Abstract
Test parameter variations were evaluated for their effects on surotomycin MICs. Calcium concentration was the only variable that influenced MICs; therefore, 50 μg/ml (standard for lipopeptide testing) is recommended. Quality control ranges for Clostridium difficile (0.12 to 1 μg/ml) and Eggerthella lenta (broth, 1 to 4 μg/ml; agar, 1 to 8 μg/ml) were approved by the Clinical and Laboratory Standards Institute based on these data.
TEXT
In recent years, the incidence and severity of Clostridium difficile-associated diarrhea (CDAD) have increased (1–4). Despite the availability of antibiotic therapies, CDAD-related morbidity and mortality rates have remained high (5–8). In a recent study, the clinical response rates were 81.1% for vancomycin and 72.7% for metronidazole, with both drugs being associated with high rates of recurrence (∼20 to 45%) (9–11). The more recently approved fidaxomicin demonstrated comparable clinical responses and reduced rates of recurrence (∼13 to 15%), as compared with vancomycin (12, 13).
Surotomycin (CB-183,315) is an orally administered, minimally absorbed, selective, bactericidal cyclic lipopeptide being developed for treatment of patients with CDAD (14). It previously demonstrated selective potent bactericidal activity against various C. difficile strains and other Gram-positive bacteria in vitro, with minimal impact on the Gram-negative organisms of the intestinal microbiota in vitro and in CDAD patients (15–20). In a phase 2 trial, surotomycin was more sparing of gut microbes (primarily Bacteroides and Prevotella species) than vancomycin, with evidence for reduction in the risk of recurrence (20).
These studies were designed to evaluate the effects of changes in key test parameters, including pH, inoculum, calcium concentration ([Ca2+]), broth/agar lot, and incubation time, on the in vitro activity of surotomycin against three C. difficile strains (ATCC 700057 and the clinical isolates SID 7907612 and SID 2946830) and to generate MIC ranges for quality control (QC) organisms (C. difficile ATCC 700057, Eggerthella lenta ATCC 43055, and Staphylococcus aureus ATCC 29213). Broth microdilution and agar dilution were reported previously to be comparable and reproducible methods for testing surotomycin against C. difficile (21). Therefore, the test parameter and QC studies were performed using both methods, based on the data presented here.
Variations in key test parameters were tested in duplicate against C. difficile ATCC 700057 and two clinical isolates. Brucella agar, as recommended by the Clinical and Laboratory Standards Institute (CLSI) for MIC determinations, has various concentrations of divalent cations (22). The in vitro activity of surotomycin is dependent on the [Ca2+] of the test medium, which needs to be adjusted to a final value of 50 μg/ml, based on previous observations with the similarly structured lipopeptide daptomycin (23). Inoculum size (5 × 103 to 5 × 105 CFU/ml), pH (pH 6 to 8), [Ca2+] (0 to 150 μg/ml), and incubation time (24 to 72 h) were varied relative to standard conditions, which were defined as incubation time of 48 h, [Ca2+] of 50 μg/ml, inoculum size of 5 × 104 CFU/ml, pH of 7.2 to 7.4, and serum level of 0% (Table 1). An ion-selective electrode method was used to assess the concentrations of both total and ionized Ca2+ in the media. Each isolate was tested using standard anaerobic broth microdilution and agar dilution methods, according to CLSI guidelines. The effects of test parameter variations on surotomycin MICs were assessed using tigecycline as a control for broth microdilution, vancomycin as a control for agar dilution (versus C. difficile), and clindamycin for both agar dilution (versus C. difficile and E. lenta) and broth microdilution (versus E. lenta) (24).
TABLE 1.
Surotomycin MICs with various test parameters, as demonstrated by anaerobic broth microdilution and agar dilution
| Assay method and C. difficile strain | Inoculum (CFU/ml) | Duplicate MICs (μg/ml) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 24-h incubation | 72-h incubation | 0 μg/ml Ca2+ | 25 μg/ml Ca2+ | Standard conditionsa | 75 μg/ml Ca2+ | 150 μg/ml Ca2+ | Inoculum of 5 × 105 CFU/ml | Inoculum of 5 × 103 CFU/ml | pH 8 | pH 6 | 5% human serum | 10% human serum | ||
| Broth microdilution | ||||||||||||||
| ATCC 700057 | 2 × 104 | 0.25/0.5 | 1/1 | 4/4 | 2/2 | 1/1 | 0.5/0.5 | 0.25/0.25 | 2/2 | 1/1 | 1/1 | —b | 0.5/1 | —c |
| SID 2907612 | 9 × 103 | 0.25/0.25 | 0.5/0.5 | 2/2 | 2/2 | 0.5/0.5 | 0.5/0.5 | 0.25/0.25 | 1/1 | 0.5/0.5 | 0.5/0.5 | —b | 1/1 | —c |
| SID 2946830 | 1.8 × 104 | 0.5/0.5 | 0.5/0.5 | 2/2 | 2/2 | 0.5/0.5 | 0.5/0.5 | 0.25/0.25 | 1/1 | 0.5/0.5 | 0.5/0.5 | —b | 0.5/1 | —c |
| Agar dilution | ||||||||||||||
| ATCC 700057 | 1.2 × 105 | 0.5/0.5 | 0.5/0.5 | 4/4 | 2/2 | 0.5/0.5 | 0.5/0.5 | 0.25/0.25 | 0.5/0.5 | 0.5/0.5 | 0.5/0.5 | 0.5/0.5 | —d | —d |
| SID 2907612 | 4 × 104 | 0.25/0.25 | 0.25/0.25 | 4/4 | 1/1 | 0.5/0.5 | 0.25/0.25 | 0.25/0.12 | 0.25/0.25 | 0.25/0.25 | 0.25/0.25 | 0.25/0.25 | —d | —d |
| SID 2946830 | 1.8 × 105 | 0.25/0.25 | 0.25/0.25 | 4/4 | 1/1 | 0.25/0.25 | 0.25/0.25 | 0.25/0.25 | 0.5/0.5 | 0.25/0.25 | 0.25/0.25 | 0.5/0.5 | —d | —d |
The standard test conditions included an incubation time of 48 h, [Ca2+] of 50 μg/ml, inoculum of 5 × 104 CFU/ml, pH of 7.2 to 7.4, and serum level of 0%.
No growth.
Difficult to read due to substantial background debris.
Agar dilution was not performed with 5% and 10% human serum.
A CLSI M23-A3 tier 2 QC study (25) was performed in eight laboratories (see Acknowledgments). The methods described in CLSI document M11-A7 were used (22). Each laboratory tested 10 replicates of each QC strain on three lots of each medium, with the broth microdilution and agar dilution methods. Colony counts were performed at all test sites and were within the parameters of the CLSI guidelines for broth microdilution and agar dilution tests for anaerobic MICs (22, 24). All study strains were incubated under anaerobic conditions. Medium lot preparation information can be found in the supplemental material. For the QC study, tier 2 MIC ranges were selected based on CLSI M23-A3 guidelines (25) and confirmed by the alternate RangeFinder method described in the guideline (26) (see the supplemental material).
Based on surotomycin MICs obtained for each of the three C. difficile isolates, the only parameter that affected surotomycin activity was [Ca2+] (Table 1). The largest shift in surotomycin MICs was an increase from 0.12 to 4 μg/ml (32-fold MIC shift) for C. difficile ATCC 700057 as the [Ca2+] was decreased from 150 to 0 μg/ml. During broth microdilution, no organism growth occurred at pH 6, and it was not possible to determine MICs at 10% serum concentrations, due to substantial background turbidity. Parameter variations did not appear to notably affect the MICs of the control drugs (clindamycin, tigecycline, and vancomycin). At the time of testing, there were no resistant isolates available; therefore, the performance for detection of resistance was not addressed.
Surotomycin MICs were all within 1 doubling dilution of each other for all three isolates tested in each of the three broth medium lots (Table 2). For all strains, minimal lot-to-lot variations were noted for surotomycin MICs, as determined by agar dilution; for the two clinical isolates, however, surotomycin MICs were 1 to 2 doubling dilutions lower in medium lot 1 when tested by agar dilution. The MICs of control drugs were not affected by different medium lots.
TABLE 2.
Medium lot comparisons of surotomycin MICs for anaerobic broth microdilution and agar dilution
| C. difficile strain | Inoculum (CFU/ml) | Duplicate MICs (μg/ml) |
|||||
|---|---|---|---|---|---|---|---|
| Broth microdilution |
Agar dilution |
||||||
| Lot 1 | Lot 2 | Lot 3 | Lot 1 | Lot 2 | Lot 3 | ||
| ATCC 700057 | 2.5 × 104 | 0.5/0.5 | 1/1 | 0.5/0.5 | 0.5/0.5 | 0.5/0.5 | 0.5/0.5 |
| SID 2907612 | 1.0 × 104 | 1/1 | 1/1 | 0.5/0.5 | 0.12/0.25 | 0.5/0.5 | 0.5/0.5 |
| SID 2946830 | 2.2 × 104 | 0.5/0.5 | 1/0.5 | 0.5/0.5 | 0.12/0.25 | 0.5/0.5 | 0.5/0.5 |
For the QC organism C. difficile ATCC 700057, all test results were within the range of 0.12 to 1 μg/ml (Table 3). For E. lenta ATCC 43055, ranges of 1 to 4 μg/ml (98.1% within range) and 2 to 8 μg/ml (96.7% within range) were approved for microdilution and agar dilution, respectively (Table 4). A range of 0.25 to 1 μg/ml (99.6% within range) was approved for broth microdilution for S. aureus ATCC 29213 incubated anaerobically (Table 5). For agar dilution, a range of 0.5 to 2 μg/ml (100% within range) was approved for S. aureus ATCC 29213 incubated anaerobically, with the exclusion of laboratory 2 as a statistical outlier (Table 5). All surotomycin QC ranges determined by methods proposed by the CLSI were identical to ranges determined by the RangeFinder method (Table 6) (26). CLSI-approved surotomycin QC ranges plus the percentages of tests within range for testing under anaerobic conditions using supplemented Brucella broth or agar are also summarized in Table 6.
TABLE 3.
Number of strains with surotomycin-specified QC ranges for anaerobic broth microdilution and agar dilution for C. difficile ATCC 700057a
Shading indicates quality control (QC) ranges.
TABLE 4.
Number of strains with surotomycin-specified QC ranges for anaerobic broth microdilution and agar dilution for E. lenta ATCC 43055a
Shading indicates quality control (QC) ranges.
Unable to obtain sufficient growth.
TABLE 5.
Number of strains with surotomycin-specified QC ranges for anaerobic broth microdilution and agar dilution for S. aureus ATCC 29213a
Shading indicates quality control (QC) ranges.
Laboratory 2 was identified as an outlier for both the mean and the mode by the RangeFinder method.
TABLE 6.
MIC ranges determined using confirmatory RangeFinder method and CLSI-approved surotomycin MIC QC ranges
| QC organism and methoda | MIC range (μg/ml) | No. of dilutions | % of tests in range |
|---|---|---|---|
| RangeFinder | |||
| C. difficile ATCC 700057b | |||
| Broth microdilution | 0.12–1 | 4 | 100 |
| Agar dilution | 0.12–1 | 4 | 100 |
| E. lenta ATCC 43055b | |||
| Broth microdilution | 1–4 | 3 | 98.1 |
| Agar dilution | 2–8 | 3 | 96.7 |
| S. aureus ATCC 29213b | |||
| Broth microdilution | 0.25–1 | 3 | 100 |
| Agar dilution | 0.25–2c | 4 | 100 |
| CLSI approved | |||
| C. difficile ATCC 700057b,d | |||
| Broth microdilution | 0.12–1 | 4 | 100 |
| Agar dilution | 0.12–1 | 4 | 100 |
| E. lenta ATCC 43055b,d | |||
| Broth microdilution | 1–4 | 3 | 98.1 |
| Agar dilution | 2–8 | 3 | 96.7 |
| S. aureus ATCC 29213b,d | |||
| Broth microdilution | 0.25–1 | 3 | 99.6 |
| Agar dilution | 0.5–2e | 3 | 100 |
QC, quality control.
Incubated under anaerobic conditions.
Laboratory 2 was identified as an outlier for both the mean and the mode.
Supplemental QC, not to be published in CLSI tables.
Excluding laboratory 2 as a statistical outlier; however, including laboratory 2 and accepting a 56% shoulder as a bimodal distribution resulted in a range of 0.25 μg/ml to 2 μg/ml.
No lot-to-lot variations in Brucella base medium were observed with any of the broth or agar media, and the tests were highly reproducible among the eight laboratories involved. The QC ranges for the control agents with C. difficile ATCC 700057 and E. lenta ATCC 43055 listed in Table 6 were presented to the CLSI Subcommittee on Antimicrobial Susceptibility Testing in January 2012 and were approved, based on the data presented here. These ranges were published in document M100-S24 in January 2014 (24). S. aureus ATCC 29213 anaerobic ranges were approved as supplemental QC ranges and are not listed in document M100-S24.
In summary, the in vitro activity of surotomycin against C. difficile was not affected by altered pH, inoculum size, incubation time, or medium lot variations. The only parameter that appeared to change the in vitro activity of surotomycin notably with the three C. difficile strains studied was [Ca2+]. This result suggests that accurate [Ca2+] determination is crucial for reliable surotomycin susceptibility testing and that a Ca2+ concentration of 50 μg/ml is necessary.
Supplementary Material
ACKNOWLEDGMENTS
The quality control study was performed by the Clinical Microbiology Institute and funded by Merck and Co., Inc. (Kenilworth, NJ). Medical writing assistance was provided by Dan Rigotti and Cara L. Hunsberger of StemScientific (Lyndhurst, NJ), an Ashfield Company, part of UDG Healthcare plc. This assistance was funded by Merck and Co., Inc.
M.M.T. and S.D.B. are employees of the Clinical Microbiology Institute. L.C. is an employee and stock holder of Merck and Co., Inc. J.D. and D.S. were employees at Eurofins Medinet at the time that the study was performed.
We thank Laura Koeth and Jeanna DiFranco-Fisher from Laboratory Specialists, Inc., for their assistance in quantifying and adjusting calcium levels in the media (broth and agar) used for this study.
We thank the individual laboratories that participated in this study, i.e., Alden Research Center (Culver City, CA), Clinical Microbiology Institute (Wilsonville, OR), Laboratory Specialists, Inc. (Westlake, OH), Loyola University (Maywood, IL), TREK Diagnostic Systems (Cleveland, OH), Tufts-New England Medical Center (Boston, MA), University of Alberta Hospital (Alberta, Canada), and University of Rochester Medical Center (Rochester, NY).
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02881-15.
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