (15) 6.3 6.3 0.8 3.1 25 25 P. morganu (26) 0.8 0.8 1.6 3.1 6.3 6.3 Providencia (29) 6.3 12.5 1.6 1.6 12.5 12.5 a 5-Epi, 5-Episisomicin; Gent, gentamicin; Net, netilmicin; Tobra, tobramycin; Ami, amikacin.
5-Epi 0.2 12.5 0.8 0.4 0.4 6.3 0.4 0.8 0.8 0.8 0.8 0.8
Ami
6.3 >50 1.6 3.1 3.1 25 1.6 25 25 6.3 12.5 3.1
TABLE 5. Comparative activity of 5-episisomicin against gentamicin-resistant organisms Medin MIC (range)a
Organism (no. of isolates)
S. aureus (1) E. coli (7)
Klebsiella (4) Enterobacter (2) Serratia (11)
Citrobacter (2) P. vulgaris (3) P. rettgeri (7) P. morganii (1) Providencia (5)
Acinetobacter (4) P. aeruginosa (13)
5-Episisomicin
Gentamicin
Netiiaicin
Amikacin
12.5 2.2 (0.4-12.5) 1.4 (0.2-6.3) (1.6-12.5) 8.3
>50 45 (12.5-250) 42.5
3.1 2.5 (0.8-250) 2.4 (0.4- .50) (1.6-25) 41.5 (25-250) (0.4-250) 25
6.3 1.1 (0.4-1.6) 1.2 (0.8-3.1) (1.6-3.1) 28.8 (6.3-250) (1.6-250) 3.1
(1.6--50) (0.4--50) 1.2 (0.4-1.6) 1.5 (0.4-3.1) 0.8 0.9 (0.8-1.6) 21 (0.8-25) 6.3
(25-2.50) (25-2.50) 41.5 (25-250) (250) 32.5
(12.5-2.50)
(12.5-2.50)
(1.6-6.3)
21.3 12.5-250) 25 20
13.8 (3.1-25) 25 20 (6.3-250) 32.5 (12.5-250) 21
2.9 (0.8-12.5) 1.6 2.1 (0.8-6.3) 9.5 (1.6-250) 17.5
(12.5-2.50) 32.5 (12.5->50) 30
(0.8-Dm50) (12.5-2s50) (1.6-e50) (1.6t50) aData are in micrograms per milliliter.
ing units. In the presence of 5 mM MgS04, the effect of 5-episisomicin was completely abolished. The comparative in vitro activity of 5-episisomicin and other aminoglycosides-amikacin, gentamicin, netilmicin, and tobramycin-is given in Table 4. Comprison to tobramycin is given only for S. aureus, enterococci and Pseudomonas because the tobramycin show little difference in activity against most other organisms (2, 8). Against S. aureus 5-episisomicin and netilnicin were the most active agents inhibiting more than 90% at concentrations below 0.8,ug/ml. Against enterococci at concentrations
of 3.1 ,ug/ml or less, 5-episisomicin, gentamicin, netilmicin, and tobramycin all had a similar low degree of activity. The activity of 5-episisomicin, amikacin, and netilmicin against the E. coli isolates were similar at concentrations below 3.1 ug/ml. However, amikacin inhibited all of the E. coli isolates at 3.1 ,ug/ml. Against Kiebsiella the four agents, 5episisomicin, gentamicin, netilmicin, and amikacin, inhibited 80% or more of isolates at concentrations of 1.6 pg/ml. Against Enterobacter and Citrobacter the compounds had similar activity. A major proportion of the S. marcescens isolates were resistant to all of the aminoglyco-
198
FU AND NEU
sides. But 5-episisomicin was the most active agent at concentrations of 3.1 to 12.5 ,jg/ml. 5Episisomicin, gentamicin, and amikacin had comparable activity against isolates of Acinetobacter, whereas netilmicin was less active than the other agents. Against P. aeruginosa tobramycin and 5-episisomicin were the agents which showed greatest activity at concentrations below 1.6 ,ug/ml. Tobramycin and 5-episisomicin were twofold more active than the other three agents. 5-episisomicin was the most active agent tested against P. rettgeri and Providencia, whereas, tested against P. morganii, all of the agents had similar inhibitory activity. The comparative activity of 5-episisomicin and gentamicin, netilmicin, and amikacin against gentamicin-resistant isolates is shown in
ANTIMICROB. AGENTS CHEMOTHER.
Table 5. The isolates came from patients at the Columbia-Presbyterian Medical Center and from affiliated hospitals. Most of the gentamicinresistant E. coli, Klebsiella, indole-positive Proteus, and Providencia were inhibited by 5-episisomicin. 5-Episisomicin inhibited most of the gentamicin-resistant Pseudomonas, but there
were isolates resistant to it and to the other aminoglycosides as well. Many of the gentamicin-resistant Serratia and Acinetobacter were resistant to 5-episisomicin, but these isolates also were often resistant to amikacin and netilmicin as well. The comparative bactericidal activity of 5-episisomicin, gentamicin, and amikacin against Pseudomonas is shown in Fig. 2. When tested at the MIC of each agent, there was only a slight
Hm FIG. 2. Bactericidal activity of 5-episisomicin compared to amikacin and gentamicin against P. aeruginosa 3886. (A) Drugs added at the MIC value: control (0), amikacin (0), 5-episisomicin (0), gentamicin (X). (B) Drugs added at 2 x MIC. The MIC of amikacin is 3.1 pg/ml, that of gentamicin is 3.1 pg/ml, and that of 5episisomicin is 0.2 pg/ml. CFU, Colony-forming units.
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5-EPISISOMICIN
VOL. 14, 1978
TABLE 6. Activity of 5-episisomicin against organismns with known inactivating enzymt., MIC (MBC)a Organism Enzyme 5 ..sioi m5-Epcisim Gentamicin Sisomicin Netilmicin Tobramycin Kanamycin Amikacin
Klebsiella
ANT (2")
E. coli
ANT (2")
Klebsiella
APH (3') II
Serratia
APH (3') II
E. coli
APH (3') I
Providencia
AAC (2')
E. coli
AAC (6')
P. aeruginosa
AAC (3) II
0.4 (0.4) 0.1 (1.6) 0.2 (0.8) 0.1 (0.8) 1.6 (6.3) 0.8 (3.1) 12.5 (25) 25
12.5 (12.5) 12.5 (12.5) 0.4 (3.1) 0.4 (0.8) 25 (25) 100 (100) 1.6 (3.2) >100
(>100)
(>100)
Serratia None 25 a Data are in micrograms per milliliter.
>50
TABLE 7. Synergistic activity of 5-epiisomicin combined with carbenicillin Organism (no. of ioSoynergy (%) Partial synergy (%) lates) E. coli (32) 3 25 Klebsiella (31) 0 10 Enterobacter (31) 7 16 Serratia (32) 16 38 Providencia (15) 0 7 Acinetobacter (16) 0 44 P. aeruginosa (31) 20 77
difference in the activity of the agents. But when tested at two times the MIC, which for 5-episisomicin was at a concentration 16-fold less than those of amikacin and gentamicin, the most rapid killing occurred with 5-episisomicin. The activity of 5-episisomicin was compared with the activity of other aminoglycosides against isolates with known inactivating enzymes in Table 6. 5-Episisomicin was active against isolates with phosphorylating and adenylating enzymes and some bacteria with acetylating enzymes. Specifically, E. coli and K. pneumoniae with enzyme ANT(2"), which are resistant to gentamicin and tobramycin, were inhibited by 5-episisomicin. Kiebsiella, Serratia, and E. coli with APH(3') I and II and Providencia with AAC(2') were inhibited. The activity of 5-episisomicin against Pseudomonas and E. coli which contained AAC(6') and against Pseudomonas with AAC(3)-II was markedly reduced. 5-Episisomicin did not inhibit Serratia or Pseudomonas which were resistant to all other aminoglycosides due to a permeability barrier, al-
6.3 (6.3) 6.3 (25) 0.4 (0.4) 0.2 (0.2) 12.5 (25) 50 (100) >100
0.4 (0.8) 0.4 (0.8) 0.4 (0.4) 0.8 (0.8) 1.6 (1.6) 100 (100) 100 (100) >100
25 (25) 25 (100) 0.8 (0.8) 3.1 (6.3) >100 (>100) 50 (100) 100 (100) >50
(>100)
(>100)
(100)
>50
>50
>50
>100 (>100) 50 (100) >100 (>100) >100
(>100) >100 (>100) >100 (>100) >100
(>100) >50
0.8 (1.6) 0.4 (0.8) 3.1 (6.3) 0.8 (0.8) 6.3 (12.5) 12.5 (25) 50 (100) 0.8
(3.1)
>50
though it had an MIC of 25 ug/ml against such isolates that often had MICs greater than 400 ,ug/ml to the other aminoglycosides. 5-Episisomicin was combined with carbenicillin and tested against 188 isolates (Table 7). The drugs were combined with the highest concentration of carbenicillin, 100 ,Lg/ml, and the higher concentration of 5-episisomicin of 6.3 ,ug/ml. Serial twofold dilutions in a checkerboard fashion were tested. Complete synergy as defined by a fourfold reduction in the MICs of both agents was infrequently achieved against most of the Enterobacteriaceae tested. This may have been due to the low 5-episisomicin MICs against these bacteria and to their resistance to carbenicillin. However, 20% of Pseudomonas tested showed synergy, and 77% of the isolates showed partial synergy with a fourfold reduction in MIC of one of the agents. An example of MICs for such a Pseudomonas were 1.6,ug/ml (5-episisomicin) and 200 ,ug/ml (carbenicillin), and MICs of 0.8 ,tg/ml (5-episisomicin) and 50 ,Lg/ml (carbencillin) when the agents were combined. DISCUSSION 5-Episisomicin has been shown to have excellent in vitro activity against gram-negative organisms and against S. aureus. Similar results have recently been reported by Waitz et al. (10). Like other aminoglycosides, the activity of 5episisoricin was affected by factors such as pH of medium and cation content (5). 5-Episisomicin was equal in activity or more active than gentamicin against many members of the Enter-
200
FU AND NEU
obacteriaceae. Recent studies by Weinstein et al. (7) have indicated that there is more rapid uptake of sisomicin by many bacteria. 5-Episisomicin was the most active aminoglycoside tested against Providencia and indole-positive Proteus, and it was as active as tobramycin against Pseudomonas. Concentrations of 6.3 ,Ag/ml would be obtained with gentamicin, tobramycin, netilmicin, and sisomicin (thus probably also with 5-episisomicin) if the agents are administered intramuscularly or intravenously at a dose of 1.5 mg/kg. The half-life of these agents, is 2 h in normal individuals, and they are not protein bound. Thus, 5-episisomicin probably would be an effective agent in the treatment of infections due to gentamicin-sensitive organisms and also be effective against a number of organisms which have gentamicin MICs beyond that which is readily achieved clinically. It should be noted, however, that amikacin was the overall most active agent against gentamicin-resistant bacteria. 5-Episisomicin was active against many bacteria carrying adenylating and phosphorylating enzymes and against some bacteria with acetylating enzymes. Although it had the best activity of the agents tested against bacteria resistant by virtue of a permeability barrier, the concentrations could only be achieved in urine and not in blood. The recent studies by Waitz et al. (10) indicate that the in vitro observations concern-
ANTIMICROB. AGENTS CHEMOTHER.
ing 5-episisomicin are confirned in protection experiments in animals. However, it appears that 5-episisomicin is not less toxic in the experimental animal. LITERATURE CITED 1. Benveniste, R., and J. Davis. 1973. Mechanism of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471-506. 2. Dienstag, J., and U. C. Neu. 1972. In vitro studies of tobramycin, an aminoglycoside antibiotic. Antimicrob. Agents Chemother. 1:4145. 3. Fu, K. P., and H. C. Neu. 1976. In vitro synergistic effect of netilmicin, a new aminoglycoside antibiotic. Antimicrob. Agents Chemother. 10:511-518. 4. Fu, K. P., and H. C. Neu. 1976. In vitro study of netilmicin compared with other aminoglycosides. Antimicrob. Agents Chemother. 10:526-534. 5. Gilbert, D. N., E. Kutsher, P. Ireland, J. A. Barnett, and J. P. Sanford. 1971. Effect of the concentrations of magnesium and calcium on the in vitro susceptibility of Pseudomonas aeruginosa to gentamicin. J. Infect. Dis. 124(Suppl.):36-45. 6. Haas, M. J., and J. E. Dowding. 1975. Aminoglycosidemodifying enzymes. Methods Enzymol. 43:611-628. 7. Lee, B. K., R. G. Condon, H. Munayyer, and M. J. Weinstein. 1978. Uptake of methyl '4C-sisomicin and methyl '4C-gentamicin into bacterial cells. J. Antibiot. 31:141-146. 8. Neu, H. C. 1976. Tobramycin: an overview. J. Infect. Dis.
134(Suppl.):3-19. 9. Testa, R. H., G. H. Wagman, P. J. L Daniels, and M. J. Weinstein. 1974. Mutamicins: biosynthetically created new sisomicin analogues. J. Antibiot. 27:917-921. 10. Waitz, J. A., G. H. Miller, E. Moss, Jr., and P. J. S. Chiu. 1978. Chemotherapeutic evaluation of 5-episisomicin (Sch 22591), a new semisynthetic aminoglycoside. Antimicrob. Agents Chemother. 13:41-48.
0066-4804/78/0014-0194$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 14, No. 2
Printed in U.S.A.
Activity of 5-Episisomicin Compared with That of Other Aminoglycosides KWUNG P. FU AND HAROLD C. NEU* Division of Infectious Diseases, Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Received for publication 6 February 1978
5-Episisomicin, a semisynthetic aminoglycoside, has been shown to inhibit many members of the Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus. At a concentration of 3.1 ,ug/ml, more than 90% of Escherichia coli, Klebsiella pneumoniae, Enterobacter, Citrobacter, indole-positive Proteus, and Providencia were inhibited. 5-Episisomicin had activity against S. aureus, E. coli, Klebsiella, Enterobacter, and Citrobacter similar to gentamicin and netilmicin. It had activity similar to amikacin and netilmicin against many gentamicinresistant bacteria. The activity of 5-episisomicin was greatest at an alkaline pH and in medium of low magnesium content. Synergy of 5-episisomicin and carbenicillin was found to occur most often against Pseudomonas. The increasing incidence of gram-negative infections and particularly of gram-negative bacteremia due to bacteria resistant to the aminoglycosides currently in clinical use prompted us to evaluate the in vitro activity of 5-episisomicin, the 5-epimer of sisomicin (9). We compared the in vitro activity of 5-episisomicin with the in vitro activity of other aminoglycosides against clinical isolates. (This material was presented at the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, N.Y., 12-14 October 1977.)
MATERLALS AND METHODS Antibiotics. Gentamicin, sisomicin, netilmicin, and 5-episisomicin were supplied by Schering Corp. Kanamycin and amikacin were obtained from Bristol Laboratories, and carbenicillin was a gift from Beecham Laboratories. Bacterial isolates. The majority of bacterial isolates were obtained from patients hospitalized at the Columbia-Presbyterian Medical Centerduring the last 2 years. Only single isolates from a patient were used, and isolates felt to be from an outbreak were not used. Gentamicin-resistant isolates, listed in Tables 5 and 6 and used for the analysis of comparative activity of the aminoglycosides, had been collected over the past 7 years. Susceptibility tests. An inoculum of 105 colonyforming units was used except where specified. Minimal inhibitory concentrations (MICs) were determined by twofold dilution of the antibiotics in MuellerHinton agar (Baltimore Biological Laboratory [BBL]) or broth (BBL) by using a multiple-inoculating device as described in detail previously (4). The activity of 5-episisomicin against Bacteroides was deter-
mined by using an anaerobe gas pack jar (BBL) with Mueller-Hinton agar which contained sheep blood and vitamin K. All incubations were for 18 h at 35°C, with the exception of those against Bacteroides, which were for 18 h at 35°C. The MBC was the lowest concentration at which less than five colonies grew. The effect tubes into Columbia blood agar (BBL) and incubating for 18 h at 35°C. the MBC was the lowest concentration at which less than five colonies grew. The effect of growth medium upon the MICs and MBCs was determined with Trypticase soy (BBL), brain heart infusion (BBL), and nutrient broths (BBL). The effect of pH of the medium was determined by use of Mueller-Hinton broth adjusted with sodium hydroxide or hydrochloric acid. The concentration of sodium was less than that which would have affected the results of the assay. The effect of divalent cations on the MIC was determined by addition of magnesium sulfate to nutrient broth. Bacterial killing studies were carried out in Mueller-Hinton broth (BBL) or nutrient broth (BBL) by using organisms incubating in a gyratory shaker at 35°C, with samples of 0.1 ml removed for dilution in broth and plated on Mueller-Hinton agar at varying dilutions. Colony counts were read after 18 h of incubation at 35°C. The activity of 5-episisomicin was determined against isolates with known inactivating enzymes which had been given to us by various investigators in the United States and abroad or which we had earlier characterized through use of the techniques of Davies (1, 6) by using radioactive substrates. Synergy test8. Synergy was defined according to the following criteria: (i) a reduction of the MIC of both antibiotics of fourfold or more and (ii) a decrease of 1 log or more in viable cell count occurring at the end of 4 h with an antibiotic combination that contained the agents at one-fourth the MICs of the individual agents. Partial synergy was defined as a fourfold reduction in MICs of one agent and a twofold or no
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5-EPISISOMICIN
VOL. 14, 1978 reduction in the MIC of the other agent (3). The concentration of carbenicillin ranged from 400 to 1 Ag/ml, and that of 5-episisomicin ranged from 10 to 0.1 pLg/ml. These concentrations would be achieved by administration of 100 mg of carbenicillin per kg and 2 mg of 5-episisomicin per kg.
RESULTS Table 1 gives the overall activity of 5-episisomicin against 506 gram-positive and gram-negative isolates. 5-episisomicin inhibited 97% of Staphylococcus aureus at 0.8 ,ug/ml, but at the concentration of 6.3 ug/ml it inhibited only 22% of Streptococcus fecalis and S. fecium (true enterococci) and inhibited only 9% of S. pyogenes and S. agalactiae. Bacteroides fragilis was not inhibited by 5-episisomicin. By using 6.3 ,ug/ml as a cut-off point, 97% of Escherichia coli, 97% of Enterobacter, 96% of Citrobacter, and 100% of Shigella, Klebsiella, Proteus mirabilis, P. rettgeri, P. vulgaris, P. morganii, and Providencia were inhibited. Indeed, at 6.3 ug/ml 81% of Acinetobacter 87% of Pseudomonas aeruginosa, and 73% of Serratia marcescens were inhibited. It is noteworthy that 5-episisomicin at 1.6 ,ug/ml inhibited 90% of E. coli, 91% of Klebsiellapneumoniae, 58% of P. mirabilis, and 78% of P aeruginosa, the organisms which account for most episodes of gram-negative bacteremia. The effect of growth medium on the activity of 5-episisomicin was tested against organisms recently isolated from the blood or urine of hospitalized patients. Organisms were selected TABLE 1. Activity of 5-episisomicin against gramnegative and gram-positive microorganisms MIC (ug/ml) required to
inhibit:
Organism (no. of isolates) 50%
90%
S. aureus (34) 0.2 Enterococcia (30) 12.5 50 Streptococcib (32) E. coli (34) 0.8 Shigella (23) 3.1 K. pneumoniae (32) 0.4 Enterobacter (37) 0.4 S. marcescens (37) 3.1 Salmonella (22) 0.8 Citrobacter (24) 0.4 P. mirabilis 31) 1.6 P. rettgeri (15) 0.4 P. vulgaris (6) 1.6 P. morganii (26) 0.8 Providencia (29) 0.8 P. aeruginosa (46) 0.8 0.8 Acinetobacter (16) B. fragilis (32) 'S. fecalis and S. fecium. b Group A and B streptococci.
0.8 25
TABLE 2. Effects ofgrowth medium on the 5episisomicin MICs and MBCs Organism
E. coli 3939 Klebsiella 3929 P. rettgeri 3919 P. mirabilis 3378 S. marcescens 3915 P. aerugi-
MIC (MBC) in: Mueller-Hin- Brain heart inNutrient broth ton broth fusion
0.8 (3.1) 0.4 (0.4)
3.1 (3.1) 0.4 (0.4)
6.3 (6.3) 1.6 (3.1)
0.8 (3.1)
0.4 (0.4)
6.3 (6.3)
0.8 (6.3)
3.1 (6.3)
12.5 (12.5)
0.8 (250)
6.3 (250)
12.5 (250)
0.4 (1.6)
0.4 (0.4)
25 (250)
0.2 (1.6)
0.2 (1.6)
0.8 (3.1)
0.8 (250)
6.3 (250)
12.5 (250)
0.2 (0.8)
0.2 (0.2)
0.8 (6.3)
0.4 (3.1)
0.4 (0.4)
6.3 (12.5)
(0.8)
0.2 (0.2)
0.4 (1.6)
0.8 (25)
0.8 (0.8)
0.8 (250)
0.2 (0.8)
0.2 (0.4)
0.4 (0.8)
nosa
3696
P. aeruginosa
3886 P. aeruginosa
3901 P. aeruginosa
3902 P. aeruginosa
3903
P. aerugi-
50.1
nosa
3904 P. aeruginosa
3950
P. aeruginosa
3948
0.6 (3.1) 3.1 (6.3) 6.3 (6.3) P. maltaphilia 3941 a MICs and MBCs are in micrograms per milliliter.
TABLE 3. Effect ofpH upon the activity of 5episisomicina MIC (MBC)b at:
1.6
3.1 1.6 1.6 25 0.8 1.6 6.3 1.6 6.3 3.1 1.6 12.5 12.5 0
195
Organism
pH 7
pH 8
12.5 (25)
3.1 (6.3)
1.6 (3.1)
12.5 (250)
6.3 (250)
3.1 (250)
1.6 (1.6)
0.4 (0.4)
0.2 (0.4)
6.3 (6.3) 12.5 (250)
3.1 (3.1) 6.3 (250)
0.4 (0.8) 3.1 (250)
0.8 (1.6)
0.2 (0.2)
0.2 (0.4)
pH 6
P. mirabilis 3378 S. marcescens 3915 K. pneunoniae 3929 E. coli 3939 P. aeruginosa 3901 P. aeruginosa 3902
Assays
were performed in Mueller-Hinton broth. bMICs and MBCs are in micrograms per milliliter.
a
196
ANTIMICROB. AGENTS CHEMOTHER.
FU AND NEU
so as to be certain that they did not represent the same strain. Three media-were tested: nutrient broth, Mueller-Hinton broth, and brain heart infusion broth. The data are given in Table 2. There was minimal difference in either MICs or MBCs between nutrient broth and MuellerHinton broth, whereas, both MICs and MBCs often were higher in the brain heart infusion medium. The E. coli, Klebsiella, P. mirabilis, and P. rettgeri had MBCs which were miniimally greater than the MICs. Some Serratia (for example, isolate 3915 listed in Table 2) and some Pseudomonas (for example, 3901 and 3950) had an MBC which was 25 ,ug/ml or greater even tht)ugh the MIC was less than 1 ,tg/ml.
Table 3 shows that 5-episisomicin had greatest activity in a basic medium. It was 4- to 16-foldmore active at pH 8 than at pH 6. Nonetheless, a 16-fold greater MBC than MIC was noted with the Serratia and Pseudomonas tested at pH 8. The inhibitory activity of 5-episisomicin against Pseudomonas could be prevented by magnesium. Figure 1 shows the effect of adding varying concentrations of magnesium sulfate to an exponential-phase culture of Pseudomonas to which 5-episisomicin at two times the MIC had been added. In the presence of 0.5 mM MgSO4, there was a reduction in the killing rate, and in the presence of 2.5 mM MgSO4 there was a lag in growth but no reduction in colony-form-
9
S-
*1
N-
Houm FIG. 1. Effect of magnesium upon the bactericidal activity of 5-episisomicin against P. aeruginosa 3948. Magnesium sulfate was added at zero time to a log-phase culture. Symbols: 0, Control; El, control + 5 mM MgS04; *, 5-episisomicin (0.4 pg/ml); I, 5-episisomicin (0.4 pAg/ml) + 0.5 mM MgS04; A, 5-episisomicin (0.4 pg/ml) + 2.5 mM MgS04; A, 5-episisomicin (0.4 pg/ml) + 5 mM MgSO4. The MIC of 5-episisomicin is 0.2 pg/ml. CFU, Colony-forming units.
197
5-EPISISOMICIN
VOL. 14, 1978
TABLE 4. Comparative activity of 5 episieomicin with known aminoglycoside antibiotics MIC (gag/ml) requred for: of strair) (no.Organis
50% of strains
90% of strains
Gent Net Tobra Ami 5-Epi Gent Net Tobra S. aureus (34) 0.4 0.4 1.6 3.1 0.8 1.6 1.6 1.6 Enterococci (32) 12.5 6.3 12.5 50 25 25 12.5 25 E. coli (34) 0.8 0.8 0.8 1.6 25 1.6 K. pneumoniae (32) 0.4 0.4 0.8 3.1 25 0.8 Enterobacter (37) 0.8 0.8 1.6 1.6 1.6 0.8 S. marcescens (37) 3.1 50 12.5 25 >50 >50 Citrobacter (24) 0.4 0.4 0.8 1.6 1.6 1.6 Acinetobacter (16) 1.6 1.6 1.6 12.5 >50 >50 P. aeruginosa (39) 3.1 3.1 0.8 3.1 12.5 50 50 12.5 P. rettgeri (15) 6.3 6.3 0.8 3.1 25 25 P. morganu (26) 0.8 0.8 1.6 3.1 6.3 6.3 Providencia (29) 6.3 12.5 1.6 1.6 12.5 12.5 a 5-Epi, 5-Episisomicin; Gent, gentamicin; Net, netilmicin; Tobra, tobramycin; Ami, amikacin.
5-Epi 0.2 12.5 0.8 0.4 0.4 6.3 0.4 0.8 0.8 0.8 0.8 0.8
Ami
6.3 >50 1.6 3.1 3.1 25 1.6 25 25 6.3 12.5 3.1
TABLE 5. Comparative activity of 5-episisomicin against gentamicin-resistant organisms Medin MIC (range)a
Organism (no. of isolates)
S. aureus (1) E. coli (7)
Klebsiella (4) Enterobacter (2) Serratia (11)
Citrobacter (2) P. vulgaris (3) P. rettgeri (7) P. morganii (1) Providencia (5)
Acinetobacter (4) P. aeruginosa (13)
5-Episisomicin
Gentamicin
Netiiaicin
Amikacin
12.5 2.2 (0.4-12.5) 1.4 (0.2-6.3) (1.6-12.5) 8.3
>50 45 (12.5-250) 42.5
3.1 2.5 (0.8-250) 2.4 (0.4- .50) (1.6-25) 41.5 (25-250) (0.4-250) 25
6.3 1.1 (0.4-1.6) 1.2 (0.8-3.1) (1.6-3.1) 28.8 (6.3-250) (1.6-250) 3.1
(1.6--50) (0.4--50) 1.2 (0.4-1.6) 1.5 (0.4-3.1) 0.8 0.9 (0.8-1.6) 21 (0.8-25) 6.3
(25-2.50) (25-2.50) 41.5 (25-250) (250) 32.5
(12.5-2.50)
(12.5-2.50)
(1.6-6.3)
21.3 12.5-250) 25 20
13.8 (3.1-25) 25 20 (6.3-250) 32.5 (12.5-250) 21
2.9 (0.8-12.5) 1.6 2.1 (0.8-6.3) 9.5 (1.6-250) 17.5
(12.5-2.50) 32.5 (12.5->50) 30
(0.8-Dm50) (12.5-2s50) (1.6-e50) (1.6t50) aData are in micrograms per milliliter.
ing units. In the presence of 5 mM MgS04, the effect of 5-episisomicin was completely abolished. The comparative in vitro activity of 5-episisomicin and other aminoglycosides-amikacin, gentamicin, netilmicin, and tobramycin-is given in Table 4. Comprison to tobramycin is given only for S. aureus, enterococci and Pseudomonas because the tobramycin show little difference in activity against most other organisms (2, 8). Against S. aureus 5-episisomicin and netilnicin were the most active agents inhibiting more than 90% at concentrations below 0.8,ug/ml. Against enterococci at concentrations
of 3.1 ,ug/ml or less, 5-episisomicin, gentamicin, netilmicin, and tobramycin all had a similar low degree of activity. The activity of 5-episisomicin, amikacin, and netilmicin against the E. coli isolates were similar at concentrations below 3.1 ug/ml. However, amikacin inhibited all of the E. coli isolates at 3.1 ,ug/ml. Against Kiebsiella the four agents, 5episisomicin, gentamicin, netilmicin, and amikacin, inhibited 80% or more of isolates at concentrations of 1.6 pg/ml. Against Enterobacter and Citrobacter the compounds had similar activity. A major proportion of the S. marcescens isolates were resistant to all of the aminoglyco-
198
FU AND NEU
sides. But 5-episisomicin was the most active agent at concentrations of 3.1 to 12.5 ,jg/ml. 5Episisomicin, gentamicin, and amikacin had comparable activity against isolates of Acinetobacter, whereas netilmicin was less active than the other agents. Against P. aeruginosa tobramycin and 5-episisomicin were the agents which showed greatest activity at concentrations below 1.6 ,ug/ml. Tobramycin and 5-episisomicin were twofold more active than the other three agents. 5-episisomicin was the most active agent tested against P. rettgeri and Providencia, whereas, tested against P. morganii, all of the agents had similar inhibitory activity. The comparative activity of 5-episisomicin and gentamicin, netilmicin, and amikacin against gentamicin-resistant isolates is shown in
ANTIMICROB. AGENTS CHEMOTHER.
Table 5. The isolates came from patients at the Columbia-Presbyterian Medical Center and from affiliated hospitals. Most of the gentamicinresistant E. coli, Klebsiella, indole-positive Proteus, and Providencia were inhibited by 5-episisomicin. 5-Episisomicin inhibited most of the gentamicin-resistant Pseudomonas, but there
were isolates resistant to it and to the other aminoglycosides as well. Many of the gentamicin-resistant Serratia and Acinetobacter were resistant to 5-episisomicin, but these isolates also were often resistant to amikacin and netilmicin as well. The comparative bactericidal activity of 5-episisomicin, gentamicin, and amikacin against Pseudomonas is shown in Fig. 2. When tested at the MIC of each agent, there was only a slight
Hm FIG. 2. Bactericidal activity of 5-episisomicin compared to amikacin and gentamicin against P. aeruginosa 3886. (A) Drugs added at the MIC value: control (0), amikacin (0), 5-episisomicin (0), gentamicin (X). (B) Drugs added at 2 x MIC. The MIC of amikacin is 3.1 pg/ml, that of gentamicin is 3.1 pg/ml, and that of 5episisomicin is 0.2 pg/ml. CFU, Colony-forming units.
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5-EPISISOMICIN
VOL. 14, 1978
TABLE 6. Activity of 5-episisomicin against organismns with known inactivating enzymt., MIC (MBC)a Organism Enzyme 5 ..sioi m5-Epcisim Gentamicin Sisomicin Netilmicin Tobramycin Kanamycin Amikacin
Klebsiella
ANT (2")
E. coli
ANT (2")
Klebsiella
APH (3') II
Serratia
APH (3') II
E. coli
APH (3') I
Providencia
AAC (2')
E. coli
AAC (6')
P. aeruginosa
AAC (3) II
0.4 (0.4) 0.1 (1.6) 0.2 (0.8) 0.1 (0.8) 1.6 (6.3) 0.8 (3.1) 12.5 (25) 25
12.5 (12.5) 12.5 (12.5) 0.4 (3.1) 0.4 (0.8) 25 (25) 100 (100) 1.6 (3.2) >100
(>100)
(>100)
Serratia None 25 a Data are in micrograms per milliliter.
>50
TABLE 7. Synergistic activity of 5-epiisomicin combined with carbenicillin Organism (no. of ioSoynergy (%) Partial synergy (%) lates) E. coli (32) 3 25 Klebsiella (31) 0 10 Enterobacter (31) 7 16 Serratia (32) 16 38 Providencia (15) 0 7 Acinetobacter (16) 0 44 P. aeruginosa (31) 20 77
difference in the activity of the agents. But when tested at two times the MIC, which for 5-episisomicin was at a concentration 16-fold less than those of amikacin and gentamicin, the most rapid killing occurred with 5-episisomicin. The activity of 5-episisomicin was compared with the activity of other aminoglycosides against isolates with known inactivating enzymes in Table 6. 5-Episisomicin was active against isolates with phosphorylating and adenylating enzymes and some bacteria with acetylating enzymes. Specifically, E. coli and K. pneumoniae with enzyme ANT(2"), which are resistant to gentamicin and tobramycin, were inhibited by 5-episisomicin. Kiebsiella, Serratia, and E. coli with APH(3') I and II and Providencia with AAC(2') were inhibited. The activity of 5-episisomicin against Pseudomonas and E. coli which contained AAC(6') and against Pseudomonas with AAC(3)-II was markedly reduced. 5-Episisomicin did not inhibit Serratia or Pseudomonas which were resistant to all other aminoglycosides due to a permeability barrier, al-
6.3 (6.3) 6.3 (25) 0.4 (0.4) 0.2 (0.2) 12.5 (25) 50 (100) >100
0.4 (0.8) 0.4 (0.8) 0.4 (0.4) 0.8 (0.8) 1.6 (1.6) 100 (100) 100 (100) >100
25 (25) 25 (100) 0.8 (0.8) 3.1 (6.3) >100 (>100) 50 (100) 100 (100) >50
(>100)
(>100)
(100)
>50
>50
>50
>100 (>100) 50 (100) >100 (>100) >100
(>100) >100 (>100) >100 (>100) >100
(>100) >50
0.8 (1.6) 0.4 (0.8) 3.1 (6.3) 0.8 (0.8) 6.3 (12.5) 12.5 (25) 50 (100) 0.8
(3.1)
>50
though it had an MIC of 25 ug/ml against such isolates that often had MICs greater than 400 ,ug/ml to the other aminoglycosides. 5-Episisomicin was combined with carbenicillin and tested against 188 isolates (Table 7). The drugs were combined with the highest concentration of carbenicillin, 100 ,Lg/ml, and the higher concentration of 5-episisomicin of 6.3 ,ug/ml. Serial twofold dilutions in a checkerboard fashion were tested. Complete synergy as defined by a fourfold reduction in the MICs of both agents was infrequently achieved against most of the Enterobacteriaceae tested. This may have been due to the low 5-episisomicin MICs against these bacteria and to their resistance to carbenicillin. However, 20% of Pseudomonas tested showed synergy, and 77% of the isolates showed partial synergy with a fourfold reduction in MIC of one of the agents. An example of MICs for such a Pseudomonas were 1.6,ug/ml (5-episisomicin) and 200 ,ug/ml (carbenicillin), and MICs of 0.8 ,tg/ml (5-episisomicin) and 50 ,Lg/ml (carbencillin) when the agents were combined. DISCUSSION 5-Episisomicin has been shown to have excellent in vitro activity against gram-negative organisms and against S. aureus. Similar results have recently been reported by Waitz et al. (10). Like other aminoglycosides, the activity of 5episisoricin was affected by factors such as pH of medium and cation content (5). 5-Episisomicin was equal in activity or more active than gentamicin against many members of the Enter-
200
FU AND NEU
obacteriaceae. Recent studies by Weinstein et al. (7) have indicated that there is more rapid uptake of sisomicin by many bacteria. 5-Episisomicin was the most active aminoglycoside tested against Providencia and indole-positive Proteus, and it was as active as tobramycin against Pseudomonas. Concentrations of 6.3 ,Ag/ml would be obtained with gentamicin, tobramycin, netilmicin, and sisomicin (thus probably also with 5-episisomicin) if the agents are administered intramuscularly or intravenously at a dose of 1.5 mg/kg. The half-life of these agents, is 2 h in normal individuals, and they are not protein bound. Thus, 5-episisomicin probably would be an effective agent in the treatment of infections due to gentamicin-sensitive organisms and also be effective against a number of organisms which have gentamicin MICs beyond that which is readily achieved clinically. It should be noted, however, that amikacin was the overall most active agent against gentamicin-resistant bacteria. 5-Episisomicin was active against many bacteria carrying adenylating and phosphorylating enzymes and against some bacteria with acetylating enzymes. Although it had the best activity of the agents tested against bacteria resistant by virtue of a permeability barrier, the concentrations could only be achieved in urine and not in blood. The recent studies by Waitz et al. (10) indicate that the in vitro observations concern-
ANTIMICROB. AGENTS CHEMOTHER.
ing 5-episisomicin are confirned in protection experiments in animals. However, it appears that 5-episisomicin is not less toxic in the experimental animal. LITERATURE CITED 1. Benveniste, R., and J. Davis. 1973. Mechanism of antibiotic resistance in bacteria. Annu. Rev. Biochem. 42:471-506. 2. Dienstag, J., and U. C. Neu. 1972. In vitro studies of tobramycin, an aminoglycoside antibiotic. Antimicrob. Agents Chemother. 1:4145. 3. Fu, K. P., and H. C. Neu. 1976. In vitro synergistic effect of netilmicin, a new aminoglycoside antibiotic. Antimicrob. Agents Chemother. 10:511-518. 4. Fu, K. P., and H. C. Neu. 1976. In vitro study of netilmicin compared with other aminoglycosides. Antimicrob. Agents Chemother. 10:526-534. 5. Gilbert, D. N., E. Kutsher, P. Ireland, J. A. Barnett, and J. P. Sanford. 1971. Effect of the concentrations of magnesium and calcium on the in vitro susceptibility of Pseudomonas aeruginosa to gentamicin. J. Infect. Dis. 124(Suppl.):36-45. 6. Haas, M. J., and J. E. Dowding. 1975. Aminoglycosidemodifying enzymes. Methods Enzymol. 43:611-628. 7. Lee, B. K., R. G. Condon, H. Munayyer, and M. J. Weinstein. 1978. Uptake of methyl '4C-sisomicin and methyl '4C-gentamicin into bacterial cells. J. Antibiot. 31:141-146. 8. Neu, H. C. 1976. Tobramycin: an overview. J. Infect. Dis.
134(Suppl.):3-19. 9. Testa, R. H., G. H. Wagman, P. J. L Daniels, and M. J. Weinstein. 1974. Mutamicins: biosynthetically created new sisomicin analogues. J. Antibiot. 27:917-921. 10. Waitz, J. A., G. H. Miller, E. Moss, Jr., and P. J. S. Chiu. 1978. Chemotherapeutic evaluation of 5-episisomicin (Sch 22591), a new semisynthetic aminoglycoside. Antimicrob. Agents Chemother. 13:41-48.
