The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA
Received 11 January 2005; returned 6 March 2005; revised 9 March 2005; accepted 21 March 2005
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Abstract |
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Methods: The in vitro susceptibilities of 2137 bacterial isolates, representing 28 different species, to doripenem, imipenem and meropenem were determined by the NCCLS broth microdilution and disc diffusion testing methods.
Results: The doripenem MIC50s/90s were (in mg/L) for Enterobacteriaceae, 0.06/0.25; Pseudomonas aeruginosa, 0.25/1; Haemophilus influenzae, 0.12/0.5; streptococci, 0.016/0.5 and for staphylococci, 0.06/4. Like other carbapenems tested, doripenem MIC50s/90s were >32/>32 and 0.5/32 mg/L for the enterococci and non-fermentative Gram-negative bacilli (excluding P. aeruginosa), respectively. Against members of the Enterobacteriaceae and H. influenzae, doripenem was generally more active than imipenem and the same as or slightly less active than meropenem. Values for the non-fermentative Gram-negative bacilli excluding P. aeruginosa were comparable for all three carbapenems. Doripenem MICs increased with increasing resistance to methicillin (staphylococci), penicillin (streptococci) and strains that were ß-lactamase-negative ampicillin-resistant (Haemophilus). Doripenem exhibits excellent activity against extended-spectrum ß-lactamase-producing strains of Escherichia coli and Klebsiella spp. The NCCLS disc diffusion test was performed simultaneously on all organisms.
Conclusions: Assuming the MIC susceptible breakpoints for doripenem are 1 mg/L for the streptococci and
2 mg/L for all other genera, then disc diffusion zone diameter breakpoints can be proposed. In addition, MIC and/or disc diffusion quality control ranges of doripenem were determined for 10 ATCC reference strains.
Keywords: spectrum , QC , breakpoints
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Introduction |
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The present study was designed to: (i) compare the in vitro antibacterial activity of doripenem with that of imipenem and meropenem against a broad range of bacterial pathogens for which doripenem might be considered for therapy; (ii) determine preliminary doripenem disc diffusion interpretive criteria for these microorganisms; and (iii) propose MIC and disc diffusion quality control ranges for 10 different aerobic and anaerobic quality control strains.
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Materials and methods |
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A total of 2137 recent clinical bacterial isolates were selected as representative pathogens that cause infections for which doripenem might be considered for therapy. These included 590 streptococci, 330 enterococci, 154 Staphylococcus aureus, 148 coagulase-negative staphylococci, 317 Enterobacteriaceae, 300 non-fermentative Gram-negative bacilli and 298 Haemophilus influenzae. This collection includes isolates with a variety of resistance profiles to other antimicrobial agents. The susceptibility of these isolates to other antibiotics has been predetermined to reflect various resistant profiles in the same species.
Antimicrobial susceptibility testing
Doripenem was provided as a sterile powder (lot no. D2X003) by Peninsula Pharmaceuticals. The comparison drugs, imipenem and meropenem, were acquired from their respective US manufacturers (imipenem from Merck & Co.; meropenem from AstraZeneca). Disc diffusion susceptibility tests used commercially prepared 10 µg discs of doripenem (Oxoid Limited, Basingstoke, UK), imipenem and meropenem (BD Microbiologic Systems, Cockeysville, MD, USA).
All aerobic organisms were tested by the broth microdilution method recommended by the NCCLS8 using cation-adjusted MuellerHinton broth. The medium was supplemented with 3% lysed horse blood for testing the streptococci or made up as Haemophilus Test Medium (HTM) for testing H. influenzae. All organisms were tested simultaneously by the disc diffusion method outlined by the NCCLS9 using MuellerHinton agar + 5% sheep blood (streptococci), HTM agar (H. influenzae) or plain MullerHinton agar (all other genera). For the quality control portion of the study, anaerobic bacteria were tested by the microbroth dilution method, as described by the NCCLS.10 The medium used was Brucella broth supplemented with 5% lysed horse blood, 1 mg/L of vitamin K1 and 5 mg/L of haemin.
MIC versus zone diameter scattergrams were prepared for each of the major groups of microorganisms. Using an error minimization approach,11 disc diffusion interpretive criteria are proposed. The zone diameter breakpoints suggested were designed to minimize the interpretive discrepancies between the two types of susceptibility testing methods. The tentative MIC breakpoints were those put forward by the sponsor based upon a conservative interpretation of previous in vivo studies and Monte Carlo simulations.1214
Quality control studies
An eight-laboratory study was undertaken in order to propose quality control ranges for MIC (aerobic and anaerobic strains) and disc diffusion (aerobic strains only) methodologies. The eight testing laboratories included both hospital and commercial microbiology laboratories in the USA. This study closely followed the protocol described by the NCCLS15 with the exception that eight testing facilities were used rather than the required seven. The quality control organisms were those recommended by the NCCLS810 and included S. aureus ATCC 29213 and ATCC 25923, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, H. influenzae ATCC 49247 and ATCC 49766, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741 and Eubacterium lentum ATCC 43055. Internal quality control results for the control drugs, imipenem or meropenem, were within published ranges available10,15 for the majority of tests. When any control value was out of the established ranges, all of the doripenem data associated with that day's testing were discarded. This study involved replicate tests on three lots of MuellerHinton broth or agar and two lots of 10 µg discs. This exercise generated 240 MICs and 480 disc diffusion zone diameters with each appropriate quality control strain. Zone diameters were evaluated using the statistics of Gavan et al.16
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Results and discussion |
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For the non-fermentative Gram-negative bacilli other than P. aeruginosa, the MIC90s of all three carbapenems were nearly identical in the respect that all values were within ±1 doubling dilution of each other (Table 1). The single exception to this rule was Burkholderia cepacia, in which the doripenem MIC90 was 2 log2 dilutions more active than imipenem, but equal to that of meropenem. Doripenem was more potent than imipenem and meropenem against P. aeruginosa isolates; with MIC90s of 1, 2 and 4 mg/L, respectively.
Doripenem was highly potent against the population of streptococci tested. Although doripenem MICs increased with increasing penicillin resistance, only one of 290 strains of S. pneumoniae and two of 300 strains of non-pneumococcal streptococci had a doripenem MIC 1 mg/L.
As expected for carbapenem class antibiotics, staphylococcal MICs of doripenem increased with resistance to methicillin or vancomycin. Doripenem MICs against methicillin-susceptible strains were low (MIC90=0.06 mg/L), while methicillin-resistant strains of S. aureus and coagulase-negative staphylococci showed higher MIC90s (8 and 4 mg/L, respectively). The Clinical Laboratory Standards Institute (CLSI, formerly the NCCLS) currently recommends that carbapenem results for oxacillin-resistant staphylococci be reported as resistant or not reported at all.15
The enterococci were generally resistant to doripenem and other carbapenems with an MIC90 of >32 mg/L for all enterococcal strains combined. E. faecalis doripenem MICs were generally lower than those for Enterococcus faecium (MIC90=8 versus >32 mg/L).
For H. influenzae, doripenem was 12 log2 dilutions more active than imipenem and 2 log2 dilutions less active than meropenem regardless of the production of ß-lactamase (Table 1). Strains that were ß-lactamase negative but ampicillin-intermediate or -resistant produced doripenem MIC90 s that were 48-fold higher than for other strains.
Figure 1 (a-f) displays the scattergrams of doripenem MICs versus disc diffusion zone diameters. Assuming MIC breakpoints for susceptible of 1 mg/L for the streptococci and
2 mg/L for all other species, satisfactory zone size breakpoints are proposed for all species under consideration.
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The error rates for Streptococcus spp. other than S. pneumoniae were all zero (Figure 1c). S. pneumoniae produced no very major and major error rates and only 5/290 (1.7%) minor errors (Figure 1d). All of the minor errors were from penicillin-resistant strains. Since the MIC90 for all enterococci combined was >32 mg/L, no enterococcal MIC breakpoints are proposed.
Proposing breakpoints for the carbapenems versus staphylococci has always been somewhat problematic. Error rates for carbapenems compared with staphylococci have traditionally been rather high, and doripenem is no exception. The CLSI disc-diffusion susceptible breakpoints for other carbapenems range from 1315 mm.15
If these breakpoints were to be proposed for doripenem, the major and minor error rates would be unacceptably high. Breakpoints of 9 mm for resistant produce much more desirable error rates, but these breakpoints are much lower than other drugs in this class. A more reasonable compromise is proposed as
10 mm for resistant, 1113 mm for intermediate and
14 mm for susceptible (Figure 1e). All of the errors observed with Staphylococcus spp. were from methicillin-resistant strains. There were no errors associated with methicillin-susceptible strains of Staphylococcus. As mentioned earlier, the CLSI currently recommends that all carbapenems be reported as resistant for oxacillin-resistant staphylococci, regardless of the carbapenem MIC, or not reported at all.
Figure 1(f) presents the activity of doripenem against 292 strains of H. influenzae, including 38 strains that are ß-lactamase-negative ampicillin-resistant (ßLNAR). All of the strains with a doripenem MIC of 4 mg/L and nine of the 11 strains with a doripenem MIC of 2 mg/L were ß-lactamase-negative ampicillin-intermediate or -resistant. The remaining two strains with a doripenem MIC of 2 mg/L were ß-lactamase-negative but ampicillin-susceptible. Since doripenem-resistant strains of H. influenzae were encountered, an intermediate MIC category of 4 mg/L should be seriously considered. With a susceptible category of 2 mg/L, intermediate of 4 mg/L and resistant of
8 mg/L, then disc diffusion breakpoints of
21 mm for susceptible, 1820 mm for intermediate and
17 mm for resistant can be proposed. This results in a minor error rate of 1.4% with no very major or major errors.
Quality control studies
Quality control ranges for MIC testing were proposed on the basis of the modal MIC values observed ± one log2 dilution. Disc diffusion zone diameter ranges were proposed using the method of Gavan et al.16 with adjustments as needed in order to encompass at least 95% of observed values. The proposed MIC and zone diameter ranges are presented in Tables 2 and 3. These quality control ranges were accepted by the Antimicrobial Susceptibility Testing Subcommittee of the NCCLS at their June 2004 meeting.
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The in vitro activity of doripenem against Gram-positive strains was similar to that of imipenem and slightly more active than meropenem. Against members of the Enterobacteriaceae and H. influenzae, doripenem was generally more active in vitro than imipenem and the same as meropenem. Values for the non-fermentative Gram-negative bacilli were comparable for all three carbapenems, with the exception that doripenem was the most active drug against most strains of P. aeruginosa. Doripenem MICs increased with increasing resistance to methicillin (staphylococci), penicillin (streptococci) and strains that are ß-lactamase-negative ampicillin-resistant (Haemophilus). Doripenem exhibits excellent activity against ESBL-producing strains of E. coli and Klebsiella spp. Disc-diffusion breakpoints are proposed based upon conservative interpretations of tentative MIC breakpoints proposed elsewhere. Final breakpoint determinations will be based upon the evaluation of pharmacokinetics, regression line analysis, overall discrepancy rates and clinical verification of breakpoints by clinical and bacteriological response rates as specified by the NCCLS.11 Quality control ranges for both MIC and disc diffusion methodologies have been accepted by the CLSI.
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Acknowledgements |
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References |
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