Abstract
In 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology of Clostridium difficile isolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample of C. difficile isolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90 was 0.5 μg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 μg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile of tcdA, tcdB, and cdtA/B positive with a tcdC 18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity against C. difficile isolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.
INTRODUCTION
In the past decade the incidence and severity of Clostridium difficile associated diarrhea has increased significantly. Outbreaks in North America, namely, Canada, followed by the United States, the United Kingdom, and Europe, have emerged, caused by the BI/NAP1/027 epidemic strain (1, 2). Most microbiology laboratories do not culture diarrheal stools for C. difficile, and virtually no laboratory performs antibiotic susceptibility on these isolates. There has been little incentive to perform susceptibility on such isolates since all isolates have been thought to be susceptible to the two main agents used for treatment, namely, metronidazole and vancomycin. In addition, evidence suggests that response to treatment is not related to the degree of susceptibility to agents in vitro, given the high concentrations in the gastrointestinal tract (3).
Recently, it is evident that some isolates of C. difficile are becoming less susceptible to metronidazole (4). In addition, there are some data that the epidemic BI strains may have higher MICs by two dilutions than non-BI strains for vancomycin and metronidazole (4, 5).
With the licensure of the first new agent for the treatment of C. difficile-associated diarrhea, fidaxomicin, the U.S. Food and Drug Administration (FDA) mandated surveillance for antimicrobial susceptibility testing of C. difficile isolates from stool, as well as isolate strain typing (6). There have been limited surveillance efforts to date to characterize C. difficile isolates nationally, according to restriction endonuclease analysis (REA) type or ribotype (7, 8). There are few U.S. susceptibility data on testing against a battery of antimicrobial compounds for C. difficile. This report characterizes the results of the first 2 years of such surveillance efforts in 2011 and 2012.
Our specific aims in establishing this network were (i) to monitor and analyze the resistance patterns of C. difficile against antibiotics used to treat such infections and to establish baseline and ongoing surveillance, (ii) to provide epidemiologic data on the prevalence of toxin profiles and REA typing of C. difficile with analysis by region of the United States among our participating centers, and (iii) to provide the scientific community (researchers, practitioners, clinical laboratories, and regulatory agencies) with accurate information on the changing epidemiology and prevalence of C. difficile resistance and toxin profiles.
(This study was presented in part at the 52nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, California, on 9 to 12 September 2012, and at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, Colorado, on 10 to 13 September 2013.)
MATERIALS AND METHODS
Medical centers.
From 2011 to 2012 a total of 925 C. difficile isolates were referred by seven medical centers for processing to the Special Studies Laboratory at Tufts Medical Center. The medical centers were the Duke University Medical Center, Durham, NC; Hines VA Hospital, Chicago, IL; Mayo Clinic, Rochester, MN; New York Presbyterian Hospital/Weill Cornell Medical Center, New York, NY; Tufts Medical Center, Boston, MA; RM Alden Research Laboratory, Culver City, CA; and the VCU Medical Center, Medical College of Virginia, Richmond, VA.
Bacterial isolates.
A convenience sample of isolates of C. difficile were obtained from seven different locations around the United States, from institutions that had excellent anaerobic bacteriology laboratories and with investigators willing to collaborate (Table 1). In 2012, Duke University Medical Center dropped out of the survey and was replaced by the VCU Medical Center, Medical College of Virginia, Richmond, VA. The isolates from toxin-positive stool samples were forwarded to the Special Studies Laboratory at Tufts Medical Center for susceptibility testing at prearranged intervals. Each institution that performed strain isolation of C. difficile was instructed to send an average of 75 isolates collected throughout the year. These isolates were sent periodically in chopped meat broth by these institutions. Other centers only sent stools from toxin positive patients. Those stools were processed for isolation of C. difficile at the reference laboratory by the method outlined below.
TABLE 1.
Isolates referred, medical centers, and investigators from 2011 to 2012
| Medical Center | Investigator | No. of isolates referreda |
|---|---|---|
| Duke University Medical Center | S. Mirrett | 61 |
| Hines VA Hospital | D. N. Gerding, S. Johnson | 137 |
| Mayo Clinic | R. Patel | 139 |
| Weill Cornell Medical College/NY Presbyterian Hospital | S. Jenkins | 179 |
| R. M. Alden Research Laboratories | E. J. C. Goldstein | 163 |
| Tufts Medical Center | D. R. Snydman | 208 |
| Virginia Medical College | B. A. Forbes | 38 |
| Total | 925 |
Isolates were referred as toxin-positive stool (frozen on dry ice) or as a culture in chopped meat broth.
Processing and identification of isolates.
Standardized testing of the isolates was performed at the Special Studies Laboratory at Tufts Medical Center. After arrival of the referred isolate, its purity and identification was confirmed. Confirmation of the isolate as C. difficile was accomplished by plating on selective C. difficile selective medium (cycloserine-cefoxitin-fructose agar with taurocholate) and observing the sample for characteristic colonial morphology (9, 10). This was followed by using the rapid identifying methods API 20A (bioMérieux, Inc., Durham, NC) and/or Rapid ANA II (Remel Products, Lenexa, KS). If identification with rapid methodology was not conclusive, the methods outlined in the Wadsworth Anaerobic Bacteriology Laboratory Manual were followed (10). The isolates were kept in chopped meat broth until tested, and a cell paste swabbed from fresh plates was later suspended directly into skim milk and frozen at −80°C for future reference (11).
Susceptibility testing.
The MICs of the isolates were determined by agar dilution, following Clinical and Laboratory Standards Institute (CLSI) recommendations (M11 A8 [11]), against the following antimicrobial agents: fidaxomicin, vancomycin, metronidazole, rifaximin, rifampin, tigecycline, imipenem, moxifloxacin, clindamycin, and chloramphenicol. Fidaxomicin (Cubist) was diluted in dimethyl sulfoxide (DMSO) and further diluted with distilled water to a final concentration in the agar plate of 0.5% DMSO. Rifampin (Sigma-Aldrich, St. Louis, MO) was dissolved in methanol and further diluted with distilled water. Rifaximin (Sigma-Aldrich, St. Louis, MO) was dissolved in methanol and then diluted in 0.1 M phosphate buffer (pH 7.4) plus 0.45% sodium dodecyl sulfate. All other antibiotics, provided by their respective manufacturers, were dissolved in distilled water.
Antibiotic containing plates were prepared on the day of the test. The medium was brucella agar (supplemented with 5 μg of hemin, 1 μg of vitamin K1/ml, and 5% [vol/vol] laked sheep blood). Serial 2-fold dilutions of the antibiotics were added to the molten agar, poured into round petri dishes, and allowed to solidify and dry. Non-antibiotic-containing controls were prepared in the same manner. The isolates were grown to log phase in supplemented brain heart infusion and adjusted to a0.5 McFarland standard (for C. difficile ATCC 700057, the density was 1 × 107 to 4 × 107 CFU/ml). The inocula were deposited on the agar surface using a Steer replicator, resulting in a final concentration of 104 CFU/spot. The plates were incubated for 48 h at 35 to 37°C in an anaerobic chamber with an atmosphere of 85% N2, 5% CO2, and 10% H2. The MICs were determined after 48 h of incubation. The following ATCC reference organisms were used as controls in all tests: Clostridium difficile ATCC 700057, Bacteroides fragilis ATCC 25825, Bacteroides thetaiotaomicron ATCC 29742, and Staphylococcus aureus ATCC 29213.
The rates of resistance of the antimicrobial agents were determined using currently accepted breakpoints recommendations by the CLSI and/or by the European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) (12, 13). Resistance breakpoints for tigecycline were those recommended by the FDA (14). The antibiotics tested, range of concentrations, and breakpoints are listed in Table 2.
TABLE 2.
Antimicrobial agents tested, MIC ranges, and the MIC breakpoints applieda
| Antimicrobial agent | MIC (μg/ml) |
||
|---|---|---|---|
| Range | Resistance breakpointb |
||
| CLSI | EUCAST | ||
| Fidaxomicinc | 0.004–4 | NA | NA |
| Rifaximind | 0.004–4 | NA | NA |
| Rifampine | 0.004–4 | NA | >0.004 |
| Vancomycin | 0.25–64 | NA | >2 |
| Metronidazole | 0.06–16 | ≥32 | >2 |
| Moxifloxacin | 0.5–16 | ≥8 | >4 |
| Clindamycin | 0.5–16 | ≥8 | NA |
| Imipenem | 0.12–16 | ≥16 | NA |
| Tigecycline | 0.06–64 | ≥16 | >0.25 |
| Chloramphenicol | 0.5–16 | ≥32 | NA |
NA, not applicable (a resistance breakpoint was not established when the study was performed).
That is, the CLSI recommended breakpoints for resistance. For tigecycline, the resistance breakpoint was established by the FDA for anaerobes. The EUCAST values are considered epidemiologic cutoff values (ECOFF) and were used for epidemiologic purposes.
Dissolved in DMSO and added to a final concentration of 0.5% DMSO.
Dissolved in methanol and diluted in water.
Dissolved in methanol and diluted 0.1 M phosphate buffer (pH 7.4) and 0.45% sodium dodecyl sulfate.
Toxin determination.
Toxin gene profiles for C. difficile were performed using PCR methodology as described by Persson et al. (15, 16). To determine the toxin profile, isolates were tested in duplicate, and nonconcordant tests were repeated. The following controls were included with each test: (i) a nontoxigenic strain of C. difficile VPI 11186 (ATCC 700057), with a PCR profile of tcdA negative, tcdB negative, and cdtA/B negative in which the 19.6-kb pathogenicity locus that encodes TcdA and TcdB (PaLoc) is absent (17) and in which no functional cdt locus is present; (ii) VPI 10463, with a PCR profile of tcdA positive, tcdB positive, and cdtA/B negative, with no detected deletion in tcdC region (18, 19); and (iii) R20291 with a PCR profile tcdA positive, tcdB positive, and cdtA/B positive, with an 18-bp deletion in the tcdC hypervariable region (20). Loss of PaLoc was confirmed in nontoxigenic strains by performing a PCR using Lok1/Lok3 primers as previously described (21).
REA typing.
A randomly selected sample of isolates, stratified by center (∼30 isolates from each center per year), were referred to the Microbiology Reference Laboratory at the Hines Veterans Administration Hospital, Chicago, IL for REA strain typing. After purification, the total cellular DNA was cut with the HindIII restriction enzyme, and fragments were separated by electrophoresis on a 0.7% agarose gel as previously described (7, 22). HindIII restriction patterns with a 90% similarity index were included in the same REA group (letter designation).
Data storage and analysis.
Results from the various tests were sent to the Reference Laboratory for analysis. All data were stored and analyzed using Microsoft Excel spreadsheets.
RESULTS
Susceptibility of the isolates.
The results from the susceptibility testing of all 925 isolates are presented in Table 3 and expressed as an MIC range, MIC50, and MIC90 and as percent resistance based on CLSI and/or EUCAST breakpoint recommendations. All isolates were inhibited at a fidaxomicin concentration ≤1 μg/ml, and the MIC90 was 0.5 μg/ml. According to CLSI breakpoints, 33.5% of isolates were resistant to moxifloxacin, and 23.8% were resistant to clindamycin. Although there is no resistance breakpoint for vancomycin by the CLSI, the MIC90 was 4 μg/ml. Using EUCAST breakpoints, which are intended for epidemiologic purposes, 17.9% of isolates had decreased susceptibility to vancomycin and 3.6% had decreased susceptibility to metronidazole. For moxifloxacin, the EUCAST results on the proportion of isolates with decreased susceptibility were identical to those of the CLSI. Rifampin also demonstrated reduced susceptibility in 65.3% of isolates according to the EUCAST breakpoints.
TABLE 3.
Activities of the antimicrobial agents versus 925 isolates of C. difficile from 2011 to 2012a
| Antimicrobial agent | MIC (μg/ml) |
% resistant |
|||
|---|---|---|---|---|---|
| Range | MIC50 | MIC90 | CLSI | EUCAST | |
| Fidaxomicin | ≤0.004 to 1 | 0.25 | 0.5 | NA | NA |
| Rifaximin | ≤0.004 to >4 | 0.015 | 0.06 | NA | NA |
| Rifampin | ≤0.004 to >4 | 0.008 | 0.015 | NA | 65.3 |
| Tigecycline | ≤0.06 to 1 | 0.12 | 0.25 | 0 | 1.3 |
| Vancomycin | ≤0.25 to 4 | 2 | 4 | NA | 17.9 |
| Imipenem | ≤0.12 to 16 | 4 | 8 | 2.3 | NA |
| Moxifloxacin | ≤0.5 to >16 | 2 | >16 | 33.5 | 33.5 |
| Metronidazole | ≤0.06 to 4 | 1 | 2 | 0 | 3.6 |
| Clindamycin | ≤0.5 to >16 | 2 | 16 | 23.8 | NA |
| Chloramphenicol | ≤0.5 to 16 | 4 | 8 | 0 | NA |
A total of 925 isolates were obtained from all of the participating centers. NA, not applicable.
Determination of toxin gene profiles.
Of the 925 isolates included in the present study, 915 (98.9%) could be assayed for a toxin gene profile (Table 4). Among isolates with toxigenic profile characterization, toxin A and toxin B genes (tcdA and tcdB) were detected in 97.3% (890/915) of isolates, while 2.7% (25/915) were nontoxigenic (tcdA negative, tcdB negative). Binary toxin genes (cdtA/B positive) were present in 33.5% (298/890) of the toxigenic isolates (tcdA positive, tcdB positive). In-frame mutations attributed to an 18-bp deletion of tcdC were found in 85.9% (256/298) of isolates with binary toxin genes (cdtA/B positive).
TABLE 4.
Distribution of toxin gene profiles among 925 isolates of C. difficile from 2011 to 2012a
| Toxin gene profile (tcdC deletion [bp])b | No. (%) of isolates |
|---|---|
| tcdA tcdB tcdC (0) | 539 (58.9) |
| tcdA tcdB tcdC (18) | 53 (5.8) |
| tcdA tcdB cdtA/B tcdC (0) | 19 (2.1) |
| tcdA tcdB cdtA/B tcdC (18) | 256 (28.0) |
| tcdA tcdB cdtA/B tcdC (39) | 21 (2.3) |
| tcdA tcdB cdtA/B tcdC (54) | 2 (0.2) |
| Nontoxigenic | 25 (2.7) |
Ten isolates could not be extracted for assay.
tcdC deletions are indicated parenthetically in base pairs.
REA strain typing.
A random sample subset, stratified by center, of 322 (34.8%) isolates was referred for REA typing. The distribution of the isolates by REA group is shown in Table 5. Of the 322 isolates referred for REA typing, 71.1% (229/322) could be assigned to one of the ten most common REA groups, while 28.9% (93/322) were cataloged as a “nonspecific REA type.” BI (NAP1/027) was the most common group and represented 25.5% (82/322) of the isolates tested. In 2011, the proportion of BI isolates was 24.3%, and in 2012 the proportion was 26.6%. There was a very strong correlation between the toxin gene profile and the REA typing result (Fig. 1). The BI REA group was almost exclusively toxin gene profile tcdA/B positive and cdtA/B positive, with an 18-bp tcdC deletion. There was more toxin gene variability among the other groups.
TABLE 5.
REA typing of 322 strains C. difficile from 2011 to 2012
| REA group | No. (%) of isolates |
|---|---|
| A | 13 (4.0) |
| BI | 82 (25.5) |
| DH | 33 (9.8) |
| G | 14 (4.3) |
| J | 12 (3.7) |
| Y | 51 (15.8) |
| CF | 8 (2.5) |
| BK | 7 (2.2) |
| K | 6 (1.9) |
| W | 3 (0.9) |
| Nonspecific | 93 (28.9) |
FIG 1.
Comparison of toxin gene profile by REA typing for strains REA typed.
Susceptibility profile by toxin gene profile.
Analysis of susceptibility results, stratified by toxin gene profile showed some notable associations (Table 6). Isolates with binary toxin had the highest MICs to many agents (Table 6). Fidaxomicin susceptibility remained unchanged, but moxifloxacin, vancomycin, and metronidazole had a rise in resistance rates according to EUCAST breakpoints. Almost 80% of the binary toxin isolates were resistant to moxifloxacin compared to 33.5% as a whole. The proportion of isolates resistant to clindamycin was 2-fold higher in the binary toxin positive strains, as well compared to the isolates without binary toxin.
TABLE 6.
Activity of the antimicrobial agents against 913 isolates of C. difficile stratified by toxin gene profilea
| Toxin gene profile (tcdC deletion [bp])b | No. of isolates | Antimicrobial agent | MIC (μg/ml) |
% resistant |
|||
|---|---|---|---|---|---|---|---|
| Range | MIC50 | MIC90 | CLSI | EUCAST | |||
| tcdA tcdB cdtA/B tcdC (0) | 19 | Fidaxomicin | 0.06 to 1 | 0.25 | 0.5 | NAc | NA |
| Rifaximin | 0.015 to 2 | 0.015 | 0.03 | NA | NA | ||
| Rifampin | ≤0.004 to 1 | 0.008 | 0.008 | NA | 68.4 | ||
| Tigecycline | 0.12 to 0.25 | 0.12 | 0.25 | 0 | 0 | ||
| Vancomycin | 0.5 to 4 | 2 | 4 | NA | 15.8 | ||
| Imipenem | 0.5 to 8 | 4 | 4 | 0 | NA | ||
| Moxifloxacin | 1 to >16 | 2 | 15.8 | 0 | 0 | ||
| Metronidazole | 0.25 to 2 | 1 | 2 | 0 | 0 | ||
| Clindamycin | ≤0.5 to >16 | 2 | 4 | 9.1 | NA | ||
| Chloramphenicol | 2 to 8 | 4 | 8 | 0 | NA | ||
| tcdA tcdB cdtA/B tcdC (18) | 256 | Fidaxomicin | ≤0.004 to 1 | 0.25 | 0.5 | NA | NA |
| Rifaximin | ≤0.004 to >4 | 0.03 | >4 | NA | NA | ||
| Rifampin | ≤0.004 to >4 | 0.008 | >4 | NA | 82.9 | ||
| Tigecycline | ≤0.06 to 0.5 | 0.12 | 0.25 | 0 | 1.6 | ||
| Vancomycin | 0.25 to 4 | 2 | 4 | NA | 24.9 | ||
| Imipenem | ≤0.12 to 16 | 4 | 8 | 1.9 | NA | ||
| Moxifloxacin | ≤0.5 to >16 | 16 | >16 | 79.4 | 79.4 | ||
| Metronidazole | ≤0.06 to 4 | 2 | 2 | 0 | 8.9 | ||
| Clindamycin | ≤0.5 to >16 | 4 | 16 | 37.4 | NA | ||
| Chloramphenicol | 2 to 8 | 4 | 8 | 0 | NA | ||
| tcdA tcdB cdtA/B tcdC (39) | 21 | Fidaxomicin | 0.008 to 1 | 0.25 | 0.5 | NA | NA |
| Rifaximin | 0.015 to 0.12 | 0.015 | 0.03 | NA | NA | ||
| Rifampin | ≤0.004 to 0.03 | 0.008 | 0.015 | NA | 71.4 | ||
| Tigecycline | ≤0.06 to 0.5 | 0.25 | 0.25 | 0 | 9.5 | ||
| Vancomycin | 0.5 to 4 | 1 | 2 | NA | 4.8 | ||
| Imipenem | 0.25 to 4 | 4 | 4 | 0 | NA | ||
| Moxifloxacin | ≤0.5−16 | 1 | 2 | 9.5 | 9.5 | ||
| Metronidazole | ≤0.06 to 4 | 0.5 | 2 | 0 | 4.8 | ||
| Clindamycin | 1 to >16 | 4 | 16 | 33.3 | NA | ||
| Chloramphenicol | 1 to 16 | 4 | 8 | 0 | NA | ||
| tcdA tcdB tcdC (0) | 539 | Fidaxomicin | ≤0.004 to 1 | 0.25 | 0.5 | NA | NA |
| Rifaximin | ≤0.004 to >4 | 0.015 | 0.03 | NA | NA | ||
| Rifampin | ≤0.004 to >4 | 0.008 | 0.008 | NA | 55.7 | ||
| Tigecycline | ≤0.06 to 1 | 0.12 | 0.25 | 0 | 1.1 | ||
| Vancomycin | ≤0.25 to 4 | 2 | 4 | NA | 15.6 | ||
| Imipenem | ≤0.12 to 16 | 4 | 8 | 2.2 | NA | ||
| Moxifloxacin | ≤0.5 to >16 | 2 | 16 | 15.8 | 15.8 | ||
| Metronidazole | ≤0.06 to 4 | 0.5 | 2 | 0 | 1.5 | ||
| Clindamycin | ≤0.5 to >16 | 2 | 16 | 17.4 | NA | ||
| Chloramphenicol | ≤0.5 to 16 | 4 | 8 | 0 | NA | ||
| tcdA tcdB tcdC (18) | 53 | Fidaxomicin | ≤0.004 to 0.5 | 0.25 | 0.5 | NA | NA |
| Rifaximin | 0.008 to >4 | 0.03 | 0.03 | NA | NA | ||
| Rifampin | ≤0.004 to >4 | 0.008 | 0.015 | NA | 73.6 | ||
| Tigecycline | ≤0.06 to 0.25 | 0.12 | 0.25 | 0 | 0 | ||
| Vancomycin | 0.5 to 4 | 2 | 4 | NA | 11.3 | ||
| Imipenem | 2 to 16 | 4 | 8 | 5.7 | NA | ||
| Moxifloxacin | 1 to 16 | 2 | 2 | 7.5 | 7.5 | ||
| Metronidazole | 0.25 to 4 | 0.5 | 2 | 0 | 1.9 | ||
| Clindamycin | ≤0.5 to 16 | 2 | 4 | 9.4 | NA | ||
| Chloramphenicol | 2 to 8 | 4 | 8 | 0 | NA | ||
| Nontoxigenic | 25 | Fidaxomicin | ≤0.004 to 1 | 0.25 | 0.5 | NA | NA |
| Rifaximin | ≤0.004 to >4 | 0.015 | 0.03 | NA | NA | ||
| Rifampin | ≤0.004 to >4 | 0.008 | 0.015 | NA | 65.2 | ||
| Tigecycline | ≤0.06 to 0.25 | 0.12 | 0.25 | 0 | 0 | ||
| Vancomycin | ≤0.25 to 4 | 2 | 4 | NA | 13.0 | ||
| Imipenem | 0.5 to 8 | 4 | 8 | 0 | NA | ||
| Moxifloxacin | ≤0.5 to >16 | 2 | >16 | 21.7 | 21.7 | ||
| Metronidazole | 0.25 to 2 | 1 | 2 | 0 | 0 | ||
| Clindamycin | ≤0.5 to 16 | 2 | 16 | 21.7 | NA | ||
| Chloramphenicol | 1 to 8 | 4 | 8 | 0 | NA | ||
Two isolates with tcdA tcdB cdtA/B tcdC (54-bp deletion) toxin profiles were not included in the table.
tcdC deletions are indicated parenthetically in base pairs.
NA, not applicable.
REA typing susceptibility results.
Of the BI strains, 84% were resistant to moxifloxacin, and 49% were resistant to clindamycin. Resistance to vancomycin among these strains according to EUCAST breakpoints was 25.6% (similar to the results seen by toxin gene profiling) and metronidazole resistance according to EUCAST breakpoints was 9.8% (data not shown).
Center differences.
There were some differences in REA strain type proportions and toxin gene profiles proportions by center when one examines binary toxin genes (Fig. 2). The range was 13 to 42% (median, 25%). As expected, susceptibility differences by center correlated strongly with REA and toxin gene type proportions (data not shown).
FIG 2.
Map of sites and C. difficile toxin gene profile distributions among seven medical centers in 2011 and 2012.
DISCUSSION
This report provides baseline results for U.S. national surveillance of fidaxomicin activity against isolates of C. difficile obtained from seven medical centers in 2011 and 2012. No fidaxomicin resistance was noted among these 925 isolates, and no differences in fidaxomicin activity were present among binary toxin-producing isolates. Our in vitro activity data for fidaxomicin, metronidazole, and vancomycin are in accordance with those presented by other investigators (3, 23–28) and are similar to those recently published in a pan-European surveillance effort (23). This study also confirms the very high resistance rates of C. difficile for moxifloxacin and clindamycin, especially for binary toxin-producing strains, again in accord with recently published data from Europe (1, 24–26).
In our surveillance study we see an increase in the MICs of C. difficile isolates to vancomycin. In contrast, to a study published about 8 years ago, encompassing isolates of C. difficile from 1984 to 2003 in which the MIC90 for vancomycin was 1 μg/ml, the MIC90 in the present study was 4 μg/ml (27). The MIC90 of 4 μg/ml is a value considered epidemiologically different from wild type by EUCAST epidemiologic cutoff values. This trend has been also noted by Goldstein et al. (24). Resistance to all agents correlated with both toxin gene profile and/or REA type. Centers with a higher proportion of these isolates had higher rates of resistance.
In comparison to other studies of resistance in C. difficile, our results for clindamycin and moxifloxacin show slightly lower rates of resistance (25, 26). Our data on binary toxin presence and the BI REA type are comparable to other recent analysis (24, 25, 28–31). There is distinct variation among centers, with a range from as low as 13% to as high as 42%. The reasons for these differences among centers is unclear and may reflect some strain selection bias, differences in antimicrobial usage in institutions, or other factors not well understood. Variation of strain prevalence is evident in both Europe and the United States (7, 29–32).
Limitations to this surveillance include the lack of information on antimicrobial exposure prior to the onset of C. difficile, information on the treatment for C. difficile disease, or information on the clinical outcome and the fact that this is a convenience sample of isolates without clinical information. Furthermore, we have no specific information on fidaxomicin usage in any patients or centers. We also do not have information on gradient testing in comparison to agar dilution. This was outside the scope of this surveillance.
Our study shows that fidaxomicin has excellent in vitro activity against a large number of C. difficile isolates that include BI isolates or those possessing a binary toxin gene profile. For 2011 to 2012, fidaxomicin usage was minimal so the impact of usage on in vitro activity of fidaxomicin remains to be determined. Surveillance for toxin gene profiles, REA typing or ribotyping for isolates of C. difficile remains very important, given the clinical data that fidaxomicin may not reduce recurrence in patients infected with the BI strains (6, 33).
This surveillance forms a baseline for future comparisons as fidaxomicin use becomes more common and other new agents are introduced for the treatment of C. difficile-associated disease (34, 35).
ACKNOWLEDGMENTS
We thank Carolina Baez-Giangreco, Barbara Rapino, and Roselia Martinez for their assistance.
We also thank Farah Babakhani and Diane Citron for their support of the study, as well as Adam Cheknis and Susan Sambol for REA typing.
This study was supported by Optimer Pharmaceuticals, La Jolla, CA. S.J. and D.N.G. are also supported by the VA Merit Review Program.
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