Journal of Antimicrobial Chemotherapy (1991) 27, 599-606
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
Meropenem was compared with imipenem and nine other antimicrobial agents, against 101 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 imipenem. The activity of meropenem was similar to that of metronidazole, and greater than that of the other antimicrobial agents tested. The bactericidal activity of meropenem 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 meropenem was rapidly bactericidal and reduced the initial inoculum significantly during the first 4-6 h. The postantibiotic effect of meropenem (2-4 h) and a sub-inhibitory concentration of i x MIC suggested that meropenem interferes with the normal growth of B. fragilis, even when administered concentrations fall below the MIC. MICs of meropenem 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 meropenem is a promising antimicrobial agent which may be useful in the treatment of problematic mixed infections.
Introduction Meropenem is a new carbapenem which resembles imipenem in terms of structure and a broad spectrum of antimicrobial activity (Jones, 1985). It also possesses great stability to /Mactamase hydrolysis (Edwards et ai, 1989; Jones et al., 1989a, b; Sanders et al., 1989) and is more stable than imipenem to human renal dehydropeptidase I (Wise, 1986; Edwards et al, 1989; Jones et al., 19896). The purpose of the present study was to determine the in-vitro activity of meropenem against members of the Bacteroides fragilis group, and the kinetics of activity against two strains of B. fragilis. We report the antimicrobial activity of meropenem compared with nine other drugs, the bactericidal activity (MBC), the postantibiotic effect (PAE), and the sub-MIC effects of meropenem. Also studied were the influence of a variable inoculum size, the effect of medium pH on MICs of meropenem, and the activity of meropenem against a strain of B. fragilis induced for the production of chromosomal /Mactamase. 599 0305-7453/91/050599+08 $02.00/0
© 1991 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on July 4, 2015
Department of Microbiology, Hospital Clinico Universitario, Paseo de San Vicente 108, 37007 Salamanca, Spain
600
J. A. Garcia-Rodrigoez et at. Materials and methods
Organisms A total of 99 recent clinical isolates belonging to the B. fragilis group (59 B. fragilis, 20 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 32 A system (bioMerieux, Montalieu Vercieu, France). B. fragilis strains NCTC 9343, 1-19422, and B. thetaiotaomicron ATCC 29741 were used for additional tests. Antibiotics
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 (1989). The organisms were cultured on Wilkins-Chalgren agar plates, supplemented with 5% sheep blood, and incubated for 24-48 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 24 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 1 x 105 cfu/spot since the inoculum replicator (Steer's replicator) deposited 0001 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 4°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 48 h. Each test was monitored by including B. fragilis NCTC 9343 and B. thetaiotaomicron ATCC 29741. 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 (1987). A broth macrodilution method was used to establish the MBC values for selected strains (B. fragilis NCTC 9343 and 1-19422) prior to
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on July 4, 2015
The antibiotics used were gifts from the various manufacturers. These antibiotics were: meropenem (ICI), imipenem and cefoxitin (Merck, Sharp & Dohme), piperacillin (Lederle), mezlocillin (Bayer), cefotaxime (Roussel), latamoxef (Eli Lilly), clindamycin (Upjohn), chloramphenicol (Parke-Davis), and metronidazole (Rhone-Poulenc). Stocks of meropenem and imipenem 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 0008-128 mg/1.
Meropenem activity against B. fragiUs group
601
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 meropenem and imipenem was confirmed by time-kill curve assays (National Committee for Clinical Laboratory Standards, 1987). PAE and sub-MIC determinations
Effect of inoculum size and pH of the medium The influence of inoculum size on MIC results was assessed with inoculum sizes of 10", 105 or 106 cfu/spot (photospectrometric adjustment of a culture derived from a 24 h plate, verified by colony counts) on Wilkins-Chalgren agar (Edwards et ai, 1989). The effect of pH on MICs of meropenem was assessed by using Wilkins-Chalgren agar which had been adjusted during preparation to pH 6, 7 or 8 (Edwards et al., 1989). The pH was measured with a pH meter (PHM 83 Autocal Radiometer) and a surface electrode (Tecan) and, whenever necessary, adjusted with 1 N HO or 1 N NaOH. MICs of meropenem with 104, 105 or 106 cfu/spot and pH 6, 7 or 8, were determined as recommended by the National Committee for Clinical Laboratory Standards (1989). Activity against a B. fragilis strain induced for the production of cephalosporinase Induction of chromosomal /3-lactamase by meropenem in a strain of B. fragilis (1-19422) was achieved by diluting a 24 h Wilkins-Chalgren broth culture 1:20 in the
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on July 4, 2015
Four or five colonies from a 24-48 h Wilkins-Chalgren blood agar culture of each test strain were inoculated into a tube containing 5-6 ml of Wilkins-Chalgren broth. After 4-6 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 1:200 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 1 h. After 2-5 h exposure to meropenem or imipenem at 8 x MIC, the drug concentration was reduced to sub-MIC levels by a 1000-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 meropenem resumed logarithmic growth. Viable counts were determined at 1, 2, 4 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, 1986). The sub-MIC concentrations (Lorian, 1980) 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 1, i, i and £xMIC to determine the MAC 'inhibition' effect.
Table I. In-vitro activity of meropenem and other antimicrobial agents against 101 strains from the Bacteroides fragilis group
Organism B. fragilis
B. thetaiotaomicron
B. vulgatus
B. distasonis
60
21
10
5
5
meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole
range 012-2 O06-0-5 4->128 4->128 4-32 2->128 025-128 O03->128
1-8 012-2 O06-4 O06-4 1—> 128 l - > 128 4-64 2-128 2->128 O06->128 1-4 O03-2 O06-O5 O06-2 8-> 128 16-> 128 4-32 4-128 4->128 025-64 2-16 012-1 006-2 O06-2 4->128 4->128 4-32 16-64
1-8 0125 2-4 05-4 O06-O-5 O06-4 8->128 8-> 128 4-64
4->128 2-64
025-> 128 1-8 025-1
MIC (mg/1) 50 012 025 16 32
8 16 2
025
90
05 05 >128
128 16 >128 32 16
2
8
0-5
2 1 1
025
025 32 16 16 16 16
05 4 05 012
025 32 64
8 16 16 05 4 025 025 025 8 8 4 16 2 012 4 05 012
025 32 64 8
32 8 05 4
05
32 32 32 64 64 2 4 2 05 1 >128 >128 32 128 64
8 8 1 05 1 128 128 8 64
%R° — — 13 13
8 40 12 17 — — — —
9 10 38 52 30
9 — — — — 30 40 20 40
50 20 10 — — — 40 40 20
60
8 012 4 1 05 1 128 128 32 64 64
8 4 1
— — — — — 40 40 40 80 40 40 — —
"% Resistant as defined by the recommended criteria (National Committee for Clinical Laboratory Standards, 1985, 1988). A provisional value of > 8 mg/1 of meropenem has been proposed.
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B. ovatus
No. of strains Compound
Meropenem activity against B.fragilisgroup
603
same medium and incubating at 35°C in anaerobic jars for 2-5-3 h, after which inducer was added to a final concentration equal to i x MIC. Incubation was then continued for an additional 2 h (Sanders & Sanders, 1986). The activity of meropenem against this induced strain was assessed by the agar dilution method (National Committee for Clinical Laboratory Standards, 1989). Results
Time ( h )
Figure 1. Killing curves of B.fragilis strains exposed to the MBC of meropenem or imipenem. B.fragilis NCTC 9343: control • , meropenem • , imipenem • ; B.fragilis 1-19422: control Q, meropenem O, imipenem O-
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The susceptibilities of 101 strains from the B.fragilis group to meropenem and other agents are presented in Table I. The activity of meropenem (MICs of 006-4 mg/1) was similar to that of imipenem (MICs of 006-4 mg/1) and metronidazole (MICs of 003-4 mg/1), and greater than that of the other antimicrobial agents tested. Meropenem (MIC*, of 0-5 mg/1) was more active than clindamycin (MIC,,, of 16 mg/1), chloramphenicol (MIC,*, of 8 mg/1) and metronidazole (MIC,,, of 2 mg/1) against B.fragilis. Meropenem and imipenem had similar activity against B.fragilis and B. thetaiotaomicron (MIC,,, of 1 mg/1). All isolates of B. ovatus, B. vulgatus and B. distasonis were sensitive to meropenem and imipenem. Both of these carbapenems had similar activity and resistance was not observed. MBCs of meropenem were only two-fold higher than the corresponding MICs {B.fragilis NCTC 9343: MIC = 0125 mg/1, MBC = 0-25 mg/1; B.fragilis 1-19422: MIC = 0-125 mg/1, MBC = 0-25 mg/1), and were consistently bactericidal. The MBCs of imipenem were two-fold higher than the measured MICs (B. fragilis NCTC 9343: MIC = 0-125 mg/1, MBC = 0-25 mg/1; B.fragilis 1-19422: MIC = 0-25 mg/1, MBC = 0-5 mg/1). The bactericidal activity was confirmed by time-kill curve assays which indicated a rapid bactericidal activity (Figure 1). In each instance, there was a three-log
604
J. A. Garcia-Rodriguez et al.
6
Figure 2. Postantibiotic effect following exposure of two strains of B. fragilis to 8 x MIC of meropencm or imipenem. B. fragilis NCTC 9343: meropenem • , imipenem • ; B. fragilis 1-19422: meropenem D, imipenem O-
decrease in viability within 6 h of exposure to the MBC of meropenem or imipenem. Identical results were obtained when the experiment was repeated. Meropenem showed an interesting PAE (Figure 2). After removal of the antibiotic (8 x MIC), both B. fragilis strains tested were found to require 2-4 h to increase their counts ten-fold. A similar PAE was observed with imipenem. 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 8 did not significantly alter the MICs of meropenem, which were 0125, 0125 and 0-25 mg/1, respectively, for both B. fragilis strains tested. Similarly, a variation in the inoculum size (104, 105 or 107 cfu/spot) had no significant effect on the MICs of meropenem (0125, 0125 and 0-25 mg/1, respectively, for B. fragilis NCTC 9343; 0125, 0125 and 05 mg/1, respectively, for B. fragilis 1-19422). Meropenem remained active against B. fragilis 1-19422 following induction of the chromosomal cephalosporinase produced by this strain. MICs before and after induction were 0125 and 05 mg/1 respectively. Discussion The penem and carbapenem classes of antimicrobial agents have rarely produced viable candidate compounds for the chemotherapy of serious infections (Wise, 1986). The only agent currently available is imipenem, which possesses the widest spectrum of antibacterial activity among the /Mactams (Jones, 1985). However, the metabolism of imipenem by human renal dehydropeptidase I (DHP-I) requires the co-administration of cilastatin, a DHP-I inhibitor (Kropp et al, 1982). The development of DHP-I-stable carbapenems such as meropenem removes the need for the co-drug.
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2 4 Time ( h ) following exposure
Meropenem activity against B. fragilis group
605
References Craig, W. A. & Gudmundsson, S. (1986). The postantibiotic effect. In Antibiotics in Laboratory Medicine, 2nd edn (Lorian, V., Ed.), pp. 515-36. Williams & Wilkins, Baltimore, MD. Edwards, J. R., Turner, P. J., Wannop, C, Withnell, E. S., Grindey, A. J. & Nairn, K. (1989).
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Meropenem has a spectrum of antimicrobial activity very similar to that described for imipenem (Jones, 1985; Edwards et al., 1989; Jones et al., 1989a, b; King, Boothman & Phillips, 1989). In the present study, meropenem was active against all the bacteria from the B. fragilis group that were tested and its activity was similar to that of imipenem. All strains included were inhibited by meropenem at < 4 mg/1. Like imipenem, meropenem appears to be unaffected by various types of /Mactamases, including the new expanded-spectrum enzymes (Edwards et al., 1989; Sanders et al., 1989). The good activity of meropenem 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, 1985; Williams, Yang & Livermore, 1986; Jones et al, 1989a). The bactericidal activity of meropenem and imipenem 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 2 x MIC (MBC) produced a significant reduction in the viable cell count after 6 h. This bactericidal effect was slightly greater with meropenem, 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, 1986). In addition to strong and rapid bactericidal activity, meropenem and imipenem can produce a PAE in vitro on B. fragilis. The PAE obtained with meropenem and imipenem (2-4 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, 1980). Both meropenem and imipenem showed a good inhibitory MAC, while meropenem MICs were affected minimally by variations in pH or changes in the inoculum size. Although meropenem, like imipenem, is a potent inducer of Class I /Mactamases, its activity was unaffected by the induction of this enzyme in B. fragilis strain 1-19422. The activity of meropenem against strains possessing specific /Mactamases, its resistance to hydrolysis and activity as an enzyme inhibitor have been reported previously (Edwards et al., 1989; Sanders et al., 1989). Our results confirm the high degree of stability of meropenem to this enzyme and its excellent activity against cephalosporinase-induced strains of B. fragilis. The in-vitro data presented, and the fact that meropenem combines the broadspectrum of activity and resistance to /Mactamases intrinsic to imipenem with enhanced activity against a variety of Gram-negative organisms and increased stability to human renal DHP-I (Jones et al., 19896), indicates that meropenem is a promising antimicrobial agent which may be useful as an alternative in the treatment of problematic mixed infections.
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(Received 10 September 1990; revised version accepted 28 December 1990)
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In vitro antibacterial activity of SM-7338, a carbapenem antibiotic with stability to dehydropeptidase 1. Antimicrobial Agents and Chemotherapy 33, 215-22. Jones, R. N. (1985). Review of the in vitro spectrum of activity of imipenem. American Journal of Medicine 78, Suppl. 6A, 22-32. Jones, R. N., Aldridge, K. E., Allen, S. D., Barry, A. L., Fuchs, P. C , Gerlach, E. H. el al. (1989a). Multicenter in vitro evaluation of SM-7338, a new carbapenem. Antimicrobial Agents and Chemotherapy 33, 562-5. Jones, R. N., Barry, A. L. & Thornsberry, C. (1989A). In-vitro studies on meropenem. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 9-29. King, A., Boothman, C. & Phillips, I. (1989). Comparative in-vitro activity of meropenem on clinical isolates from the United Kingdom. Journal of Antimicrobial Chemotherapy 24, Suppl. ,4, 31-45. Kropp, H., Sundelof, J. G., Hajdu, R. & Kahan, F. M. (1982). Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase. Antimicrobial Agents and Chemotherapy 11, 62-70. Lorian, V. (1980). Effects of subminimum inhibitory concentrations of antibiotics in bacteria. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 342-408. Williams & Wilkins, Baltimore, MD. National Committee for Clinical Laboratory Standards. (1985). Standard Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria which Grow Aerobically. Approved Standard Ml-A. NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1987). Methods for Determining Bactericidal Activity of Antimicrobial Agents; Proposed Guideline M26-P. NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1988). Performance Standards for Antimicrobial Susceptibility Testing; Second Informational Supplement, M100-S2 (Revised). NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1989). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 2nd edn; Tentative Standard Ml 1-T2. NCCLS, Villanova, PA. Sanders, C. C. & Sanders, W. E. (1986). Type I /Mactamases of gram-negative bacteria: interactions with /?-lactam antibiotics. Journal of Infectious Diseases 154, 792-800. Sanders, C. C , Sanders, W. E., Thomson, K. S. & Iaconis, J. P. (1989). Meropenem: activity against resistant Gram-negative bacteria and interactions with /Mactamases. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 187-96. Williams, R. J., Yang, Y. J. & Livermore, D. M. (1986). Mechanisms by which imipenem may overcome resistance in Gram-negative bacilli. Journal of Antimicrobial Chemotherapy 18, Suppl. E, 9-13. Wise, R. (1986). In vitro and pharmacokinetic properties of the carbapenems. Antimicrobial Agents and Chemotherapy 30, 343-9. , Yoshimura, F. & Nikaido, H. (1985). Diffusion of /Mactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrobial Agents and Chemotherapy 27, 84-92.
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
Meropenem was compared with imipenem and nine other antimicrobial agents, against 101 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 imipenem. The activity of meropenem was similar to that of metronidazole, and greater than that of the other antimicrobial agents tested. The bactericidal activity of meropenem 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 meropenem was rapidly bactericidal and reduced the initial inoculum significantly during the first 4-6 h. The postantibiotic effect of meropenem (2-4 h) and a sub-inhibitory concentration of i x MIC suggested that meropenem interferes with the normal growth of B. fragilis, even when administered concentrations fall below the MIC. MICs of meropenem 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 meropenem is a promising antimicrobial agent which may be useful in the treatment of problematic mixed infections.
Introduction Meropenem is a new carbapenem which resembles imipenem in terms of structure and a broad spectrum of antimicrobial activity (Jones, 1985). It also possesses great stability to /Mactamase hydrolysis (Edwards et ai, 1989; Jones et al., 1989a, b; Sanders et al., 1989) and is more stable than imipenem to human renal dehydropeptidase I (Wise, 1986; Edwards et al, 1989; Jones et al., 19896). The purpose of the present study was to determine the in-vitro activity of meropenem against members of the Bacteroides fragilis group, and the kinetics of activity against two strains of B. fragilis. We report the antimicrobial activity of meropenem compared with nine other drugs, the bactericidal activity (MBC), the postantibiotic effect (PAE), and the sub-MIC effects of meropenem. Also studied were the influence of a variable inoculum size, the effect of medium pH on MICs of meropenem, and the activity of meropenem against a strain of B. fragilis induced for the production of chromosomal /Mactamase. 599 0305-7453/91/050599+08 $02.00/0
© 1991 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on July 4, 2015
Department of Microbiology, Hospital Clinico Universitario, Paseo de San Vicente 108, 37007 Salamanca, Spain
600
J. A. Garcia-Rodrigoez et at. Materials and methods
Organisms A total of 99 recent clinical isolates belonging to the B. fragilis group (59 B. fragilis, 20 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 32 A system (bioMerieux, Montalieu Vercieu, France). B. fragilis strains NCTC 9343, 1-19422, and B. thetaiotaomicron ATCC 29741 were used for additional tests. Antibiotics
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 (1989). The organisms were cultured on Wilkins-Chalgren agar plates, supplemented with 5% sheep blood, and incubated for 24-48 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 24 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 1 x 105 cfu/spot since the inoculum replicator (Steer's replicator) deposited 0001 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 4°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 48 h. Each test was monitored by including B. fragilis NCTC 9343 and B. thetaiotaomicron ATCC 29741. 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 (1987). A broth macrodilution method was used to establish the MBC values for selected strains (B. fragilis NCTC 9343 and 1-19422) prior to
Downloaded from http://jac.oxfordjournals.org/ at University of Otago on July 4, 2015
The antibiotics used were gifts from the various manufacturers. These antibiotics were: meropenem (ICI), imipenem and cefoxitin (Merck, Sharp & Dohme), piperacillin (Lederle), mezlocillin (Bayer), cefotaxime (Roussel), latamoxef (Eli Lilly), clindamycin (Upjohn), chloramphenicol (Parke-Davis), and metronidazole (Rhone-Poulenc). Stocks of meropenem and imipenem 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 0008-128 mg/1.
Meropenem activity against B. fragiUs group
601
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 meropenem and imipenem was confirmed by time-kill curve assays (National Committee for Clinical Laboratory Standards, 1987). PAE and sub-MIC determinations
Effect of inoculum size and pH of the medium The influence of inoculum size on MIC results was assessed with inoculum sizes of 10", 105 or 106 cfu/spot (photospectrometric adjustment of a culture derived from a 24 h plate, verified by colony counts) on Wilkins-Chalgren agar (Edwards et ai, 1989). The effect of pH on MICs of meropenem was assessed by using Wilkins-Chalgren agar which had been adjusted during preparation to pH 6, 7 or 8 (Edwards et al., 1989). The pH was measured with a pH meter (PHM 83 Autocal Radiometer) and a surface electrode (Tecan) and, whenever necessary, adjusted with 1 N HO or 1 N NaOH. MICs of meropenem with 104, 105 or 106 cfu/spot and pH 6, 7 or 8, were determined as recommended by the National Committee for Clinical Laboratory Standards (1989). Activity against a B. fragilis strain induced for the production of cephalosporinase Induction of chromosomal /3-lactamase by meropenem in a strain of B. fragilis (1-19422) was achieved by diluting a 24 h Wilkins-Chalgren broth culture 1:20 in the
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Four or five colonies from a 24-48 h Wilkins-Chalgren blood agar culture of each test strain were inoculated into a tube containing 5-6 ml of Wilkins-Chalgren broth. After 4-6 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 1:200 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 1 h. After 2-5 h exposure to meropenem or imipenem at 8 x MIC, the drug concentration was reduced to sub-MIC levels by a 1000-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 meropenem resumed logarithmic growth. Viable counts were determined at 1, 2, 4 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, 1986). The sub-MIC concentrations (Lorian, 1980) 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 1, i, i and £xMIC to determine the MAC 'inhibition' effect.
Table I. In-vitro activity of meropenem and other antimicrobial agents against 101 strains from the Bacteroides fragilis group
Organism B. fragilis
B. thetaiotaomicron
B. vulgatus
B. distasonis
60
21
10
5
5
meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole meropenem imipenem piperacillin mezlocillin cefoxitin cefotaxime latamoxef clindamycin chloramphenicol metronidazole
range 012-2 O06-0-5 4->128 4->128 4-32 2->128 025-128 O03->128
1-8 012-2 O06-4 O06-4 1—> 128 l - > 128 4-64 2-128 2->128 O06->128 1-4 O03-2 O06-O5 O06-2 8-> 128 16-> 128 4-32 4-128 4->128 025-64 2-16 012-1 006-2 O06-2 4->128 4->128 4-32 16-64
1-8 0125 2-4 05-4 O06-O-5 O06-4 8->128 8-> 128 4-64
4->128 2-64
025-> 128 1-8 025-1
MIC (mg/1) 50 012 025 16 32
8 16 2
025
90
05 05 >128
128 16 >128 32 16
2
8
0-5
2 1 1
025
025 32 16 16 16 16
05 4 05 012
025 32 64
8 16 16 05 4 025 025 025 8 8 4 16 2 012 4 05 012
025 32 64 8
32 8 05 4
05
32 32 32 64 64 2 4 2 05 1 >128 >128 32 128 64
8 8 1 05 1 128 128 8 64
%R° — — 13 13
8 40 12 17 — — — —
9 10 38 52 30
9 — — — — 30 40 20 40
50 20 10 — — — 40 40 20
60
8 012 4 1 05 1 128 128 32 64 64
8 4 1
— — — — — 40 40 40 80 40 40 — —
"% Resistant as defined by the recommended criteria (National Committee for Clinical Laboratory Standards, 1985, 1988). A provisional value of > 8 mg/1 of meropenem has been proposed.
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B. ovatus
No. of strains Compound
Meropenem activity against B.fragilisgroup
603
same medium and incubating at 35°C in anaerobic jars for 2-5-3 h, after which inducer was added to a final concentration equal to i x MIC. Incubation was then continued for an additional 2 h (Sanders & Sanders, 1986). The activity of meropenem against this induced strain was assessed by the agar dilution method (National Committee for Clinical Laboratory Standards, 1989). Results
Time ( h )
Figure 1. Killing curves of B.fragilis strains exposed to the MBC of meropenem or imipenem. B.fragilis NCTC 9343: control • , meropenem • , imipenem • ; B.fragilis 1-19422: control Q, meropenem O, imipenem O-
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The susceptibilities of 101 strains from the B.fragilis group to meropenem and other agents are presented in Table I. The activity of meropenem (MICs of 006-4 mg/1) was similar to that of imipenem (MICs of 006-4 mg/1) and metronidazole (MICs of 003-4 mg/1), and greater than that of the other antimicrobial agents tested. Meropenem (MIC*, of 0-5 mg/1) was more active than clindamycin (MIC,,, of 16 mg/1), chloramphenicol (MIC,*, of 8 mg/1) and metronidazole (MIC,,, of 2 mg/1) against B.fragilis. Meropenem and imipenem had similar activity against B.fragilis and B. thetaiotaomicron (MIC,,, of 1 mg/1). All isolates of B. ovatus, B. vulgatus and B. distasonis were sensitive to meropenem and imipenem. Both of these carbapenems had similar activity and resistance was not observed. MBCs of meropenem were only two-fold higher than the corresponding MICs {B.fragilis NCTC 9343: MIC = 0125 mg/1, MBC = 0-25 mg/1; B.fragilis 1-19422: MIC = 0-125 mg/1, MBC = 0-25 mg/1), and were consistently bactericidal. The MBCs of imipenem were two-fold higher than the measured MICs (B. fragilis NCTC 9343: MIC = 0-125 mg/1, MBC = 0-25 mg/1; B.fragilis 1-19422: MIC = 0-25 mg/1, MBC = 0-5 mg/1). The bactericidal activity was confirmed by time-kill curve assays which indicated a rapid bactericidal activity (Figure 1). In each instance, there was a three-log
604
J. A. Garcia-Rodriguez et al.
6
Figure 2. Postantibiotic effect following exposure of two strains of B. fragilis to 8 x MIC of meropencm or imipenem. B. fragilis NCTC 9343: meropenem • , imipenem • ; B. fragilis 1-19422: meropenem D, imipenem O-
decrease in viability within 6 h of exposure to the MBC of meropenem or imipenem. Identical results were obtained when the experiment was repeated. Meropenem showed an interesting PAE (Figure 2). After removal of the antibiotic (8 x MIC), both B. fragilis strains tested were found to require 2-4 h to increase their counts ten-fold. A similar PAE was observed with imipenem. 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 8 did not significantly alter the MICs of meropenem, which were 0125, 0125 and 0-25 mg/1, respectively, for both B. fragilis strains tested. Similarly, a variation in the inoculum size (104, 105 or 107 cfu/spot) had no significant effect on the MICs of meropenem (0125, 0125 and 0-25 mg/1, respectively, for B. fragilis NCTC 9343; 0125, 0125 and 05 mg/1, respectively, for B. fragilis 1-19422). Meropenem remained active against B. fragilis 1-19422 following induction of the chromosomal cephalosporinase produced by this strain. MICs before and after induction were 0125 and 05 mg/1 respectively. Discussion The penem and carbapenem classes of antimicrobial agents have rarely produced viable candidate compounds for the chemotherapy of serious infections (Wise, 1986). The only agent currently available is imipenem, which possesses the widest spectrum of antibacterial activity among the /Mactams (Jones, 1985). However, the metabolism of imipenem by human renal dehydropeptidase I (DHP-I) requires the co-administration of cilastatin, a DHP-I inhibitor (Kropp et al, 1982). The development of DHP-I-stable carbapenems such as meropenem removes the need for the co-drug.
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2 4 Time ( h ) following exposure
Meropenem activity against B. fragilis group
605
References Craig, W. A. & Gudmundsson, S. (1986). The postantibiotic effect. In Antibiotics in Laboratory Medicine, 2nd edn (Lorian, V., Ed.), pp. 515-36. Williams & Wilkins, Baltimore, MD. Edwards, J. R., Turner, P. J., Wannop, C, Withnell, E. S., Grindey, A. J. & Nairn, K. (1989).
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Meropenem has a spectrum of antimicrobial activity very similar to that described for imipenem (Jones, 1985; Edwards et al., 1989; Jones et al., 1989a, b; King, Boothman & Phillips, 1989). In the present study, meropenem was active against all the bacteria from the B. fragilis group that were tested and its activity was similar to that of imipenem. All strains included were inhibited by meropenem at < 4 mg/1. Like imipenem, meropenem appears to be unaffected by various types of /Mactamases, including the new expanded-spectrum enzymes (Edwards et al., 1989; Sanders et al., 1989). The good activity of meropenem 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, 1985; Williams, Yang & Livermore, 1986; Jones et al, 1989a). The bactericidal activity of meropenem and imipenem 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 2 x MIC (MBC) produced a significant reduction in the viable cell count after 6 h. This bactericidal effect was slightly greater with meropenem, 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, 1986). In addition to strong and rapid bactericidal activity, meropenem and imipenem can produce a PAE in vitro on B. fragilis. The PAE obtained with meropenem and imipenem (2-4 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, 1980). Both meropenem and imipenem showed a good inhibitory MAC, while meropenem MICs were affected minimally by variations in pH or changes in the inoculum size. Although meropenem, like imipenem, is a potent inducer of Class I /Mactamases, its activity was unaffected by the induction of this enzyme in B. fragilis strain 1-19422. The activity of meropenem against strains possessing specific /Mactamases, its resistance to hydrolysis and activity as an enzyme inhibitor have been reported previously (Edwards et al., 1989; Sanders et al., 1989). Our results confirm the high degree of stability of meropenem to this enzyme and its excellent activity against cephalosporinase-induced strains of B. fragilis. The in-vitro data presented, and the fact that meropenem combines the broadspectrum of activity and resistance to /Mactamases intrinsic to imipenem with enhanced activity against a variety of Gram-negative organisms and increased stability to human renal DHP-I (Jones et al., 19896), indicates that meropenem is a promising antimicrobial agent which may be useful as an alternative in the treatment of problematic mixed infections.
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(Received 10 September 1990; revised version accepted 28 December 1990)
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In vitro antibacterial activity of SM-7338, a carbapenem antibiotic with stability to dehydropeptidase 1. Antimicrobial Agents and Chemotherapy 33, 215-22. Jones, R. N. (1985). Review of the in vitro spectrum of activity of imipenem. American Journal of Medicine 78, Suppl. 6A, 22-32. Jones, R. N., Aldridge, K. E., Allen, S. D., Barry, A. L., Fuchs, P. C , Gerlach, E. H. el al. (1989a). Multicenter in vitro evaluation of SM-7338, a new carbapenem. Antimicrobial Agents and Chemotherapy 33, 562-5. Jones, R. N., Barry, A. L. & Thornsberry, C. (1989A). In-vitro studies on meropenem. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 9-29. King, A., Boothman, C. & Phillips, I. (1989). Comparative in-vitro activity of meropenem on clinical isolates from the United Kingdom. Journal of Antimicrobial Chemotherapy 24, Suppl. ,4, 31-45. Kropp, H., Sundelof, J. G., Hajdu, R. & Kahan, F. M. (1982). Metabolism of thienamycin and related carbapenem antibiotics by the renal dipeptidase, dehydropeptidase. Antimicrobial Agents and Chemotherapy 11, 62-70. Lorian, V. (1980). Effects of subminimum inhibitory concentrations of antibiotics in bacteria. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 342-408. Williams & Wilkins, Baltimore, MD. National Committee for Clinical Laboratory Standards. (1985). Standard Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria which Grow Aerobically. Approved Standard Ml-A. NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1987). Methods for Determining Bactericidal Activity of Antimicrobial Agents; Proposed Guideline M26-P. NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1988). Performance Standards for Antimicrobial Susceptibility Testing; Second Informational Supplement, M100-S2 (Revised). NCCLS, Villanova, PA. National Committee for Clinical Laboratory Standards. (1989). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 2nd edn; Tentative Standard Ml 1-T2. NCCLS, Villanova, PA. Sanders, C. C. & Sanders, W. E. (1986). Type I /Mactamases of gram-negative bacteria: interactions with /?-lactam antibiotics. Journal of Infectious Diseases 154, 792-800. Sanders, C. C , Sanders, W. E., Thomson, K. S. & Iaconis, J. P. (1989). Meropenem: activity against resistant Gram-negative bacteria and interactions with /Mactamases. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 187-96. Williams, R. J., Yang, Y. J. & Livermore, D. M. (1986). Mechanisms by which imipenem may overcome resistance in Gram-negative bacilli. Journal of Antimicrobial Chemotherapy 18, Suppl. E, 9-13. Wise, R. (1986). In vitro and pharmacokinetic properties of the carbapenems. Antimicrobial Agents and Chemotherapy 30, 343-9. , Yoshimura, F. & Nikaido, H. (1985). Diffusion of /Mactam antibiotics through the porin channels of Escherichia coli K-12. Antimicrobial Agents and Chemotherapy 27, 84-92.
