Comparative in vitro antimicrobial activity of a new carbapenem, doripenem: tentative disc diffusion criteria and quality control

Steven D. Brown* and Maria M. Traczewski

The Clinical Microbiology Institute, 9725 SW Commerce Circle, Wilsonville, OR 97070, USA


* Corresponding author. Tel: +1-503-682-3232; Fax: +1-503-682-2065; Email: sbrown{at}clinmicroinst.com

Received 11 January 2005; returned 6 March 2005; revised 9 March 2005; accepted 21 March 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To determine the spectrum of activity of doripenem and to propose tentative MIC and disc diffusion breakpoints.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Doripenem (formerly S-4661) is a new parenteral 1-ß-methyl carbapenem with a broad spectrum of antibacterial activity.17 These preliminary studies have indicated that doripenem has a spectrum of activity that is similar to imipenem against Gram-positive strains and closely related to meropenem against Gram-negative strains. Doripenem is currently under clinical development for the treatment of serious bacterial infections in hospitals.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacteria tested

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 Mueller–Hinton 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 Mueller–Hinton agar + 5% sheep blood (streptococci), HTM agar (H. influenzae) or plain Muller–Hinton 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 Mueller–Hinton 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


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Table 1 summarizes the MICs of doripenem, meropenem and imipenem against all bacterial isolates tested. The activity of doripenem against Gram-positive strains was similar to that of imipenem and slightly more active than meropenem. For the majority of the Gram-positive strains, the MIC90s of doripenem were either the same as imipenem or were 1 log2 dilution less active than imipenem. The only instances in which more than a 1 log2 dilution was noted were for vancomycin-susceptible E. faecalis and methicillin-susceptible coagulase-negative staphylococci. Doripenem MIC90s were either the same as or 1 log2 dilution more active than meropenem.


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Table 1. Susceptibility of aerobic bacteria to doripenem and other carbapenems against aerobic organisms (mg/L)

 
Against members of the Enterobacteriaceae, doripenem was generally more active than imipenem and the same as or slightly less active than meropenem. The MIC90 for all Enterobacteriaceae combined was 3 log2 dilutions more active than imipenem and 1 log2 dilution less active than meropenem (Table 1). Considerable variation occurred among the various species of Enterobacteriaceae. Doripenem was 5 log2 dilutions more active than imipenem against Citrobacter freundii and 4 log2 dilutions more active than imipenem against extended-spectrum ß-lactamase (ESBL)-positive strains of E. coli. Numerous instances occurred when the doripenem activity was 1 or 2 log2 dilutions more active than imipenem. Conversely, doripenem was 3 log2 dilutions less active than meropenem against strains of Proteus mirabilis. For the remainder of the species, doripenem MIC90s differed by ≤2 log2 dilutions from those of meropenem.

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 1–2 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 4–8-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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Figure 1. Scattergrams of doripenem MICs versus zone diameters (10 µg discs). (a) Enterobacteriaceae, (b) non-fermentative Gram-negative bacilli, (c) non-pneumococcal streptococci, (d) S. pneumoniae, (e) Staphylococcus spp., (f) H. influenzae. Horizontal lines represent proposed susceptible (lower line) and resistant (upper line) MIC breakpoints; vertical lines represent proposed susceptible (right-hand line) and resistant (left-hand line) zone diameter breakpoints. Abbreviations: n, number of strains tested; VM, very major errors; M, major errors; m, minor errors; I+2, intermediate MIC breakpoint plus 2 log2 dilutions; I–1 to I+1, intermediate MIC breakpoint plus or minus 1 log2 dilution; I–2, intermediate MIC breakpoint minus 2 log2 dilutions; R + 1, resistant MIC breakpoint plus 1 log2 dilution; R + S, resistant plus susceptible MIC breakpoints; R–1, resistant MIC breakpoint minus 1 log2 dilution.

 
The error rates for the Enterobacteriaceae and non-fermentative Gram-negative bacilli are within acceptable ranges (Figure 1a and b, respectively). The profound activity of doripenem against all members of the Enterobacteriaceae is readily apparent. Included in these strains are isolates known to produce ESBLs. The activity against the non-fermentative Gram-negative bacilli was also acceptable.

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 13–15 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, 11–13 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, 18–20 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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Table 2. Doripenem MIC quality control

 

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Table 3. Doripenem disc diffusion quality control

 
Conclusions

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.


    Acknowledgements
 
Financial support for this project was provided by Peninsula Pharmaceuticals, Alameda, CA, USA.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Brown S, Traczewski M. Doripenem: in vitro potency, spectrum of activity, MIC and disc breakpoints. In: Abstracts of the Forty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004. Abstract E-2016, p. 175. American Society for Microbiology, Washington, DC, USA.

2 . Traczewski MM, Brown SD. In vitro activity of doripenem versus 194 strains of P. aeruginosa and B. cepacia from both cystic fibrosis and non-cystic fibrosis patients. In: Abstracts of the One Hundred-fourth General Meeting of the American Society for Microbiology, New Orleans, LA, 2004. Abstract A-141, p. 28. American Society for Microbiology, Washington, DC, USA.

3 . Tsuji M, Ishii Y, Ohno A et al. In vitro and in vivo antibacterial activities of S-4661, a new carbapenem. Antimicrob Agents Chemother 1998; 42: 94–9.[Abstract/Free Full Text]

4 . Mushtaq S, Ge Y, Livermore DM. Comparative activities of doripenem versus isolates, mutants, and transconjugants of Enterobacteriaceae and Acinetobacter spp. with characterized ß-lactamases. Antimicrob Agents Chemother 2004; 48: 1313–9.[Abstract/Free Full Text]

5 . Mikamo H, Izumi K, Hau YZ et al. In vivo and in vivo antibacterial activities of a new injectable carbapenem, S-4664, against gynaecological pathogens. J Antimicrob Chemother 2000; 46: 471–4.[Abstract/Free Full Text]

6 . Mushtaq S, Ge Y, Livermore DM. Doripenem versus Pseudomonas aeruginosa in vitro: activity against characterized isolates, mutants, and transconjugants and resistance selection potential. Antimicrob Agents Chemother 2004; 48: 3086–92.[Abstract/Free Full Text]

7 . Ge Y, Wikler MA, Sahm DF et al. In vitro antimicrobial activity of doripenem, a new carbapenem. Antimicrob Agents Chemother 2004; 48: 1384–96.[Abstract/Free Full Text]

8 . National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.

9 . National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disc Susceptibility Tests: Approved Standard M2-A8. NCCLS, Wayne, PA, USA, 2003.

10 . National Committee for Clinical Laboratory Standards. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Approved Standard M11-A6. NCCLS, Wayne, PA, USA, 2004.

11 . National Committee for Clinical Laboratory Standards. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters: Approved Standard M23-A2. NCCLS, Wayne, PA, USA, 2001.

12 . Bhavnani SM, Hammel JP, Cirincione BB et al. PK-PD target attainment with Monte Carlo Simulation (MCS) as decision support of phase 2/3 dosing strategies for the clinical development of doripenem (DOR). In: Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract A-11, p. 2. American Society for Microbiology, Washington, DC, USA.

13 . Thye DA, Kilfoil T, Leighton A et al. A Phase 1 study to evaluate safety, tolerability and pharmacokinetics in a western healthy volunteer population. In: Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract A-21, p. 5. American Society for Microbiology, Washington, DC, USA.

14 . Andes DR, Keim S, Craig WA. In vivo pharmacodynamic activity of a new carbapenem, doripenem (DOR), against multiple bacteria in a murine thigh infection model. In: Abstracts of the Forty-third Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2003. Abstract A-309, p. 10. American Society for Microbiology, Washington, DC, USA.

15 . National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S10. NCCLS, Wayne, PA, USA, 1999.

16 . Gavan TL, Jones RN, Barry AL et al. Quality control limits for ampicillin, carbenicillin, mezlocillin, and piperacillin disc diffusion susceptibility tests: a collaborative study. J Clin Microbiol 1981; 14: 67–72.[ISI][Medline]





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