against Cefotaxime-Resistant Aerobic Gram-Negative Bacilli

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1986, 0066-4804/86/010155-03$02.00/0 Copyright © 1986, American Society for Microbiology

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Vol. 29, No. 1

155-157

Antibacterial Activity of Ro 17-2301 and Other Antimicrobial Agents against Cefotaxime-Resistant Aerobic Gram-Negative Bacilli CONSTANCE A. BENSON,1 FRANCES E. WALTON,2 AND GORDON M. TRENHOLMEl2* Section of Infectious Diseases, Department of Medicine,l and Section of Clinical Microbiology, Office of Consolidated Laboratory Services,2 Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612 Received 1 July 1985/Accepted 20 October 1985

The in vitro activity of Ro 17-2301 was determined and compared with those of aztreonam, ceftazidime, amikacin, and piperacillin against 141 cefotaxime-resistant gram-negative bacilli. Ro 17-2301 was bactericidal, and its activity against the majority of these oganisms was equal or superior to those of ceftazidime, aztreonam, and piperacillin. An inoculum effect of Ro 17-2301 and aztreonam for many Pseudomonas aeruginosa and Enterobacter spp. isolates was demonstrated.

automated microdilution method (American MicroScan, Mahwah, N.J.). Antimicrobial activity was measured by the National Committee for Clinical Laboratory Standards agar dilution technique, using Mueller-Hinton agar (pH 7.4). Plates were prepared with various concentrations of Ro 17-2301, aztreonam, ceftazidime, amikacin, piperacillin, and cefotaxime. Isolates stored in skim milk at -70°C were retrieved and cultured onto Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% sheep blood. All inocula were prepared by transferring four to five colonies into Trypticase soy broth. After 4 h of incubation at 37°C, turbidity was adjusted to a 0.5 McFarland standard. The standardized suspensions were then diluted 1:10 to achieve a cell density of approximately 107 CFU/ml. A multipoint replicator (Cathra, St. Paul, Minn.), designed to deliver 0.001 ml, was used to inoculate approximately 104 CFU per spot to the surface of the antibiotic-containing agar. Plates were inoculated from the lowest to the highest concentration of antibiotic to avoid carry-over. The MIC, defined as the lowest concentration of antibiotic at which there was no visible growth, was determined after overnight incubation at 37°C. MICs of Ro 17-2301 and aztreonam for 57 Enterobacter spp. and 34 Pseudomonas aeruginosa isolates were also determined by using a microtiter broth dilution technique to determine the inoculum effect. Briefly, bacterial suspensions were obtained as described above and adjusted to contain 108 CFU/ml. Standardized suspensions were then diluted to final concentrations of either 107 or 104 CFU/ml. National Committee for Clinical Laboratory Standards bulk broth dilution schemes were used to prepare the antimicrobial solutions, and 100-pl aliquots were added to each well in 96-well microtiter trays. The wells were then inoculated with 100 [LI of bacterial suspension at a final concentration of either 107 or 104 CFU/ml. The same bacterial inocula were added to media containing 50 p.g of amikacin per ml in Mueller-Hinton broth to allow for the comparison of visual turbidity and bacterial button size in the wells when bacteria did not grow or lyse. The MIC was determined visually after incubation for 24 h at 37°C. The MIC was defined as the lowest concentration of the antibiotic that prevented visible growth in the wells. The MBCs of Ro 17-2301 and aztreonam for 57 Enterobacter spp. and 34 P. aeruginosa isolates were determined by inoculating Trypticase soy agar plates containing 5% sheep blood with an appropriate subculture volume to assure

A number of bacterial species have been found to produce a family of naturally occurring and structurally similar monocyclic beta-lactam compounds (6). The first of the synthetic monobactams, aztreonam, has been reported to show a high degree of activity against beta-lactamaseproducing aerobic gram-negative bacilli, including Pseudomonas spp., and to be stable to the majority of betalactamases produced by these organisms (4-7). It has little or no activity against gram-positive organisms and anaerobes (4, 6). Ro 17-2301, a more recently developed monobactam, distinct from aztreonam in the deletion of two methyl groups in the side chain at the C-7 position and the addition of an aminocarboxy group in the side chain at the C-4 position of the monobactam structure, appears to be more active in vitro than aztreonam against the Enterobacteriaceae. In contrast to aztreonam, Ro 17-2301 also demonstrates modest activity against Streptococcus pneumoniae and Bacteroides fragilis (8). In addition, Ro 17-2301 may possess a greater degree of stability than aztreonam to a wide range of plasmid and chromosomally mediated beta-lactamases (K. Okonogi and M. Kuno, Program Abstr. 23rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 325, 1983). Potential targets for the monobactams are multiply resistant gramnegative bacteria increasingly associated with nosocomial infections (5). This report summarizes our investigation of the in vitro activity of Ro 17-2301 compared with those of aztreonam and other similarly targeted antimicrobial agents

against cefotaxime-resistant aerobic gram-negative bacilli. Ro 17-2301 was supplied by Hoffman-La Roche, Inc., Nutley, N.J.; amikacin was from Bristol Laboratories, Syracuse, N.Y.; aztreonam was from E. R. Squibb and Sons, Inc., Princeton, N.J.; cefotaxime was from HoechstRoussel Pharmaceuticals, Inc., Somerville, N.J.; ceftazidime was from Glaxo, Inc., Research Triangle Park, N.C.; and piperacillin was from Lederle Laboratories, Pearl River, N.Y. A total of 141 cefotaxime-resistant aerobic gram-negative bacilli isolated from patients hospitalized at RushPresbyterian-St. Luke's Medical Center were collected from the clinical microbiology laboratory between 15 November 1983 and 15 July 1984. Approximately 11.4% of all nonduplicate gram-negative bacilli isolated during this period were cefotaxime resistant. Resistance to cefotaxime was defined as an MIC of greater than 8 jig/ml by an *

Corresponding author. 155

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ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Antibacterial activities of Ro 17-2301 and other antimicrobial agents against cefotaxime-resistant aerobic gram-negative bacilli Organism tested 1-of isolates) tno.

~~~~Antibiotic

(,ug/ml) MIC

range

MIC50

Pseudomonas Ro 17-2301 aeruginosa (34) Aztreonam Ceftazidime Amikacin Piperacillin Cefotaxime

2-64 2-32 s0.5-64 2-16 4-256 16-.256

4 8 2 4 4 32

Pseudomonas Ro 17-2301 maltophilia (19) Aztreonam Ceftazidime Amikacin Piperacillin Cefotaxime

8-128 -256 2-256 16->256 64->256 32->256

32 >256 64 256 256 128

Ro 17-2301 Aztreonam Ceftazidime Amikacin Piperacillin Cefotaxime

4-128 4->256 2-64 0.5-128 2-128 8-128

32 32 8 32 8 16


-0.063-32 s0.5-4 2-128 256 8-256

16 16 32 1 64 64

Citrobacter freundii (14)


2-256 256 2-256 1-256 16->256 16->256

Other Pseudomonas spp.

(8)"

Enterobacter aerogenes

(25)

Acinetobacter calcoaceticus subsp. anitratus (9)

s0.5-32 s0.5-128

0.063->128

s0.5-32 s0.5-256 s0.5-16

MICgo

8 16 64 1 64 32

" P. cepacia (six isolates), P. putida (one isolate), and P. putrefaciens (one isolate).

The antibacterial activities of Ro 17-2301, aztreonam, ceftazidime, amikacin, piperacillin, and cefotaxime are shown in Table 1. Against the 34 cefotaxime-resistant P. aeruginosa isolates, Ro 17-2301, aztreonam, and ceftazidime demonstrated similar activities with Ro 17-2301 being slightly more active than aztreonam (MICs for 50% of strains [MIC50s] were 4 and 8, respectively). Against the 19 isolates of Pseudomonas maltophilia, Ro 17-2301 was the most active compound (MIC50, 32 ,ug/ml). Against the 57 isolates of Enterobacter spp., Ro 17-2301 and amikacin were the most active agents, with Ro 17-2301 demonstrating greater activity than both aztreonam and ceftazidime. Against the remaining 31 isolates, Ro 17-2301 and amikacin were more active than aztreonam and ceftazidime. When tested against all Enterobacter spp. and P. aeruginosa isolates, Ro 17-2301 and aztreonam were found to be bactericidal; the MBCs were equal to or one tube dilution greater than the MICs. The 57 Enterobacter spp. and 34 P. aeruginosa isolates were tested for an inoculum effect against Ro 17-2301 and aztreonam at 104 and 107 CFU/ml. An eightfold or greater rise in MIC was seen for 32 of 57 Enterobacter isolates and for 17 of 34 P. aeruginosa isolates when tested against Ro 17-2301 at 104 and 107 CFU/ml. A similar rise in MIC was noted for 43 of 57 Enterobacter spp. isolates and for 18 of 34 P. aeruginosa isolates when tested against aztreonam at 104 and 107 CFU/ml (Table 2). Ro 17-2301 is reported to have a broad spectrum of activity for the Enterobacteriaceae similar to that of aztreonam, with a greater degree of stability than that of aztreonam to a wide range of plasmid-mediated and chromosomally mediated beta-lactamases (Okonogi and Kuno, 23rd ICAAC abstr. no. 325, 1983). Our data suggest that Ro 17-2301 may be slightly more active than aztreonam against a number of cefotaxime-resistant aerobic gramnegative bacilli. The activity of Ro 17-2301 appears comparable to that of ceftazidime for these organisms except against cefotaxime-resistant Enterobacter spp., for which it appears more active than ceftazidime. The greater activity of Ro 17-2301 demonstrated in this study may reflect the previously described side chain alterations, resulting in its reportedly greater beta-lactamase stability. A marked inoculum effect was demonstrated for Ro 17-2301 and aztreonam with the Enterobacter spp. and P. aeruginosa isolates. Prior investigators have noted a similar effect of Enterobacter spp. and P. aeruginosa on aztreonam activity (1, 5). Other studies report no effect on in vitro activity of Ro 17-2301 or aztreonam when the inoculum size of P. aeruginosa was increased (3). The clinical significance of this inoculum effect remains unclear. Previously reported data suggest a poor correlation between the degree of inoculum effect demonstrated in vitro and the presence or absence of drug inactivation (1). Ro 17-2301 achieves maximum plasma levels of 78 and 150

accurate determination of the 99.9% killing endpoint. The

procedure was performed in the following manner. Quantitative colony counts were done on each initial inoculum from which MICs were determined. Based on the colony counts, a subculture volume was calculated to assure 99.9% killing. Subculture inocula ranged from 104 to 107 CFU/ml, and subculture volumes were from 0.1 to 0.001 ml. The MBC was defined as the lowest concentration of antibiotic resulting in a 99.9% decline in colony count of the subculture inoculum after overnight incubation on antibiotic-free media. P. aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were used throughout the study as control organisms.

TABLE 2. Effect of inoculum size on the activity of Ro 17-2301 and aztreonam MIC90 (pg/ml) at inoculum (CFU/ml): Antibiotic Organism tested (no. of isolates) 104b107 104/107 1O4 107 ratio Enterobacter spp. (57) P. aeruginosa (34)

Ro 17-2301 Aztreonam Ro 17-2301 Aztreonam

32 64 8 16

512

1,024 256

1,024

16 16 32 64

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VOL. 29, 1986

jLg/ml after intravenous doses of 1,000 and 2,000 mg, respectively, with a mean elimination half-life of 1.8 h (2). These plasma levels are greater than the MICg) for the majority of organisms for which Ro 17-2301 demonstrated some activity. Within 24 h after a 1-h intravenous infusion, 85% of the dose has been recovered from the urine (2). Ro 17-2301 demonstrated significant activity for a number of cefotaxime-resistant aerobic gram-negative bacilli in our study. Its activity was equivalent to or greater than those of ceftazidime, aztreonam, and piperacillin, which are also beta-lactam agents potentially targeted against multiply resistant nosocomially acquired gram-negative bacilli. The favorable pharmacokinetics and antimicrobial activity of Ro 17-2301 suggest that it may be useful in the treatment of infections in such settings and that further in vitro and in vivo investigations of this drug are warranted. LITERATURE CITED 1. Eng, R. H. K., S. M. Smith, and C. Cherubin. 1984. Inoculum effect of new r-lactam antibiotics of Pseiudotmionas aeruiginiosai. Antimicrob. Agents Chemother. 26:42-47. 2. McNulty, C. A. M., G. M. F. Garden, J. Ashby, and R. Wise.

157

1985. Pharmacokinetics and tissue penetration of Carumonam, a new synthetic monobactam. Antimicrob. Agents Chemother.

28:425-427. 3. Ng, W. W. S., P. Y. Chau, Y. K. Leung, and D. M. Livermore. 1985. In vitro activities of Ro 17-2301 and aztreonam compared with those of other new 3-lactam antibiotics against clinical isolates of Pseiudoilonacis (iruiginosa. Antimicrob. Agents Chemother. 27:872-873. 4. Phillips, I., A. King, K. Shannon, and C. Warren. 1981. SQ 26,776: in vitro antibacterial activity and susceptibility to betalactamases. J. Antimicrob. Chemother. 8(Suppl. E):103-110. 5. Sykes, R. B., D. P. Bonner, K. Bush and N. H. Georgopapadakou. 1982. Azthreonam (SQ 26,776), a synthetic monobactam specifically active against aerobic gram-negative bacteria. Antimicrob. Agents Chemother. 21:85-92. 6. Sykes, R. B., D. P. Bonner, K. Bush, N. H. Georgopapadakou, and J. S. Wells. 1981. Monobactams-monocyclic beta-lactam antibiotics produced by bacteria. J. Antimicrob. Chemother. 8(Suppl. E):1-16. 7. Wise, R., J. M. Andrews, and J. Hancox. 1981. SQ 26,776, a novel beta-lactam: an in vitro comparison with other antimicrobial agents. J. Antimicrob. Chemother. 8(Suppl. E):39-47. 8. Wise, R., J. M. Andrews, and L. J. V. Piddock. 1985. The in vitro activity of RO 17-2301, a new monobactam, compared with other antimicrobial agents. J. Antimicrob. Chemother. 15:193-200.