Minimal Antibiotic Concentrations of Aminoglycosides and,b-lactam Antibiotics for Some Gram-Negative Bacilli and Gram-Positive Cocci

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1 THE JOURNAL OF INFECTIOUS DISEASES. VOL. 139, NO.5. MAY by The University of Chicago / $.75 Minimal Antibiotic Concentrations of Aminoglycosides and,b-lactam Antibiotics for Some Gram-Negative Bacilli and Gram-Positive Cocci Victor Lorian and Cicero Carlos De Freitas From the Division of Microbiology, Department of Pathology, the Bronx-Lebanon Hospital Center; and the Department of Laboratory Medicine, The Albert Einstein College of Medicine, Bronx, New York The minimal antibiotic concentration (MAC) is the lowest concentration of an antibacterial agent that produces a decrease of I log in the number of organismsjml as compared with a control culture in drug-free medium. Various gram-negative bacilli and gram-positive cocci were grown in the presence of amikacin, gentamicin, tobramycin, ampicillin, amoxicillin, oxacillin, carbenicillin, ticarcillin, and cefamandole at concentrations varying from eight times the minimal inhibitory concentration (MIC) to 1/128 of the MIC. Colony forming units (cfu) were counted, the MIC was determined, and the MIC: MAC ratio, which indicates the magnitude of the effective range, was calculated, The MIC: MAC ratio appears to be characteristic for a given species and antibiotic. There is no relation between the MICs and the MIC:MAC ratios. The highest ratios were given by Proteus mirabilis with aminoglycosides (MIC: MAC mean, 29.2 with tobramycin), and the lowest ratios were given witl~.8-lactam antibiotics by Pseudomonas aeruginosa and Streptococcus faecalis (MIC:MAC means, 2.1 with carbenicillin and cefamandole, respectively). The morphological effects of sublethal concentrations of antibiotics on bacteria were first reported for penicillin's actions on Escherichia coli [1]. Subminimal inhibitory concentrations (submics) of various antibacterial agents have been shown to produce abnormal bacterial morphology [2] and ultrastructure [3, 4], to inhibit the rate of growth in vitro [5-8], and to influence the outcome of experimental infections in animals [9, 10]. Just as the MIC defines antibacterial activity at high concentrations of drug, the minimal antibiotic concentration (MAC) has been suggested as a means of describing antibacterial activity at low concentrations [5, 11-13] and has been used as a criterion for the evaluation of drugs in vitro [14, 15]. It is defined as the lowest concentration of an antibacterial agent that affects the rate of growth and/or the structure of bacteria [5]. As with the Received for publication August 15, 1978, and in revised form November 13, Cicero Carlos De Freitas is the recipient of a career development award from the National Research Council of Brazil. Please address requests for reprints to Dr. Victor Lorian, Division of Microbiology and Epidemiology, Bronx-Lebanon Hospital Center, Fulton Avenue at 169th Street, Bronx, New York determination of the MIC, both cfu counts and turbidimetry have been used for measuring the MAC [5-8]. Materials and Methods Strains. The following isolates obtained from specimens received at the clinical laboratory of the Bronx-Lebanon Hospital Center were used: E. coli (12 strains), Klebsiella pneumoniae (10 strains), Proteus mirabilis (12 strains), Pseudomonas aeruginosa (12 strains), Staphylococcus aureus (11 strains), and Streptococcus faecalis (12 strains). All strains were identified by standard laboratory methods [16] and selected randomly from those sensitive to the antibiotics listed below. Antibiotic sensitivities were determined by the method of Bauer et al. [17]. Media. Trypticase soy broth (TSB; Baltimore Biological Laboratories [BBL], Cockeysville, Md.), trypticase soy agar (BBL), and MacConkey's agar (BBL) containing a final concentration of 3% agar were used. Antibiotics. The following drugs were tested: amikacin (Bristol Laboratories, Syracuse, N.Y.), amoxicillin (Hoffmann-La Roche, Nutley, N.J.), ampicillin (Bristol), carbenicillin (Pfizer, New 599

2 6 Lorian and De Freitas York, N.Y.), cefamandole (Eli Lilly and Co., Indianapolis, Ind.), cephalothin (Lilly), gentamicin (Schering Corp., Bloomfield, N.J.), oxacillin (Bristol), ticarcillin (Beecham-Massengil Pharmaceutical Co., Clifton, N.].), and tobramycin (Lilly). All drugs were dissolved in sterile distilled water and added to the media to give the desired final concentrations. The MIC was determined by placing 0.05 ml of I: 1 dilutions of an 18-hr culture in TSB on a series of trypticase soy agar plates containing twofold dilutions of the drug to be tested. The MIC was the lowest concentration of drug that produced no growth on trypticase soy agar after incubation for 24 hr at 36 C. Determination of the MAC. Each organism was incubated in TSB at 36 C for 18 hr. For each culture, a 0.5-ml aliquot of a I: 10 dilution in TSB was added to 2 ml of TSB and incubated in a water bath at 36 C for I hr (for P. aeruginosa, s. aureus, and S. faecalis, the incubation time was I hr 45 min). Samples (4.5 ml) of TSB culture were added to 0.5 ml of TSB containing antibiotic concentrations calculated to yield final concentrations of 1/128 to eight times the MIC for each strain. Controls grown in TSB with no drugs were also prepared. All tubes were incubated in a water bath at 36 C for 5~ hr. From each tube IO-fold dilutions in 0.85% NaCl were made up to 10-8 for turbid and 10-5 for clear tubes, and 0.2 ml of the last four dilutions was planted on trypticase soy agar plates (or MacConkey's agar for P. mirabilis) and incubated for 20 hr. An electric colony counter (New Brunswick Scientific, New Brunswick, N.].) was used for du counts. The MAC was defined as the lowest concentration of an antibiotic producing a I-log (90%) decrease in the number of cfujrnl as compared with the control grown in drug-free broth [5]. Each strain was tested simultaneously with the three aminoglycosides or with the f3-lactam antibiotics. Although turbidimetric techniques are valuable in the determination of MICs because only large differences in turbidity have to be detected, the MAC is much more difficult to determine by optical methods. Most organisms exposed to submics of antibiotics show changes in morphology accompanied by an increase in volume. With f3-lactam antibiotics these increases could be of great magnitude [4]. As particle size increases and cell number decreases, the optical density increases [18]. O-~O- ;e; ~ 0<> r-, \0 ~~~ ~ +1 lc')cx>lc') ~ ";' \0 \0 l"':c'1c'i 0<> 0<>0\0 ~e-j-: Cl'l~~ ~l!')"" '".. o~~. l!') """,,,, N~c0 O~""" '<j<0'" ~ ~ ""~l!') '<j<. '" '" "" r-'<j< ~Nr...: '<j< '" en' ~e-=r-o'<j< C'iNN 0l!')Cl'l LC'iNO "N' Cl'lCl'l ~~ -=-~~ ~ O'ltOl'-- ~ ~~e-.i +1 Cl'll!')0 a:! cn";'«5 l!') r-.0.0 : : d 0-.n.'" +1 '0.n r-, l!') ~ d r--: ~ 0<> '<j<r- : 0";' \0...:r...:

3 MACs oj Aminoglycosides and fj-laetams 601 ~ ~... "' ~ ~~O> OOO~ e-j,...;<q.. c-r e-r c<')...;~c'i... c<').. r...:u-)~ oo'<l'c<') ""';""';0... : :~ 0U"lC'l O""':N 0U"l <o~.n ~_CN' C'l'<l'c<') ~C'l~... ~o ~~~ :'< ~ ""';ia) C'l e-r c-r ~~'<I' ee::.. "'0>C'l "":C'ioo ~f..o '<I'~.n... N ~... ~ ~ '" ~c<')~ u:""':c<: U"l... c<') c<')'<1'... oocioci so ~...: C'l ~~ c-r O! '" +1 o e-r r-, '"o +1 '<1'0> cxi '" ~.n... Ṇ... ~ ~'" U"l~ o.'".... C'l : "''<I' <o.n +1 "': c<') ọ... x Co... x Therefore, cfu counts rather than optical methods were used in this study. Depletion of nutrients and oxygen in the culture media [19] and drug inactivation could influence the outcome of an in vitro broth susceptibility test. Therefore, an incubation period of 5Y2 hr, rather than overnight, was used to establish the MAC in this study. However, for comparison of results with the traditional standard, the MICs were determined on agar after 24 hr of incubation. Results The MAC becomes more meaningful when it IS expressed as a fraction of the MIC (MIC/MAC) rather than as the absolute value in p,g/ml. The MIC: lviac ratio indicates the relative concentration range throu~h which antibiotic efficacy can be detected: the greater the ratio, the greater the range of antibacterial activity. The means, SD values, and medians are given for the MICs in table 1 and for the MIC: MAC ratios in table 2. The MIC: MAC ratios showed a characteristic range for certain species and antibiotics. All strains of P. aeruginosa had a ratio of 6 for amikacin (the highest among all drugs studied), and the MIC:MAC ratios for E. coli and ampicillin showed a mean of 21, an SD of 6.9, and a median of 20. With few exceptions, the ratios found for each antibiotic-strain pair did not vary more than fourfold from the lowest to the highest value. For a given species and antibiotic, there was no relation between the MIC values and the corresponding MAC values. Among the three aminoglycosides tested, gentamicin had the lowest MIC, but the highest MIC: MAC ratio with E. coli. When the aminoglycosides were tested with K. pneumoniae, tobramycin showed both the highest MICs and the highest ratios. In contrast, in P. mirabilis, amoxicillin showed somewhat lower MICs than did ampicillin, but the ratios seen with amoxicillin were much lower than those with ampicillin. Some characteristics can be observed in the relation between MAC and species. P. mirabilis showed relatively high MICs for all three aminoglycosides, but the MIC:MAC ratios were the highest seen among all species and all drugs tested. The gram-positive cocci and P. aeruginosa showed low ratios for the,b-lactam drugs. Although no general pattern could be observed, the highest

4 602 Lorian and De Freitas mean ratio among the aminoglycosides was seen with gentamicin and, among the /Hactam antibiotics, with ampicillin. Cephaloridine, cephaloglycin, and cephalothin have been shown to give MIC:MAC ratios considerably lower than that of ampicillin [5]. In this study, a new cephalosporin, cefamandole, produced MIC:MAC ratios less than, but close to, those obtained with ampicillin. Simultaneous tests with both cephalosporins confirmed the previous finding with cephalothin and showed that cefamandole produces much higher MIC:MAC ratios than the older cephalosporin. Among the aminoglycosides amikacin has been shown to have the highest MIC: MAC ratio for Serratia marcescens (K. L. Den Bleyker and F. Leitner, personal communication). Discussion Studies of drugs in combination at their MACs have revealed both highly synergistic effects, such as those obtained with mecillinam plus ampicillin [4], and an unexpected antagonistic effect, such as that obtained with sul amethoxazole and trimethoprim [15]. Bacteria exposed to submics of antibiotics have also shown increased vulnerability to immunodefense mechanisms [20-23]. Ampicillin at one-fourth the MIC prevented the in vitro attachment of E. coli to epithelial cells of the human urinary tract [24]. The number of organisms present is important to the clinical outcome of infections of the urinary tract [25, 26], lung [27], and wounds [28]. Since antibiotics at submic levels can reduce bacterial populations and increase susceptibility to the immunodefense system in vitro, it is conceivable that such small concentrations of antibiotics could show clinical effects. The MAC might point to the lowest concentration, and the MIC:MAC ratio might indicate the magnitude of the concentration range for such activity. Although the criteria for the determination and expression of the MAC need further study, knowledge of the submic range of activity of an antibacterial agent adds valuable information to its characterization. References l. Gardner, A. D. Morphological effects of penicillin on bacteria. Nature 146: , Lorian, V., Atkinson, B. Abnormal forms of bacteria produced by antibiotics. Am. J. Clin. Patho!. 64: , Lorian, V., Sabath, L. D., Simionescu, M. Decrease in ribosomal density of Proteus mirabilis exposed to inhibitory concentrations of ampicillin or cephalothin. Proc. Soc. Exp. Bio!. Med. 149: , Lorian, V., Atkinson, B. Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. II : , Lorian, V. Some effects of subinhibitory concentrations of antibiotics on bacteria. Bull, N.Y. Acad. Med. (Second Series) 51: , Shah, P. M., Stille, W. Uber die antibacterielle Wirkung sub inhibitorischer Tetracyclin-Konzentrationen. Infection I:III-II2, Shah, P. M., Heetderks, G., Stille, W. Activity of amikacin at sub-inhibitory levels. ]. Antimicrob. Chemother. 2:97-1, Zanon, U. Sub-inhibitory levels of antibiotics. J. Antimicrob. Chemother. 3: , Zak, O. Effects of sub-mic of antibiotics in experimental animal infection. Curr. Chemother. 1:74, Gemmell, C. G. Effect of subinhibitory concentrations of antibiotics on experimental pyogenic infections in mice. Curr. Chemother. 1: , Il. Lorian, V., Popoola, B. The effect of nitrofurantoin on the morphology of gram-negative bacilli. ]. Infect. Dis. 125: , Lorian, V., Sabath, L. D. Penicillins and cephalosporins: differences in morphologic effects on Proteus mirabilis.]. Infect. Dis. 125: , Lorian, V. Antibiotiques a concentrations subinhibitrices. Effet sur la morphologie et la croissance. Pathol. Bio!. 25: , Greenwood, D. Activity of flumequine against Escherichia coli: in vitro comparison with nalidixic and oxolinic acids. Antimicrob. Agents Chemother. 13: , Greenwood, D. Trimethoprim plus sulphamethoxazole: synergic or antagonistic combination. In M. Woodbine [ed.], Antibiotics and antibiosis in agriculture. Butterworths, London and Boston, 1977, p Lennette, E. H., Spaulding, E. H., Truant, ]. P. [ed.]. Manual of clinical microbiology. 2nd ed. American Society for Microbiology, Washington, D.C., 1974, 970 p. 17. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., Turck, M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Patho!' 45: , Melchior, N. H., BJorn, j., Tybring, L., Birch-Andersen, A. Light and electron microscopy of the early response of Escherichia coli to a 6 {:l-amidino-penicillanic acid (FL 1060). Acta Pathol, Microbio!. Scand. [B] 81: , Brown, M. R. W. Nutrient depletion and antibiotic susceptibility. J. Antimicrob. Chemother. 3: , 1977.

5 MACs of Aminoglycosides and fj-lactams Nishida, M., Mine, Y., Nonoyama, S., Yokota, Y. Effect of antibiotics on the phagocytosis and killing of Pseudomonas aeruginosa by rabbit polymorphonuclear leukocytes. Chemotherapy 22: , Friedman, H., Warren, H. Enhanced susceptibility of penicillin-resistant staphylococci to phagocytosis after in vitro incubation with low doses of nafcillin (38177). Proc. Soc. Exp. BioI. Med. 146: , Sud, l. J., Feingold, D. S. Detection of agents that alter the bacterial cell surface. Antimicrob. Agents Chemother. 8:34-37, Dutcher, B. S., Reynard, A. M., Beck, M. E., Cunningham, R. K. Potentiation of antibiotic bactericidal activity by normal human serum. Antimicrob. Agents Chemother. 13: , Svanborg Eden, C., Sandberg, T., Stenqvist, K., Ahlstedt, S. Adhesion of E. coli to human urinary tract epithelial cells ill vitro is decreased by subinhibitory concentrations of ampicillin. A preliminary study. Infection 6(Suppl.):SI21-S124, Kunin, C. M. A ten-year study of bacteriuria in schoolgirls: final report of bacteriologic, urologic, and epidemiologic findings. J. Infect. Dis. 122: , Kass, E. H. Horatio at the orifice: the significance of bacteriuria. J. Infect. Dis. 138: , Louria, D. B. Uses of quantitative analyses of bacterial population in sputum. J.A.M.A. 182: , Lyman, I. R., Tenery, J. G., Basson, R. P. Correlation between decrease in bacterial load and rate of wound healing. Surgery 130: , 1977.