ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 6 Copyright 1988, Institute for Clinical Science, Inc. Comparison of the Inhibitory and Bactericidal Activity of Aztreonam and Amikacin Against Gram Negative Aerobic Bacilli JOAN C. FUNG-TOMC, Ph.D., WILLIAM LINCOURT, B.S., CHRISTIAN THATER, B.S., and ROBERT E. KESSLER, Ph.D. Department of Microbiology, Pharmaceutical Research and Development Division, Bristol-Myers Company, Wallingford, CT 06492 ABSTRACT Aztreonam has been compared both in vitro and in clinical trials to aminoglycosides in its activity against aerobic gram-negative bacteria. The inhibitory and killing abilities of aztreonam and amikacin were examined against five gram-negative bacillary strains. Time kill analysis was carried out at serum-achievable concentrations (25 xg per ml amikacin, 100 and 200 xg per ml aztreonam) and levels found three to five hours post-infusion (8 (Jig per ml amikacin and 25 xg per ml aztreonam). Amikacin killed all five strains faster than aztreonam at all the concentrations tested. Regrowth and the presence of persisters were observed in aztreonamtreated cultures. In the presence of amikacin, there was no detectable increase in cell mass, as measured by optical density. However, following aztreonam treatment, bacterial cell mass increased in the first two to three hours before decreasing. Long, filamentous cells were observed in aztreonam-containing cultures. Though amikacin and aztreonam are bactericidal drugs, prolonged bacterial survival, continued cell growth, regrowth, and persisters were observed only in aztreonam-treated cultures. Introduction Aztreonam (previously aztreonam and SQ 26,776) is a m onocyclic (3-lactam (monobactam). Its antibacterial spectrum is similar to that of the aminoglycosides, i.e., activity against gram-negative bacteria but not against anaerobic or grampositive bacteria.14 The major indications for both aztreonam and aminoglycoside include treatment of infections caused by susceptible gram-negative bacteria.2,7 O ne desirable and clinical pertinent property of aminoglycosides is the rapid bactericidal effect of the class against suscep tib le bacteria. In this study, the inhibitory and bactericidal activity of aztreonam and amikacin were examined against five strains of gram-negative aerobic bacteria. 463 0091-7370/88/1100-0463 $00.90 Institute for Clinical Science, Inc.
46 4 FUNG-TOMC, LINCOURT, THATER AND KESSLER Methods Five clinical isolates susceptible to aztreonam* and amikacinf were used (table I). Minimal inhibitory concentration (MIC) was determined by an agar dilution method in accordance with the procedure outlined by the National Committee for Clinical Laboratory Standards (NCCLS).6 Bactericidal activity was monitored by tim e-kill analysis. Exponential-phase cells were inoculated into prewarmed Mueller-Hinton broths to a final inoculum of 5 X 105 to 5 X 106 cfu per ml. F r e sh ly p rep ared a n tib io tics w ere added. Cultures were incubated at 35 C. Viability was determined at time intervals by plating samples and sample dilutions onto Mueller-Hinton agar with the spiral plater. Plates were incubated for 18 hours at 35 C. At each time point, a sample was removed and fixed in two percent formaldehyde. The cell lengths of bacteria in these samples were measured using an image analysis system. 1 The growth curve patterns of neat and antibiotic-containing cultures were also determ ined using the Advantage System. H Two milliliters of each time-kill analysis culture were rem oved at the time of antibiotic addition and placed in to a cham ber o f a m ulticham ber research cuvette. Biomass in each culture was m onitored and the growth curve plotted as optical density readings relative to the initial tim e point. All studies were performed in duplicate, done at various times. * Squibb, Princeton, NJ. t Bristol Laboratories, Syracuse, NY. $ Difco, Detroit, MI. Spiral System Instrum ents, Inc., Cincinnati, OH. 1Image Technology Corporation, D eer Park, NY. U Abbott, Irving, TX. T A B L E I Minimal Inhibitory Concentration of Bacterial Isolates to Amikacin and Aztreonam O r g a n i s m M I C (U g /ml) A m i k a c i n A z t r e o n a m Escherichia coli A20341 2 0.13 K l e b s i e l l a p n e u m o n i a e A15730 2 0.13 Enterobacter cloacae A15154 2 1.00 S e r r a t i a m a r c e s c e n s A22243 2 0.13 P s e u d o m o n a s a e r u g i n o s a A 9843 8 8.00 Results Kill curves o f the five strains are shown in figure 1 (A to E). The 100 and 200 fxg per ml aztreonam concentrations represent, respectively, approximate peak serum levels after one g and two g intravenous infusions; 25 xg per ml aztreonam approximates the three to five hours post-infusion concen tration.10 Similarly, an approximate peak postinfusion level of 25 juug per ml amikacin and four hour post-infusion concentration o f 8 jjlg per ml am ikacin w ere tested.5 Rapid killing was achieved with amikacin against all five gram-negative bacterial strains. By 0.5 hours of incubation in 25 xg per ml amikacin, there was > 99.9 percent kill of all bacterial strains relative to the initial inoculum. At 8 xg per ml amikacin, this same level of killing was obtained by 0.5 hour against K lebsiella pneum oniae, Pseudom onas aeruginosa, Enterobacter cloacae, Escherichia coli, and by 1.5 hour against Serratia marcescens. The bactericidal rate of aztreonam was much slower. The most rapid killing was obtained with Enterobacter cloacae and Escherichia coli, where 99.9 percent killing was achieved by 1.5 to two hours using 100 and 200 xg per ml aztreonam, and by 2.5 to three hours with 25 xg per ml aztreonam. W ith Serratia m arcescens, a 3-log reduction resulted after 2.5
ACTIVITY OF AZTREONAM AND AMIKACIN 46 5 TIME (hours) F i g u r e 1. Time-ldll studies comparing the response to amikacin and aztreonam of Escherichia coli (A), Klebsiella pneumoniae (B), Enterobacter cloacae (C), Serratia marcescens (D), and Pseudomonas aeruginosa (E). Antibiotic concentrations used were 25 [Lg per ml amikacin (O), 8 ng per ml amikacin (O), 200 (xg per ml aztreonam (A), 100 (j.g per ml aztreonam ( ), 25 xg per ml aztreonam ( ), and no antibiotic (A). hours with 200 xg per ml aztreonam and 4.5 hours with 25 and 100 )xg per ml aztreonam. Though this level of Klebsiella pneumoniae killing was reached by 1.5 hours with 100 and 200 (jug per ml aztreonam, regrowth occurred by six hours o f incubation. With Pseudomonas aeruginosa, 99.9 percent kill was never achieved with aztreonam treatment. Despite the decrease in viable counts of aztreonam-containing cultures, biomass continued to increase for all five strains during the first two to three hours of aztreonam treatment (figure 2 A to E). Only after two to three hours did cell m ass d ecrease. On the other hand, growth immediately ceased with addition of amikacin; there was no change in cell mass following addition of amikacin. Long filaments in aztreonam-treated cultures probably account for increased biomass in spite of decreased viability. In table II are listed the average cell length in cultures containing no antibiotic, amikacin, or aztreonam. There was no difference in cell length between amikacintreated cells and cells from growth control cultures. By three hours of incubation, aztreonam-treated cells reached three to 14 times the length of nontreated cells. Longer filam ents w ere observed in cultures containing lower concentrations of aztreonam. Discussion Am ikacin and aztreonam are both active against a broad spectrum of gramnegative aerobic bacilli. Though both antibiotics are bactericidal, aztreonam show ed substantially delayed killing com pared to am ikacin. In addition, P s e u d o m o n a s a e r u g in o s a d id n o t decrease more than three logs in the presence o f aztreonam and Klebsiella pneumoniae regrew within six hours following exposure to aztreonam. The presence of persisters in aztreonam-treated Pseudom onas aeruginosa cultures has been reported.8 The highest aztreonam concentrations used in that study were 2.5 and 5 yug per ml. W e observed Pseudomonas aeruginosa persisters even at 100 and 200 xg per ml peak concentra
46 6 FUNG-TOMC, LINCOURT, THATER AND KESSLER F ig u re 2. Growth curves of Escherichia coli (A), Klebsiella pneumoniae (B), Enterobacter cloacae (C), Serratia marcescens (D), and Pseudomonas aeruginosa (E) in the presence of amikacin and aztreonam. Growth curve lines represent no antibiotic (1), 8 p.g per ml amikacin (2), 25 (xg per ml amikacin (3), 25 ng per ml aztreonam (4), 100 (xg per ml aztreonam (5), and 200 xg per ml aztreonam (6). tions of aztreonam. Our results agree with previous data reporting rapid killing of Escherichia coli by aztreonam.11 However, as with our strains, prolonged survival was also noted for Klebsiella pneum o n ia e, S e r r a tia m a rcescen s, and P roteu s m ira b ilis in that study and r e g r o w th w as a ls o o b s e r v e d in aztreonam-treated Klebsiella pneumoniae and P roteus m irabilis cultures.11 These in vitro observations may reflect the 40 percent failure rate of aztreonam to eradicate Pseudomonas aeruginosa in vivo.3-13 The results from this study also show that despite the lower aztreonam MIC for three of the five strains tested, amikacin exhibited a faster rate of killing. Long filam ents w ere observed in aztreonam-treated cultures, these filaments were longer in cultures containing lower concentrations of aztreonam. This filamentous growth w ill not result in increased viable counts, thus the viable counts shown for the first three hours following exposure to aztreonam yield underestimates of the growth inhibitory activity of aztreonam for these strains. The filamentous cells which result from inhibition of penicillin-binding protein 3 (PBP 3) in E. coli or its equivalents in the other strain s,112 are in h ib ited from forming septa4 but continued to be metabolically capable of producing the various endotoxins and exotoxins of these bacteria. Prolonged bacterial survival, continued growth of cells, regrowth, and the p resen ce o f p ersisters o b served in aztreonam-containing cultures are not observed with amikacin-treated cells. As a general rule, bactericidal antibiotics are desired in the treatment of serious and deep-seated infections. Whether the delayed killing by aztreonam observed in v itro is clinically significant awaits results from more extensive experience with aztreonam in serious infections. Careful usage of aminoglycosides such as am ikacin sh ou ld contin u e to be important in therapy of severe infections due to gram-negative aerobic bacilli. W hen used in combination with ß-lactams in treatment of Pseudomonas aeruginosa, staphylococcal and enterococcal
ACTIVITY OF AZTREONAM AND AMIKACIN 46 7 TABLE I I Average Cell Length Measured at Three Hours of Incubation in Cultures Containing No Antibiotics, Amikacin, or Aztreonam Average Cell Length (ß)* Amikacin Aztreonam Organism No Antibiotic 8 ug/ml 25 lig/ml 25 \ig/ml 100 lig/ml 200 ug/ml Escherichia coli 3.9 3.8 Klebsiella pneumoniae 3.9 3.6 Enterobacter cloacae 3.5 4.8 Serratia marcescens 2.7 2.8 Pseudomonas aeruginosa 3.0 2.4 3.7 14.4 13.6 9.4 4.3 22.7 14.7 11.5 3.4 34.1 16.1 22.8 3.4 38.6 18.5 17.2 1.1 40.2 35.8 27.7 * The average cell length is estimated from measurements taken from approximately 30 cells. infections they offer the benefits of dual therapy and avoid the controversy of dual (3-lactam therapy.9,14,15 References 1. G e o r g o p a p a d a k o u, N. H., S m i t h, S. A. ClMARUSTl, C. M., and S y k e s, R. B.: Binding of monobactams to penicillin-binding proteins of Escherichia coli and Staphylococcus aureus: Relation to antibacterial activity. Antimicrob. Agents Chemother. 23:98-104, 1983. 2. G o o d i n g, P. G., B e r m a n, E., L a n d, A. Z., and AGRE, K.: A review of results of clinical trials w ith am ikacin. J. Infect. D is. 134(Supplement):S441-S445, 1976. 3. H ara, K., K o ba y a sh i, H., N is h iu r a, T., Yura, J., and Sa it o, A.: Clinical studies of aztreonam in Japan. Rev. Infect. Dis. /(S u p p lem en t 4 ):S 810 -S 824, 1985. 4. I s h i n o, E and M a t s u h a s h i, M.: Peptidogylcan synthetic enzyme activities of highly purified penicillin-binding protein 3 in Escherichia coli: A septum-focusing reaction sequence. Biochem. Biophys. Res. Commun. 101:905 911, 1981. 5. L o d e, H., G r u n e r t, K,, K o e p p e, P., and LANGMAACK, H.: Pharmacokinetic and clinical studies with amikacin, a new aminoglycoside a n tib io tic. J. I n f e c t. D is. 2 3 4 (S u p p le - ment):s316- S322, 1976. 6. N ational C om m ittee for Clinical Laboratory Standards: Approved standard M7-A. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, PA: N ational C om m ittee for Clinical Laboratory Standards, 1985. 7. N e u, H. C.: Concluding statement. Rev. Infect. Dis. /(Supplement 4):S840 S843, 1985. 8. R u s s e l l, A. D.: In vitro studies on SQ 26,776, a new monobactam. J. Antimicrob. Chemother. 8(Supplement E):81-88, 1981. 9. S a n d e r s, C. C.: Novel resistance selected by the new expanded-spectrum cephalosporins: A concern. J. Infect. Dis. 147:585-589, 1983. 10. S c u l l y, B. E., S w a b b, E. A., and N e u, H. C.: Pharmacology of aztreonam after intravenous infusion. A ntim icrob. A gents C hem o ther. 24:18-22, 1983. 11. S h a h, P. M., L o s e r t - B r u g g n e r, B., and S n L L E, W.: Bactericidal activity of SQ 26,776. Antimicrob. Chemother. 8(Supplement E):77-80, 1981. 12. S p r a t t, B. G.: Distinct penicillin-binding proteins involved in the division, elongation and shape of Escherichia coli K12. Proc. Natl. Acad. Sci. USA 72:2999-3003, 1975. 13. STILLE, W. and GlLLISSEN, J.: Clincial experience with aztreonam in Germany and Austria. Rev. Infect. Dis. /(Supplem ent 4):S825-S830, 1985. 14. S y k e s, R. B., B o n n e r, D. P., B u s h, K., and G e o r g o p a p a d a k o u, N. H.: Azthreonam (S Q 26,776), a synthetic monobactam specifically active against aerobic gram-negative bacteria. Antim icrob. Agents C hem other. 2 J:85-92, 1982. 15. W a l d v o g e l, F. A.: Future perspective of aminoglycoside therapy. J. Antimicrob. Chemother. I3(Supplement A):73-78, 1984. 16. T h e n, R. L. and A n g e h r n, P.: Trapping of nonhydrolyzable cephalosporins by cephalosporinases in Enterobacter cloacae and Pseudomonas aeruginosa as a possible resistance mechanism. Antimicrob. Agents Chem other. 21:711-717, 1982.