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Antimicrobial Agents and Chemotherapy, January 2009, p. 261-263, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.01443-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

OPT-80 Eliminates Clostridium difficile and Is Sparing of Bacteroides Species during Treatment of C. difficile Infection{triangledown}

Thomas J. Louie,* Judy Emery, Walter Krulicki, Brendan Byrne, and Manuel Mah

Division of Infectious Diseases, Departments of Medicine and Microbiology Infectious Diseases, University of Calgary, Calgary, Alberta, Canada, and Infection Prevention & Control Program, Calgary Health Region, Calgary, Alberta, Canada

Received 6 November 2007/ Returned for modification 26 March 2008/ Accepted 17 October 2008


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ABSTRACT
 
During a 10-day treatment for Clostridium difficile infection, OPT-80 and vancomycin were comparably effective in reducing C. difficile counts. Bacteroides fragilis group counts appeared unaffected with increasing dosages of OPT-80, whereas vancomycin was markedly suppressive. Retention of components of the normal microflora might lower the risk of recurrent disease.


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INTRODUCTION
 
The selectivity of OPT-80, a novel macrocycle (5, 9, 10), as treatment for Clostridium difficile infection (CDI) (MIC at which 90% of the isolates tested are inhibited [MIC90] of 0.12 µg/ml) was examined during a clinical trial (8). OPT-80 has poor activity against aerobic and facultative gram-negative bacilli, Bacteroides species (MIC90 of >1,024 µg/ml), and other gram-negative anaerobes (1, 2, 4), a feature that might result in a reduced ecologic impact (3, 6). Microbial ecologic parameters examined during treatment of CDI included the following: (i) suppression of C. difficile quantitative counts, (ii) recrudescence of C. difficile vegetative and/or spore counts after therapy, (iii) reexpression of C. difficile cytotoxin B in fecal filtrates following treatment, and (iv) changes in quantitative counts of Bacteroides species as a marker of the anaerobic microbial flora.

(This study was presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, 16 to 19 December 2005, Washington, DC, abstr. LB2-30, and at the 16th European Congress of Clinical Microbiology & Infectious Diseases, 1 to 4 April 2006, Nice, France.)

The phase 2A open-label study randomized patients to receive 50 mg, 100 mg, or 200 mg of OPT-80 orally every 12 hours (q12h) for 10 days (8). Following completion of the study, eight additional comparable patients with CDI received 125 mg of vancomycin four times a day (qid) for 10 days as standard treatment controls. Stools from five healthy volunteers served as culture controls. Serial stool samples (≥5 grams) were obtained at study entry and at days 4,10, 14, 21, 28, and 42. C. difficile cytotoxin B titers were determined by serial twofold dilution of fecal filtrates (1/20 to 1/16,000) prepared from freshly passed samples by HeLa cell tissue culture assay with neutralization (TechLab, Blacksburg, VA). C. difficile counts were determined by dilution of the sample (10–2, 10–4, 10–6, and 10–8 all per gram [wet weight] of stool) on cefoxitin cycloserine fructose agar. Spore counts were determined by alcohol shock.

Media and methods for fecal cultures are outlined in the Wadsworth-KTL Anaerobic Manual (6a). Since Bacteroides species are most easily and consistently recovered and represent 8% of the fecal biomass (7), this genus was used as a marker of the status of the normal microbiota. If samples failed to recover ≥108 CFU of Bacteroides spp. per gram of stool, later samples were processed. Quantitative counts were determined by serial dilution (10–3, 10–5, 10–7, and 10–9 [all per gram of stool]) onto selective and differential media incubated for 7 days.

Differences in microbial counts were determined after log10 transformation using a two-tailed Wilcoxon matched-pair signed-rank test (GraphPad Instat, San Diego, CA). For counts of <3 log 10 CFU/g, a value of 2.9 was used. Differences in spore counts were determined by unpaired t test, Welch corrected; spore counts of <2 log10 CFU/g were tabulated as 1.9.

Thirty of 33 phase 2A patients provided sufficient samples for determination of suppression and recrudescence of C. difficile (50-mg dose, n = 10; 100-mg dose, n = 8; 200-mg dose, n = 12); 29 were cultured for normal flora. The majority (23/30) of OPT-80 recipients were treatment naïve as were 7/8 vancomycin controls. Normal stools (all five) contained four or more Bacteroides spp. per sample, in counts from 1011 to 1012 CFU/g of stool. Multiple other genera were present but in variable numbers. Of 38 study entry samples, Bacteroides group (BG) counts were <103 CFU/g of stool in 11 patients (29%), 103 to 107 CFU/g of stool in 11 patients (29%), and 108 to 1010 CFU/g of stool in 16 patients (42%). BG counts in patients who received one or two doses of metronidazole (n = 6) or vancomycin (n = 1) did not differ from treatment naïve patients (n = 30) (mean log10 CFU/g of stool ± standard deviation [SD] of 6.6 ± 3.6 versus 7.2 ± 2.8; P = 0.86).

Table 1 summarizes changes in BG counts over the 10-day treatment period. Dose-dependent suppression of BG counts with increasing dosages of OPT-80 was not observed. Vancomycin reduced BG counts in six of seven subjects (mean log 10 CFU/g of stool ± SD of 8.2 ± 2.1 to <3); in four of six subjects, the BG count recovered by days 21 to 28. No recovery of BG was observed in two patients with samples available to days 28 and 42. The remaining vancomycin-treated patient had no Bacteroides spp. detected at study entry, and no recovery was detected within the 21 days that samples were available. Failure of detection of BG organisms at study entry and over 21 to 28 days was also observed in 5/29 (17%) OPT-80-treated patients. One such patient in the 50-mg OPT-80 group showed eradication of C. difficile and clinical response but also showed persistent absence of BG organisms from study entry onwards, reappearance of C. difficile (same clone by ribotype) by day 42, and recurrence of CDI by day 53.


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  		TABLE 1. Changes in Bacteroides group counts over the 10-day treatment perioda

In vivo activity of OPT-80 against C. difficile is shown in Fig. 1. At study entry, mean log 10 CFU/g of C. difficile ± SD quantitative paired vegetative and spore counts across all OPT-80 treatment groups (n = 28) were 7.0 ± 2.4 (95% confidence interval [95% CI] of 6.0 to 7.9) and 4.2 ± 1.6 (95% CI of 3.5 to 4.8), respectively (P < 0.0001). Between groups, counts were similar. All dosages of OPT-80 appeared to be effective in reducing C. difficile total counts.


Figure 1
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  		FIG. 1. Results of paired quantitative cultures for C. difficile at study entry (day 0) and at the end of therapy (day 10) in patients treated with OPT-80 and vancomycin (Vanco). bid, twice a day; QID, four times a day.

In the 50-mg OPT-80 group, all 10 patients were clinical responders; C. difficile counts were reduced to <102 CFU/g of stool by day 4 in 8/10 patients and remained so at day 10. Of the two remaining patients, both with persisting BG serial counts of ≥109 CFU/g of stool, one showed early complete suppression of the C. difficile with lower count reappearance from days 10 to 28 (not recovered day 42), and the other showed a 2 log10 unit reduction during and after treatment with persistence of C. difficile until recurrence at day 41.

All eight patients in the 100-mg dosage group showed reduction of C. difficile counts to <102 CFU/g of stool by days 4 and day 10. All patients were cured, with no recurrence of CDI.

In the 200-mg group (all 12 responders), C. difficile counts were ≤102 CFU/g of stool by days 4 and 10 in 11/12 patients. One patient showed a modest reduction in C. difficile (MIC of 0.06) counts from 107.2 to 104.9 CFU/g of stool by day 4, but counts were 107.5 CFU/g of stool on day 8 and 107.7 CFU/g of stool at day 10 (OPT-80/OPT-1118 fecal concentration of 3,975/1,165 µg/ml). Subsequent C. difficile cultures showed clearance of the organism by day 21 with no reappearance through day 42.

In vancomycin controls, day 0 and day 10 cultures showed that C. difficile counts were reduced to <10 2 CFU/g of stool by day 10. At day 4, five/six patients showed no recovery of C. difficile. Recurrence was observed in one patient 1 week after completing vancomycin treatment.

Reemergence of C. difficile spores and toxin is summarized in Table 2. In comparing day 21 to 28 spore counts (mean ± SD [95% CI]) for vancomycin recipients (3.6 ± 2.0 [2.4 to 4.7]), the counts in OPT-80 recipients were found to be lower (2.7 ± 1.5 [2.0 to 3.4], P = 0.21; 2.3 ± 0.9 [1.8 to 2.7], P = 0.04; and 2.3 ± 0.9 [1.8 to 2.7], P = 0.04 for 50-mg, 100-mg, and 200-mg OPT-80 treatment, respectively. Compared to all OPT-80 patients, reappearance of C. difficile cytotoxin B appeared to be more likely following vancomycin treatment (P = 0.03).


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  		TABLE 2. Reemergence of C. difficile spores in the follow-up period (days 21 to 28)

The results of this study show the following. (i) C. difficile counts are markedly reduced by both OPT-80 and vancomycin. (ii) Bacteroides group counts are highly and variably reduced by CDI itself. (iii) Increasing dosages of OPT-80 did not appear to suppress BG group counts. (iv) Lower posttreatment spore counts and infrequent reexpression of C. difficile toxin B imply a comparative beneficial ecologic effect by OPT-80 as a CDI treatment option.


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ACKNOWLEDGMENTS
 
Microbiologic aspects of the study were supported by an unrestricted grant from Optimer Pharma. T. Louie is an investigator for Optimer Pharma, Genzyme, and Wyeth. The other authors have no conflicts to declare.


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FOOTNOTES
 
* Corresponding author. Mailing address: Infection Prevention & Control, AGW-5, Foothills Medical Center, 1403 29th St. N.W., Calgary, Alberta T2N 2T9, Canada. Phone: (403) 944-4766. Fax: (403) 944-2484. E-mail: thomas.louie{at}calgaryhealthregion.ca Back

{triangledown} Published ahead of print on 27 October 2008. Back


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Antimicrobial Agents and Chemotherapy, January 2009, p. 261-263, Vol. 53, No. 1
0066-4804/09/$08.00+0     doi:10.1128/AAC.01443-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Williams, O. M., Spencer, R. C. (2009). The management of Clostridium difficile infection. Br Med Bull 91: 87-110 [Abstract] [Full Text]  

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