Meropenem: in-vitro activity and kinetics of activity against organisms of the Bacteroides fragilis group

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1 Journal of Antimicrobial Chemotherapy (99) 7, Meropenem: in-vitro activity and kinetics of activity against organisms of the Bacteroides fragilis group J. A. Garcia-Rodriguez, J. E. Garcia Sanchez, I. Trujillano and A. Sanchez de San Lorenzo Department of Microbiology, Hospital Clinico Universitario, Paseo de San Vicente 0, Salamanca, Spain Meropenem was compared with and nine other antimicrobial agents, against 0 strains of the Bacteroides fragilis group. Meropenem was active against all strains tested, and its activity was similar to, and in many cases better than, that of. The activity of was similar to that of, and greater than that of the other antimicrobial agents tested. The bactericidal activity of against B. fragilis was impressive, since the MBC to MIC ratios were no greater than two. The bactericidal activity was confirmed by time-killing curve assays with two strains which showed that was rapidly bactericidal and reduced the initial inoculum significantly during the first -6 h. The postantibiotic effect of (- h) and a sub-inhibitory concentration of i x MIC suggested that interferes with the normal growth of B. fragilis, even when administered concentrations fall below the MIC. MICs of were affected minimally by the ph of the medium or by an increase in inoculum size. Meropenem continued to have good activity against a B. fragilis strain that had been induced for the production of cephalosporinase. The in-vitro data presented in this paper indicate that is a promising antimicrobial agent which may be useful in the treatment of problematic mixed infections. Introduction Meropenem is a new carbapenem which resembles in terms of structure and a broad spectrum of antimicrobial activity (Jones, 95). It also possesses great stability to /Mactamase hydrolysis (Edwards et ai, 99; Jones et al., 99a, b; Sanders et al., 99) and is more stable than to human renal dehydropeptidase I (Wise, 96; Edwards et al, 99; Jones et al., 996). The purpose of the present study was to determine the in-vitro activity of against members of the Bacteroides fragilis group, and the kinetics of activity against two strains of B. fragilis. We report the antimicrobial activity of compared with nine other drugs, the bactericidal activity (MBC), the postantibiotic effect (PAE), and the sub-mic effects of. Also studied were the influence of a variable inoculum size, the effect of medium ph on MICs of, and the activity of against a strain of B. fragilis induced for the production of chromosomal /Mactamase /9/99+0 $0.00/0 99 The British Society for Antimicrobial Chemotherapy

2 600 J. A. Garcia-Rodrigoez et at. Organisms Materials and methods A total of 99 recent clinical isolates belonging to the B. fragilis group (59 B. fragilis, 0 B. thetaiotaomicron, ten B. ovatus,fiveb. vulgatus and five B. distasonis) were studied. These strains were isolated from clinical specimens at the Hospital Clinico Universitario of Salamanca, Spain. All strains were identified with the ATB A system (biomerieux, Montalieu Vercieu, France). B. fragilis strains NCTC 933, -9, and B. thetaiotaomicron ATCC 97 were used for additional tests. Antibiotics The antibiotics used were gifts from the various manufacturers. These antibiotics were: (ICI), and (Merck, Sharp & Dohme), (Lederle), (Bayer), (Roussel), (Eli Lilly), (Upjohn), (Parke-Davis), and (Rhone-Poulenc). Stocks of and were prepared freshly for each experiment. The other stock solutions were stored at 70 C. Once thawed, the solutions were not refrozen. The concentration range assayed for each antibiotic was 000- mg/. MICs These were determined by the agar dilution method for antimicrobial susceptibility testing of anaerobic bacteria, as recommended by the National Committee for Clinical Laboratory Standards (99). The organisms were cultured on Wilkins-Chalgren agar plates, supplemented with 5% sheep blood, and incubated for - h at 35 C in anaerobic jars (Gas Generating Kit, Anaerobic System, Oxoid Limited). Four or five colonies were then inoculated into Wilkins-Chalgren broth and incubated for h in the same conditions. The turbidity of each culture was adjusted, in the same broth, to the density of a 0-5 McFarland Standard. This resulted in a final inoculum density of x 0 5 cfu/spot since the inoculum replicator (Steer's replicator) deposited 000 ml on the agar surface. In addition to the agar plates containing antibiotic, two plates (Mueller-Hinton agar and Wilkins-Chalgren agar) not containing antimicrobial agent were inoculated in each set of tests. The Mueller-Hinton plate was incubated aerobically to check for possible contamination with aerobic bacteria, while the Wilkins-Chalgren plate was incubated anaerobically to verify good growth of the test strains. An additional plate of Wilkins-Chalgren agar was inoculated and kept at C to serve as an inoculum control. This is an aid in distinguishing slight growth from dried inoculum. The remaining plates were incubated at 35 C in anaerobic jars for h. Each test was monitored by including B. fragilis NCTC 933 and B. thetaiotaomicron ATCC 97. The MIC was defined as the lowest concentration of drug yielding no growth; a haze at the point of inoculation or one discrete colony was ignored. MBCs These were determined as recommended by the National Committee for Clinical Laboratory Standards (97). A broth macrodilution method was used to establish the MBC values for selected strains (B. fragilis NCTC 933 and -9) prior to

3 Meropenem activity against B. fragius group 60 performing time-kill curve assays. The MBC was defined as the lowest concentration of drug which reduced the starting inoculum by 99-9%. Any reduction in numbers was assessed by viable counts. The bactericidal activity of and was confirmed by time-kill curve assays (National Committee for Clinical Laboratory Standards, 97). PAE and sub-mic determinations Four or five colonies from a - h Wilkins-Chalgren blood agar culture of each test strain were inoculated into a tube containing 5-6 ml of Wilkins-Chalgren broth. After -6 h incubation at 35 C in anaerobic jars, the culture was diluted in the same broth to the turbidity of a 0-5 McFarland Standard, and then diluted :00 in Wilkins-Chalgren broth. Examination of the PAE in vitro involved exposing a broth culture in logarithmic growth to an antimicrobial concentration above the MIC for at least h. After -5 h exposure to or at x MIC, the drug concentration was reduced to sub-mic levels by a 000-fold dilution in fresh broth. Untreated control organisms were diluted similarly. After dilution, both the control organisms and the cultures of B. fragilis exposed to resumed logarithmic growth. Viable counts were determined at,, and 6 h intervals following drug exposure. The PAE was calculated by determining the difference between the time required for the number of cfu/ml in drug-treated and untreated cultures to increase ten-fold above the number present immediately after drug removal (Craig & Gudmundsson, 96). The sub-mic concentrations (Lorian, 90) were used to simulate the levels of antibiotic remaining after culture dilution, and permit detection of any inhibitory effects on growth resulting from residual drug. The minimum antibiotic concentration (MAC) has been defined as the lowest concentration of an antimicrobial agent that can affect bacterial structure, growth rate, or both. MAC effects can be divided into MAC 'morphology and ultrastructure', i.e. the minimum drug concentration producing a structural change as seen by light or electron microscopy, or MAC 'inhibition', which indicates the minimum concentration of a drug producing a 90% reduction in the viable count of a culture over 5-5 h, compared with a control culture grown in drug-free broth. We employed, i, i and xmic to determine the MAC 'inhibition' effect. Effect of inoculum size and ph of the medium The influence of inoculum size on MIC results was assessed with inoculum sizes of 0", 0 5 or 0 6 cfu/spot (photospectrometric adjustment of a culture derived from a h plate, verified by colony counts) on Wilkins-Chalgren agar (Edwards et ai, 99). The effect of ph on MICs of was assessed by using Wilkins-Chalgren agar which had been adjusted during preparation to ph 6, 7 or (Edwards et al., 99). The ph was measured with a ph meter (PHM 3 Autocal Radiometer) and a surface electrode (Tecan) and, whenever necessary, adjusted with N HO or N NaOH. MICs of with 0, 0 5 or 0 6 cfu/spot and ph 6, 7 or, were determined as recommended by the National Committee for Clinical Laboratory Standards (99). Activity against a B. fragilis strain induced for the production of cephalosporinase Induction of chromosomal /3-lactamase by in a strain of B. fragilis (-9) was achieved by diluting a h Wilkins-Chalgren broth culture :0 in the

4 Table I. In-vitro activity of and other antimicrobial agents against 0 strains from the Bacteroides fragilis group Organism No. of strains Compound range MIC (mg/) %R B. fragilis B. thetaiotaomicron B. ovatus B. vulgatus B. distasonis O > -> - -> - O03-> - 0- O06- O06- > l-> - - -> O06-> - O03- O06-O5 O06- -> -> - - -> O06- -> -> O06-O-5 O06- -> -> - -> - -> > > > > "% Resistant as defined by the recommended criteria (National Committee for Clinical Laboratory Standards, 95, 9). A provisional value of > mg/ of has been proposed.

5 Meropenem activity against B.fragilisgroup 603 same medium and incubating at 35 C in anaerobic jars for -5-3 h, after which inducer was added to a final concentration equal to i x MIC. Incubation was then continued for an additional h (Sanders & Sanders, 96). The activity of against this induced strain was assessed by the agar dilution method (National Committee for Clinical Laboratory Standards, 99). Results The susceptibilities of 0 strains from the B.fragilis group to and other agents are presented in Table I. The activity of (MICs of 006- mg/) was similar to that of (MICs of 006- mg/) and (MICs of 003- mg/), and greater than that of the other antimicrobial agents tested. Meropenem (MIC*, of 0-5 mg/) was more active than (MIC,,, of mg/), (MIC,*, of mg/) and (MIC,,, of mg/) against B.fragilis. Meropenem and had similar activity against B.fragilis and B. thetaiotaomicron (MIC,,, of mg/). All isolates of B. ovatus, B. vulgatus and B. distasonis were sensitive to and. Both of these carbapenems had similar activity and resistance was not observed. MBCs of were only two-fold higher than the corresponding MICs {B.fragilis NCTC 933: MIC = 05 mg/, MBC = 0-5 mg/; B.fragilis -9: MIC = 0-5 mg/, MBC = 0-5 mg/), and were consistently bactericidal. The MBCs of were two-fold higher than the measured MICs (B. fragilis NCTC 933: MIC = 0-5 mg/, MBC = 0-5 mg/; B.fragilis -9: MIC = 0-5 mg/, MBC = 0-5 mg/). The bactericidal activity was confirmed by time-kill curve assays which indicated a rapid bactericidal activity (Figure ). In each instance, there was a three-log Time (h) Figure. Killing curves of B.fragilis strains exposed to the MBC of or. B.fragilis NCTC 933: control,, ; B.fragilis -9: control Q, O, O-

6 60 J. A. Garcia-Rodriguez et al. 6 Time (h) following exposure Figure. Postantibiotic effect following exposure of two strains of B. fragilis to x MIC of meropencm or. B. fragilis NCTC 933:, ; B. fragilis -9: D, O- decrease in viability within 6 h of exposure to the MBC of or. Identical results were obtained when the experiment was repeated. Meropenem showed an interesting PAE (Figure ). After removal of the antibiotic ( x MIC), both B. fragilis strains tested were found to require - h to increase their counts ten-fold. A similar PAE was observed with. The Inhibitory MAC of each antibiotic was x MIC for both strains. Identical results were obtained when the experiment was repeated. Adjustment of the ph of Wilkins-Chalgren agar to ph 6, 7 or did not significantly alter the MICs of, which were 05, 05 and 0-5 mg/, respectively, for both B. fragilis strains tested. Similarly, a variation in the inoculum size (0, 0 5 or 0 7 cfu/spot) had no significant effect on the MICs of (05, 05 and 0-5 mg/, respectively, for B. fragilis NCTC 933; 05, 05 and mg/, respectively, for B. fragilis -9). Meropenem remained active against B. fragilis -9 following induction of the chromosomal cephalosporinase produced by this strain. MICs before and after induction were 05 and mg/ respectively. Discussion The penem and carbapenem classes of antimicrobial agents have rarely produced viable candidate compounds for the chemotherapy of serious infections (Wise, 96). The only agent currently available is, which possesses the widest spectrum of antibacterial activity among the /Mactams (Jones, 95). However, the metabolism of by human renal dehydropeptidase I (DHP-I) requires the co-administration of cilastatin, a DHP-I inhibitor (Kropp et al, 9). The development of DHP-I-stable carbapenems such as removes the need for the co-drug.

7 Meropenem activity against B. fragilis group 6 Meropenem has a spectrum of antimicrobial activity very similar to that described for (Jones, 95; Edwards et al., 99; Jones et al., 99a, b; King, Boothman & Phillips, 99). In the present study, was active against all the bacteria from the B. fragilis group that were tested and its activity was similar to that of. All strains included were inhibited by at < mg/. Like, appears to be unaffected by various types of /Mactamases, including the new expanded-spectrum enzymes (Edwards et al., 99; Sanders et al., 99). The good activity of in vitro against the B. fragilis group probably resulted from a combination of factors, including resistance to hydrolysis by /Mactamases, rapid permeation into the cell, and/or great affinity for targets in the cell (Yoshimura & Nikaido, 95; Williams, Yang & Livermore, 96; Jones et al, 99a). The bactericidal activity of and was impressive, with MBC to MIC ratios that were no greater than two. Evaluation of the bactericidal activity by determination of the killing curves for two strains of B. fragilis indicated that concentrations equal to x MIC (MBC) produced a significant reduction in the viable cell count after 6 h. This bactericidal effect was slightly greater with, although the time-kill curves showed that both agents were rapidly bactericidal. The clinical outcome of infections treated with /Mactam drugs may be enhanced by a PAE, which may prevent regrowth or reduce the growth rate of organisms between drug doses when the drug concentration at the site of infection decreases to sub-mic levels (Craig & Gudmundsson, 96). In addition to strong and rapid bactericidal activity, and can produce a PAE in vitro on B. fragilis. The PAE obtained with and (- h) may be related to the growth rate of B. fragilis, which is much slower than that of Escherichia coli (the organism most frequently used to determine a PAE). Several authors have demonstrated that antibiotic concentrations lower than the MIC can affect bacteria structurally, and significantly diminish the number of cfu/ml (Lorian, 90). Both and showed a good inhibitory MAC, while MICs were affected minimally by variations in ph or changes in the inoculum size. Although, like, is a potent inducer of Class I /Mactamases, its activity was unaffected by the induction of this enzyme in B. fragilis strain -9. The activity of against strains possessing specific /Mactamases, its resistance to hydrolysis and activity as an enzyme inhibitor have been reported previously (Edwards et al., 99; Sanders et al., 99). Our results confirm the high degree of stability of to this enzyme and its excellent activity against cephalosporinase-induced strains of B. fragilis. The in-vitro data presented, and the fact that combines the broadspectrum of activity and resistance to /Mactamases intrinsic to with enhanced activity against a variety of Gram-negative organisms and increased stability to human renal DHP-I (Jones et al., 996), indicates that is a promising antimicrobial agent which may be useful as an alternative in the treatment of problematic mixed infections. References Craig, W. A. & Gudmundsson, S. (96). The postantibiotic effect. In Antibiotics in Laboratory Medicine, nd edn (Lorian, V., Ed.), pp Williams & Wilkins, Baltimore, MD. Edwards, J. R., Turner, P. J., Wannop, C, Withnell, E. S., Grindey, A. J. & Nairn, K. (99).

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