ANTImiCROBIAL AGUNTS AND CHzMOTHBRAPY, Dec. 1976, p. 889-892 Copyright C) 1976 American Society for Microbiology
Vol. 10, No. 6 Printed in U.S.A.
Antibiotic Synergy and Antagonism Against Clinical Isolates of Klebsiella Species ROBERT M. D'ALESSANDRI, DAVID J. McNEELY, AND RONICA M. KLUGE* Division of Infectious Diseases, Department of Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610 Received for publication 7 July 1976
Minimal inhibitory concentrations of kanamycin, gentamicin, amikacin, cephalothin, and chloramphenicol were determined in Trypticase soy broth for 70 clinical isolates ofKlebsiella species. Gentamicin and amikacin were the most active on a weight basis. Chloramphenicol was more active than kanamycin, and cephalothin was the least active of all. Studies using a microtiter modification of the checkerboard technique were performed to evaluate the comparative activity of the three aminoglycosides in combination with either chloramphenicol or cephalothin. The cephalothin-aminoglycoside combinations demonstrated synergy in >80% of the isolates tested. No antagonism was noted. The chloramphenicol-aminoglycoside combinations showed antagonism in 35 to 45% of the isolates tested. The data suggest that the chloramphenicol-aminoglycoside combinations be used with caution when treating serious infections where Klebsiella is a potential pathogen.
The combination of a cephalosporin and an aminoglycoside antibiotic is considered the treatment of choice for serious Klebsiella infections (3, 11). This is based on the finding of antimicrobial synergy in vitro and the suggestion that combined antibiotic therapy retards the development of resistance to either antibiotic (2). In patients allergic to the cephalosporins, or in patients with polymicrobial bacteremia in whom both Enterobacteriaceae and anaerobes are potential pathogens, the combination of chloramphenicol and an aminoglycoside antibiotic is often recommended. There is some in vitro evidence to indicate that such a combination may have an antagonistic effect (2, 4, 8). Kanamycin and gentamicin are the most commonly used aminoglycosides in clinical practice today. Amikacin, an investigational aminoglycoside, is pharmacologically related to kanamycin. In a recent report (H. Standiford, B. Tatum, and F. Calia, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 263, 1975), amikacin in clinically achievable levels was found to be more active in vitro against Klebsiella strains than gentamicin. In the present study the comparative activity of kanamycin, gentamicin, amikacin, chloramphenicol, and cephalothin against Klebsiella isolates was evaluated. In addition, the activity of each aminoglycoside in combination with either chloramphenicol or cephalothin was determined.
MATERIALS AND METHODS Seventy clinical isolates ofKlebsiella species were obtained from the Veterans Administration Hospital and the Shands Teaching Hospital, Gainesville, Fla. Isolates were maintained on tryptic soy agar at 4°C until studied. Solutions of each antibiotic were prepared in concentrations of 80 ,g/ml. The solutions were divided into 4-ml portions and stored at -20°C. Fresh solutions were prepared monthly. All susceptibility testing was performed in tryptic soy broth (TSB). The concentration of calcium in the TSB ranged between 3.4 and 4.1 mg/100 ml; the magnesium concentration was between 1.2 and 1.3 mg/100 ml. The minimal inhibitory concentration (MIC) of each drug alone was measured by the standard twofold tube dilution method. The MICs of the combination of each aminoglycoside with chloramphenicol and each aminoglycoside with cephalothin were measured using a microtiter modification of the checkerboard technique (7). The final concentration of antibiotic ranged from 40 to 0.5 ,ug/ml, and the inoculum consisted of a 10-3 dilution of a 4-h culture. Pour plates were made to determine the exact inoculum. MIC results were considered valid when the inoculum fell between 105 and 5 x 105 colony-forming units. The character of the antibiotic interaction was determined by plotting an isobologram (Fig. 1) for each microtiter plate (10). The concentration of aminoglycoside formed the ordinate, and the concentration of either cephalothin or chloramphenicol formed the abscissa. The MICs of each drug alone and of the combinations as read from the microtiter plate were plotted on the isobologram. If the points fell on a straight line joining the aminoglycoside 889
890
D'ALESSANDRI, McNEELY, AND KLUGE
MIC to the cephalothin or chloramphenicol MIC, an additive interaction was recorded. If the curve bowed upward, antagonism was recorded, whereas if the curve bowed downward toward the coordinates, synergy was recorded.
RESULTS A comparison of the MICs of kanamycin, gentamicin, and amikacin against 70 Klebsiella isolates is seen in Table 1. Fifty-six isolates were sensitive to 10 ,ug or less ofkanamycin per ml, a clinically achievable level. Twelve isolates were moderately resi3tant, requiring 20 ,ug/ml. Only two isolates were resistant to more than 40 ,ug of kanamycin per ml. All 70 isolates were sensitive to 5 pug or less of gentamicin per ml. Sixty-eight of the 70 isolates were sensitive to 10 ,ug or less of amikacin per ml. One isolate was moderately resistant, with an MIC of 20 ,ug/ml, and one isolate was resistant with an MIC of 40 yg of amikacin per ml. On a weight basis gentamicin was the most active of the three aminoglycosides and kanamycin was the least active. Sixty-eight of the isolates were sensitive to 20 ,tg or less of chloramphenicol per
10.0 1.25 Z5 SO CEPHALOTHIN OR CHLORAMPIIHENICOL CONCENTRATION (mcg/mI)
O
FIG. 1. Isobologram with three isobols representing additive effect (A), synergy (B), and antagonism (C).
ANTnacROB. AGZNTS CHZMOTHZR.
ml,
an
easily achieved
serum level. more than
isolates were resistant to
Only two 40 ,ug/ml.
Only 49 isolates were sensitive to 20 jAg or less of cephalothin per ml, a clinically achievable level. However, 15 isolates were resistant to more than 40 ,ug/ml, and 6 isolates with an MIC of 40 ,ug/ml were moderately resistant. The results of combining cephalothin with each of the three aminoglycosides are seen in Table 2. The combination of cephalothin and kanamycin was synergistic in 32 of 35 isolates (92%). For the remainder of the isolates an additive effect was noted. Cephalothin with gentamicin was synergistic in 36 of 38 isolates (95%). Cephalothin and amikacin showed synergy in 26 of 38 isolates (68%). The cephalothingentamicin combination was synergistic for a' statistically greater number of isolates than was the cephalothin-amikacin combination (P < 0.01). The statistical significance was similar when comparing the cephalothin-kanamicin and cephalothin-amikacin combinations. None of the cephalothin-aminoglycoside combinations were antagonistic. In Table 3 are the results of testing the combination of chloramphenicol with each of the three aminoglycosides against Klebsiella isolates. Chloramphenicol with kanamycin and chloramphenicol with amikacin were synergistic in 14 of 39 and 13 of 40 isolates, respectively. Chloramphenicol with gentamicin was synergistic in 20 of 39 isolates. This difference in synergy between chloramphenicol with kanamycin or amikacin and chloramphenicol with gentamicin was not statistically significant (P > 0.10). It is important to note that each combination was antagonistic against 35 to 45% of the isolates tested. Synergy was demonstrated for 47 of 118 combinations using the aminoglycosides with chloramphenicol; another 47 of these 118 combinations showed antagonism. In the aminoglycosides with cephalothin group, synergy was noted with 94 of 111 combinations and no antagonism was shown. The difference in synergy between the two groups was statistically significant (P < 0.001). The difference in
TABLE 1. MICs of aminoglycosides, chloramphenicol, and cephalothin against 70 clinical isolates of Klebsiella pneumoniae Antimicrobial agent
Gentamicin Amikacin Kanamycin Chloramphenicol
No. of strains inhibited
40.0
(100) (99) (97) (79)
2 (100) 2 (100) 15 (100)
ANTIBIOTIC SYNERGY IN KLEBSIELLA
VOL. 10, 1976
891
TABLE 2. Cephalothin-aminoglycoside combination No. of isolates tested Cephalothin with.-
Additive
Kanamycin Gentamicin Amikacin Total
3 2 12 17
(8.5) (5.2) (31.5) (15.3)
Synergistic
Antagonistic
Total
(91.5) (94.8) (68.5) (84.7)
0 0 0 0
35 38 38 111
32 36 26 94
TABLE 3. Chloramphenicol-aminoglycoside combination No. of isolates tested
Chloramphenicol with:
(
Kanamycin Gentamicin Amikacin Total
10 5 9 24
Antagonistic ( 15 (38.4) 14 (35.8) 18 (45.0) 47 (39.9)
Synergistic
Additive
(
(25.6) (12.8) (22.5) (20.2)
14 20 13 47
(35.9) (51.4) (32.5) (39.9)
Total
39 39 40 118
TABLE 4. Comparison of MIC offive antibiotics against Klebsiella isolates in TSB and MHB MIC (jtglml) Organism
STH 14 STH 32 VAH 44 VAH 49 STH 68
Gentamicin
Amikacin
Kanamycin
Chloramphenicol
Cephalothin
TSB
MHB
TSB
MHB
TSB
MHB
TSB
MHB
TSB
MHB
0.625 1.25 2.50 5.00 0.625
0.156 0.156 0.625 0.313 0.313
1.25 1.25 2.50 10.0 5.00
0.313 0.313 0.625 1.25 0.313
5.00 5.00 20.0 20.0 5.00
0.156 1.25 10.0 2.5 0.313
2.50 5.00 5.00 2.50 2.50
1.25 2.50 5.00 2.50 1.25
5.00 10.0 40.0 10.0 40.0
1.25 2.50 40.0 5.00 40.0
antagonism was also statistically significant at the same level.
ity in various media are the pH of the growth medium and the electrolyte content. TSB contains added dextrose (250 mg/ml), and this is, of course, fermentable by the Klebsiella with a DISCUSSION resultant fall in pH during growth. The net Gentamicin was the most active aminoglyco- result is a diminution in the aminoglycoside side in this system, followed by amikacin and effectiveness (1). TSB has a generally higher kanamycin against 70 clinical isolates of Kleb- cation content than MHB or brain heart infusiella species. This hierarchy of in vitro activity sion broth (12), and this increased cation conagainst Klebsiella has been reported previously tent (particularly calcium and magnesium) has (1). In terms of clinically achievable serum an- been shown to adversely affect in vitro aminotimicrobial levels, gentamicin, amikacin, and glycoside activity (13). chloramphenicol have greater in vitro activity To determine if our Klebsiella isolates folthan kanamycin or cephalothin. Cephalothin lowed this pattern, we performed simultaneous was the least active, with only 68% of isolates twofold tube dilution MICs of gentamicin, amisensitive to 20 ,ug/ml or less. kacin, kanamycin, chloramphenicol, and cephA partial explanation for the relatively high alothin in TSB and MHB for 5 of our 70 KlebsiMICs of kanamycin and amikacin in this sys- ella isolates (see Table 4). There was an avertem may be the use of TSB as the test medium. age fourfold drop in the aminoglycoside MIC It has been shown that aminoglycoside MICs when MHB was used for susceptibility testing, against different Enter6bacteriaceae, including but the relative order of activity againstKlebsiKlebsiella, are four- to sixfold higher in TSB ella was similar to that determined in TSB (i.e., than in nutrient broth or Mueller-Hinton broth gentamicin > amikacin > kanamycin). No dif(MHB) (1, 2). The exact mechanism for this is ference in chloramphenicol MIC was apparent, only partially understood. Two parameters that but a trend to a lower MIC of cephalothin in have been shown to affect aminoglycoside activ- MHB was demonstrated.
ANTIMICROB. AGENTs CHZMOTHZR. 892 D'ALESSANDRI, McNEELY, AND KLUGE LITERATURE CITED To further delineate the media effect, if any, on the synergy results, microtiter synergy studG. P., and D. Stewart. 1973. In vitro effectiveies were repeated in MHB for two of the five 1. Bodey, ness of BB-K8, a new aminoglycoside antibiotic. Anorganisms surveyed in Table 4 (STH 32 and timicrob. Agents Chemother. 4:182-192. VAH 49). Whereas the absolute values for the 2. Bulger, R. J. 1967. In vitro effectiveness of kanamycin and kanamycin/cephalothin against klebsiella. Ann. combination MICs were reduced two- to fourIntern. Med. 67:523-532. fold, the isobologram curves denoting synergy W. J., and W. A. Taylor. 1973. Gram-negaHolloway, 3. or antagonism were superimposable in 11 of the tive rods, p. 104-106. In Sepsis. Futura Publishing 12 plates when compared to the corresponding Co., Mt. Kisco, N.Y. TSB synergy plate results. 4. Jawetz, E., and J. B. Gunnison. 1952. Studies on antibiotic synergism and antagonism: a scheme of comIn summary, synergy was demonstrated bined antibiotic action. Antibiot. Chemother. 2:243most commonly with the cephalothin-gertami248. cin and cephalothin-kanamycin combinations. 5. Klastersky, J., R. Cappel, and D. Daneau. 1972. CliniCephalothin-amikacin also exhibited synergy cal significance of in vitro synergism between antithat was significantly greater than the respecbiotics in gram-negative infections. Antimicrob. Agents Chemother. 2:470-475. tive aminoglycoside-chloramphenicol combinaJ., R. Cappel, G. Swings, and L. Vandention. None of the cephalothin-aminoglycoside 6. Klastersky, 1971. Bacteriological and clinical activity of borre. combinations showed antagonism as opposed to the ampicillin/gentamicin and cephalothin/gentami35 to 45% of the chloramphenicol-aminoglycocin combinatioAs. Am. J. Med. Sci. 262:283-290. side combinations. In comparing the MIC data 7. Kluge, R. M., H. C. Standiford, B. Tatum, V. M. Young, W. H. Greene, S. C. Schimpff, F. M. Calia, with the checkerboard results, there was no R. B. Hornick. 1974. Comparative activity of and with which one consistent sensitivity pattern tobramycin, amikacin, and gentamicin alone and could predict whether or not the combinations with carbenicillin against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6:442-446. ofantibiotics would be synergistic or antagonis8. Luboshitzky, R., T. Sacks, and J. Michel. 1973. Bactetic. ricidal effect of combinations of antibiotics on klebsiThe clinical importance of in vitro synergy or ella-enterobacter-serratia. Chemotherapy 19:354antagonism is uncertain in the absence of an in 366. vivo model or a clinical trial. Until such data 9. Martin, C. M., A. J. Cuomo, M. J. Geraghty, J. R. Zager, and T. C. Mandes. 1969. Gram negative rod are available, the finding of significant antagoJ. Infect. Dis. 119:506-517. bacteremia. nism using the chloramphenicol-aminoglyco- 10. Sabath, L. D. 1968. Synergy ofantibacterial substances side combinations mitigates against the indisby apparently known mechanisms, p. 210-217. Antimicrob. Agents Chemother. 1967. criminate use of this antibiotic combination in J. P. 1975. Klebsiella and other gram-negative situations in which Klebsiella is a potential 11. Sanford, pneumonias, p. 286-28a. In P. B. Beeson bacterial in 80% of The over synergy finding of pathogen. and W. McDermott (ed.), Textbook of medicine, W. B. the cephalothin-aminoglycoside combinations Saunders Co., Philadelphia. tested suggests that this combination be used in 12. Warren, E., R. J. Snyder, and J. A. Washington, II. 1972. Four-hour microbiological assay of gentamicin therapy of serious Klebsiella infections. In adin serum. Antimicrob. Agents Chemother. 1:46-48. dition, there is some clinical evidence to sup- 13. Washington, J. A., II, R. E. Ritts, Jr., and W. J. port the use of these combinations in serious Martin. 1970. In vitro susceptibility of gram-negative bacilli to gentamicin. Mayo Clin. Proc. 45:146-149. gram-negative infections (5, 6, 9).
Vol. 10, No. 6 Printed in U.S.A.
Antibiotic Synergy and Antagonism Against Clinical Isolates of Klebsiella Species ROBERT M. D'ALESSANDRI, DAVID J. McNEELY, AND RONICA M. KLUGE* Division of Infectious Diseases, Department of Medicine, College of Medicine, University of Florida, Gainesville, Florida 32610 Received for publication 7 July 1976
Minimal inhibitory concentrations of kanamycin, gentamicin, amikacin, cephalothin, and chloramphenicol were determined in Trypticase soy broth for 70 clinical isolates ofKlebsiella species. Gentamicin and amikacin were the most active on a weight basis. Chloramphenicol was more active than kanamycin, and cephalothin was the least active of all. Studies using a microtiter modification of the checkerboard technique were performed to evaluate the comparative activity of the three aminoglycosides in combination with either chloramphenicol or cephalothin. The cephalothin-aminoglycoside combinations demonstrated synergy in >80% of the isolates tested. No antagonism was noted. The chloramphenicol-aminoglycoside combinations showed antagonism in 35 to 45% of the isolates tested. The data suggest that the chloramphenicol-aminoglycoside combinations be used with caution when treating serious infections where Klebsiella is a potential pathogen.
The combination of a cephalosporin and an aminoglycoside antibiotic is considered the treatment of choice for serious Klebsiella infections (3, 11). This is based on the finding of antimicrobial synergy in vitro and the suggestion that combined antibiotic therapy retards the development of resistance to either antibiotic (2). In patients allergic to the cephalosporins, or in patients with polymicrobial bacteremia in whom both Enterobacteriaceae and anaerobes are potential pathogens, the combination of chloramphenicol and an aminoglycoside antibiotic is often recommended. There is some in vitro evidence to indicate that such a combination may have an antagonistic effect (2, 4, 8). Kanamycin and gentamicin are the most commonly used aminoglycosides in clinical practice today. Amikacin, an investigational aminoglycoside, is pharmacologically related to kanamycin. In a recent report (H. Standiford, B. Tatum, and F. Calia, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 263, 1975), amikacin in clinically achievable levels was found to be more active in vitro against Klebsiella strains than gentamicin. In the present study the comparative activity of kanamycin, gentamicin, amikacin, chloramphenicol, and cephalothin against Klebsiella isolates was evaluated. In addition, the activity of each aminoglycoside in combination with either chloramphenicol or cephalothin was determined.
MATERIALS AND METHODS Seventy clinical isolates ofKlebsiella species were obtained from the Veterans Administration Hospital and the Shands Teaching Hospital, Gainesville, Fla. Isolates were maintained on tryptic soy agar at 4°C until studied. Solutions of each antibiotic were prepared in concentrations of 80 ,g/ml. The solutions were divided into 4-ml portions and stored at -20°C. Fresh solutions were prepared monthly. All susceptibility testing was performed in tryptic soy broth (TSB). The concentration of calcium in the TSB ranged between 3.4 and 4.1 mg/100 ml; the magnesium concentration was between 1.2 and 1.3 mg/100 ml. The minimal inhibitory concentration (MIC) of each drug alone was measured by the standard twofold tube dilution method. The MICs of the combination of each aminoglycoside with chloramphenicol and each aminoglycoside with cephalothin were measured using a microtiter modification of the checkerboard technique (7). The final concentration of antibiotic ranged from 40 to 0.5 ,ug/ml, and the inoculum consisted of a 10-3 dilution of a 4-h culture. Pour plates were made to determine the exact inoculum. MIC results were considered valid when the inoculum fell between 105 and 5 x 105 colony-forming units. The character of the antibiotic interaction was determined by plotting an isobologram (Fig. 1) for each microtiter plate (10). The concentration of aminoglycoside formed the ordinate, and the concentration of either cephalothin or chloramphenicol formed the abscissa. The MICs of each drug alone and of the combinations as read from the microtiter plate were plotted on the isobologram. If the points fell on a straight line joining the aminoglycoside 889
890
D'ALESSANDRI, McNEELY, AND KLUGE
MIC to the cephalothin or chloramphenicol MIC, an additive interaction was recorded. If the curve bowed upward, antagonism was recorded, whereas if the curve bowed downward toward the coordinates, synergy was recorded.
RESULTS A comparison of the MICs of kanamycin, gentamicin, and amikacin against 70 Klebsiella isolates is seen in Table 1. Fifty-six isolates were sensitive to 10 ,ug or less ofkanamycin per ml, a clinically achievable level. Twelve isolates were moderately resi3tant, requiring 20 ,ug/ml. Only two isolates were resistant to more than 40 ,ug of kanamycin per ml. All 70 isolates were sensitive to 5 pug or less of gentamicin per ml. Sixty-eight of the 70 isolates were sensitive to 10 ,ug or less of amikacin per ml. One isolate was moderately resistant, with an MIC of 20 ,ug/ml, and one isolate was resistant with an MIC of 40 yg of amikacin per ml. On a weight basis gentamicin was the most active of the three aminoglycosides and kanamycin was the least active. Sixty-eight of the isolates were sensitive to 20 ,tg or less of chloramphenicol per
10.0 1.25 Z5 SO CEPHALOTHIN OR CHLORAMPIIHENICOL CONCENTRATION (mcg/mI)
O
FIG. 1. Isobologram with three isobols representing additive effect (A), synergy (B), and antagonism (C).
ANTnacROB. AGZNTS CHZMOTHZR.
ml,
an
easily achieved
serum level. more than
isolates were resistant to
Only two 40 ,ug/ml.
Only 49 isolates were sensitive to 20 jAg or less of cephalothin per ml, a clinically achievable level. However, 15 isolates were resistant to more than 40 ,ug/ml, and 6 isolates with an MIC of 40 ,ug/ml were moderately resistant. The results of combining cephalothin with each of the three aminoglycosides are seen in Table 2. The combination of cephalothin and kanamycin was synergistic in 32 of 35 isolates (92%). For the remainder of the isolates an additive effect was noted. Cephalothin with gentamicin was synergistic in 36 of 38 isolates (95%). Cephalothin and amikacin showed synergy in 26 of 38 isolates (68%). The cephalothingentamicin combination was synergistic for a' statistically greater number of isolates than was the cephalothin-amikacin combination (P < 0.01). The statistical significance was similar when comparing the cephalothin-kanamicin and cephalothin-amikacin combinations. None of the cephalothin-aminoglycoside combinations were antagonistic. In Table 3 are the results of testing the combination of chloramphenicol with each of the three aminoglycosides against Klebsiella isolates. Chloramphenicol with kanamycin and chloramphenicol with amikacin were synergistic in 14 of 39 and 13 of 40 isolates, respectively. Chloramphenicol with gentamicin was synergistic in 20 of 39 isolates. This difference in synergy between chloramphenicol with kanamycin or amikacin and chloramphenicol with gentamicin was not statistically significant (P > 0.10). It is important to note that each combination was antagonistic against 35 to 45% of the isolates tested. Synergy was demonstrated for 47 of 118 combinations using the aminoglycosides with chloramphenicol; another 47 of these 118 combinations showed antagonism. In the aminoglycosides with cephalothin group, synergy was noted with 94 of 111 combinations and no antagonism was shown. The difference in synergy between the two groups was statistically significant (P < 0.001). The difference in
TABLE 1. MICs of aminoglycosides, chloramphenicol, and cephalothin against 70 clinical isolates of Klebsiella pneumoniae Antimicrobial agent
Gentamicin Amikacin Kanamycin Chloramphenicol
No. of strains inhibited
40.0
(100) (99) (97) (79)
2 (100) 2 (100) 15 (100)
ANTIBIOTIC SYNERGY IN KLEBSIELLA
VOL. 10, 1976
891
TABLE 2. Cephalothin-aminoglycoside combination No. of isolates tested Cephalothin with.-
Additive
Kanamycin Gentamicin Amikacin Total
3 2 12 17
(8.5) (5.2) (31.5) (15.3)
Synergistic
Antagonistic
Total
(91.5) (94.8) (68.5) (84.7)
0 0 0 0
35 38 38 111
32 36 26 94
TABLE 3. Chloramphenicol-aminoglycoside combination No. of isolates tested
Chloramphenicol with:
(
Kanamycin Gentamicin Amikacin Total
10 5 9 24
Antagonistic ( 15 (38.4) 14 (35.8) 18 (45.0) 47 (39.9)
Synergistic
Additive
(
(25.6) (12.8) (22.5) (20.2)
14 20 13 47
(35.9) (51.4) (32.5) (39.9)
Total
39 39 40 118
TABLE 4. Comparison of MIC offive antibiotics against Klebsiella isolates in TSB and MHB MIC (jtglml) Organism
STH 14 STH 32 VAH 44 VAH 49 STH 68
Gentamicin
Amikacin
Kanamycin
Chloramphenicol
Cephalothin
TSB
MHB
TSB
MHB
TSB
MHB
TSB
MHB
TSB
MHB
0.625 1.25 2.50 5.00 0.625
0.156 0.156 0.625 0.313 0.313
1.25 1.25 2.50 10.0 5.00
0.313 0.313 0.625 1.25 0.313
5.00 5.00 20.0 20.0 5.00
0.156 1.25 10.0 2.5 0.313
2.50 5.00 5.00 2.50 2.50
1.25 2.50 5.00 2.50 1.25
5.00 10.0 40.0 10.0 40.0
1.25 2.50 40.0 5.00 40.0
antagonism was also statistically significant at the same level.
ity in various media are the pH of the growth medium and the electrolyte content. TSB contains added dextrose (250 mg/ml), and this is, of course, fermentable by the Klebsiella with a DISCUSSION resultant fall in pH during growth. The net Gentamicin was the most active aminoglyco- result is a diminution in the aminoglycoside side in this system, followed by amikacin and effectiveness (1). TSB has a generally higher kanamycin against 70 clinical isolates of Kleb- cation content than MHB or brain heart infusiella species. This hierarchy of in vitro activity sion broth (12), and this increased cation conagainst Klebsiella has been reported previously tent (particularly calcium and magnesium) has (1). In terms of clinically achievable serum an- been shown to adversely affect in vitro aminotimicrobial levels, gentamicin, amikacin, and glycoside activity (13). chloramphenicol have greater in vitro activity To determine if our Klebsiella isolates folthan kanamycin or cephalothin. Cephalothin lowed this pattern, we performed simultaneous was the least active, with only 68% of isolates twofold tube dilution MICs of gentamicin, amisensitive to 20 ,ug/ml or less. kacin, kanamycin, chloramphenicol, and cephA partial explanation for the relatively high alothin in TSB and MHB for 5 of our 70 KlebsiMICs of kanamycin and amikacin in this sys- ella isolates (see Table 4). There was an avertem may be the use of TSB as the test medium. age fourfold drop in the aminoglycoside MIC It has been shown that aminoglycoside MICs when MHB was used for susceptibility testing, against different Enter6bacteriaceae, including but the relative order of activity againstKlebsiKlebsiella, are four- to sixfold higher in TSB ella was similar to that determined in TSB (i.e., than in nutrient broth or Mueller-Hinton broth gentamicin > amikacin > kanamycin). No dif(MHB) (1, 2). The exact mechanism for this is ference in chloramphenicol MIC was apparent, only partially understood. Two parameters that but a trend to a lower MIC of cephalothin in have been shown to affect aminoglycoside activ- MHB was demonstrated.
ANTIMICROB. AGENTs CHZMOTHZR. 892 D'ALESSANDRI, McNEELY, AND KLUGE LITERATURE CITED To further delineate the media effect, if any, on the synergy results, microtiter synergy studG. P., and D. Stewart. 1973. In vitro effectiveies were repeated in MHB for two of the five 1. Bodey, ness of BB-K8, a new aminoglycoside antibiotic. Anorganisms surveyed in Table 4 (STH 32 and timicrob. Agents Chemother. 4:182-192. VAH 49). Whereas the absolute values for the 2. Bulger, R. J. 1967. In vitro effectiveness of kanamycin and kanamycin/cephalothin against klebsiella. Ann. combination MICs were reduced two- to fourIntern. Med. 67:523-532. fold, the isobologram curves denoting synergy W. J., and W. A. Taylor. 1973. Gram-negaHolloway, 3. or antagonism were superimposable in 11 of the tive rods, p. 104-106. In Sepsis. Futura Publishing 12 plates when compared to the corresponding Co., Mt. Kisco, N.Y. TSB synergy plate results. 4. Jawetz, E., and J. B. Gunnison. 1952. Studies on antibiotic synergism and antagonism: a scheme of comIn summary, synergy was demonstrated bined antibiotic action. Antibiot. Chemother. 2:243most commonly with the cephalothin-gertami248. cin and cephalothin-kanamycin combinations. 5. Klastersky, J., R. Cappel, and D. Daneau. 1972. CliniCephalothin-amikacin also exhibited synergy cal significance of in vitro synergism between antithat was significantly greater than the respecbiotics in gram-negative infections. Antimicrob. Agents Chemother. 2:470-475. tive aminoglycoside-chloramphenicol combinaJ., R. Cappel, G. Swings, and L. Vandention. None of the cephalothin-aminoglycoside 6. Klastersky, 1971. Bacteriological and clinical activity of borre. combinations showed antagonism as opposed to the ampicillin/gentamicin and cephalothin/gentami35 to 45% of the chloramphenicol-aminoglycocin combinatioAs. Am. J. Med. Sci. 262:283-290. side combinations. In comparing the MIC data 7. Kluge, R. M., H. C. Standiford, B. Tatum, V. M. Young, W. H. Greene, S. C. Schimpff, F. M. Calia, with the checkerboard results, there was no R. B. Hornick. 1974. Comparative activity of and with which one consistent sensitivity pattern tobramycin, amikacin, and gentamicin alone and could predict whether or not the combinations with carbenicillin against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 6:442-446. ofantibiotics would be synergistic or antagonis8. Luboshitzky, R., T. Sacks, and J. Michel. 1973. Bactetic. ricidal effect of combinations of antibiotics on klebsiThe clinical importance of in vitro synergy or ella-enterobacter-serratia. Chemotherapy 19:354antagonism is uncertain in the absence of an in 366. vivo model or a clinical trial. Until such data 9. Martin, C. M., A. J. Cuomo, M. J. Geraghty, J. R. Zager, and T. C. Mandes. 1969. Gram negative rod are available, the finding of significant antagoJ. Infect. Dis. 119:506-517. bacteremia. nism using the chloramphenicol-aminoglyco- 10. Sabath, L. D. 1968. Synergy ofantibacterial substances side combinations mitigates against the indisby apparently known mechanisms, p. 210-217. Antimicrob. Agents Chemother. 1967. criminate use of this antibiotic combination in J. P. 1975. Klebsiella and other gram-negative situations in which Klebsiella is a potential 11. Sanford, pneumonias, p. 286-28a. In P. B. Beeson bacterial in 80% of The over synergy finding of pathogen. and W. McDermott (ed.), Textbook of medicine, W. B. the cephalothin-aminoglycoside combinations Saunders Co., Philadelphia. tested suggests that this combination be used in 12. Warren, E., R. J. Snyder, and J. A. Washington, II. 1972. Four-hour microbiological assay of gentamicin therapy of serious Klebsiella infections. In adin serum. Antimicrob. Agents Chemother. 1:46-48. dition, there is some clinical evidence to sup- 13. Washington, J. A., II, R. E. Ritts, Jr., and W. J. port the use of these combinations in serious Martin. 1970. In vitro susceptibility of gram-negative bacilli to gentamicin. Mayo Clin. Proc. 45:146-149. gram-negative infections (5, 6, 9).
