Reassessment of Clostridium difficile Susceptibility to Metronidazole and Vancomycin

Clostridium difficile is the most frequently identified entericpathogen in patients with nosocomially acquired, antibiotic-associateddiarrhea. The drugs most commonly used to treat diseases associatedwith C. difficile are metronidazole and vancomycin. Most clinicallaboratories assume that all C. difficile isolates are susceptibleto metronidazole and vancomycin. We report on the antimicrobialsusceptibilities of 415 C. difficile isolates to metronidazoleand vancomycin over an 8-year period (1993 to 2000). The overallrate of resistance to metronidazole at the critical breakpoint(16 µg/ml) was 6.3%. Although full resistance to vancomycinwas not observed, the overall rate of intermediate resistancewas 3.1%. One isolate had a combination of resistance to metronidazoleand intermediate resistance to vancomycin. Rates of resistanceto metronidazole and vancomycin were higher among isolates fromhuman immunodeficiency virus-infected patients. Molecular typingmethods proved the absence of clonality among the isolates withdecreased susceptibilities to the antimicrobials tested.

INTRODUCTION

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Introduction

Clostridium difficile-associated diarrhea (CDAD) is the mostcommon nosocomial diarrhea in adults, occurring mainly in patientswith prior antimicrobial therapy. The disease has variable incidencesand severities in different hospital populations (2, 6, 22,25, 27, 38). Recent information also points to CDAD as a relativelyfrequent cause of community-acquired episodes of diarrhea [24;T. V. Riley, M. Cooper, B. Bell, and C. L. Golledge, abstractfrom the 5th European Congress of Clinical Microbiology andInfectious Diseases (ECCMID) 1995, Clin. Infect. Dis. 20(Suppl.2):S263-S265, 1995].

The most serious cases of CDAD require antimicrobial therapywith agents which are active against C. difficile. Metronidazoleis the drug of choice due to its in vitro activity, its efficacyby either the oral or the intravenous route of administration,its presumed lower potential for selection of vancomycin-resistantEnterococcus (VRE), and the low cost of treatment with the drug.Nowadays, vancomycin is considered a second-line drug, mainlydue to the potential for the selection of VRE and its high cost.Both drugs are considered equivalent in efficacy, but infectionrecurrence rates of 15 to 35% have been reported for both drugs(3, 7).

In vitro determination of the susceptibility of C. difficileto these antibiotics is not routinely performed, as it is broadlyaccepted that C. difficile is regularly and predictably susceptibleto metronidazole and vancomycin. The assay method is time-consuming,and the use of susceptibility breakpoints is based on the therapeuticlevels of drugs in serum and not on the levels in the intraluminalarea, where higher drug concentrations can be achieved (1, 19).At present, metronidazole resistance in C. difficile is consideredanecdotal and vancomycin resistance has been, to the best ofour knowledge, reported only once (16), although on that occasionthe method used was not the present standard.

In 1994, we sounded the alarm when we reported on the firstseries of metronidazole-resistant C. difficile isolates (T.Peláez, R. Sánchez, R. Blázquez, P. Catalán,P. Muñoz, and E. Bouza, Abstr. 34th. Intersci. Conf.Antimicrob. Agents Chemother., abstr. E-34, p. 50, 1994). Theaim of the present study is to report on the decrease in therates of susceptibility to metronidazole and vancomycin in someof the C. difficile isolates in our institution over an 8-yearperiod (1993 to 2000).

MATERIALS AND METHODS

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Materials and Methods

Patients and strains. During the study period we identified 2,384 toxigenic strainsof C. difficile from 1,787 patients. Of these, 415 isolatesfrom 415 hospitalized adults with their first CDAD episode wererandomly selected for study.

C. difficile isolates were presumptively identified by theircolony morphology, yellow color, ground-glass texture, characteristichorse-dung smell, and Gram staining and fluorescent properties.Additional biochemical tests were also used (32).

The demonstration of toxin production was carried out eitherdirectly from fecal samples or, if the result was negative,from a "second-look" assay with strains isolated on cycloserine-cefoxitin-fructoseagar medium (12, 44). The presence of C. difficile toxin B wasdetermined by demonstrating a specific cytopathic effect onMRC-5 cells, as described previously (32, 41).

MIC determination by agar dilution. MICs were determined by the agar dilution method described bythe National Committee for Clinical Laboratory Standards (35).All assays were performed in duplicate.

The antimicrobial agents used in the study, metronidazole andvancomycin, were obtained from Sigma (St. Louis, Mo.). Serialtwofold dilutions of the antimicrobials were incorporated intosupplemented brucella agar (Oxoid, Basingstoke, United Kingdom),with antibiotic concentrations ranging from 0.016 to 32 µg/ml.Inocula were prepared from supplemented brucella broth (Oxoid),in which the organisms were grown at 37°C for 24 h. Cultureswere adjusted to an optical density on the McFarland scale of0.5, and 10 µl (10⁵ CFU/spot) was applied with a Steersreplicator to prereduced brucella agar. The plates were observedafter 48 h of incubation in an anaerobic chamber (85% nitrogen,10% hydrogen, 5% carbon dioxide) at 37°C.

The MIC was defined as the lowest concentration of the agentthat inhibited growth. The appearance of one or two coloniesor a barely visible haze was disregarded. Reference strains(Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicronATCC 29741) were included as controls to monitor the antimicrobialsusceptibility testing. A collection strain of C. difficile(ATCC 9689) was also included to assess the reproducibilitiesof the assays. The breakpoints for metronidazole were $<=$ 8 µg/mlfor susceptible, 16 µg/ml for intermediate, and $>=$ 32 µg/mlfor resistant. For vancomycin we used breakpoints of $<=$ 2 µg/mlfor susceptible, 4 to 16 µg/ml for intermediate, and $>=$ 32µg/ml for resistant, as no standard is defined by theNCCLS.

Bacterial typing. A subset of 10 isolates which were representative of those obtainedfrom different areas of the hospital throughout the study periodfrom patients belonging to different risk groups and which hadphenotypes of decreased susceptibility to both study drugs (metronidazoleand vancomycin) were studied for clonal relatedness. Randomamplification of polymorphic DNA (RAPD) analysis was performedas described previously (3). Oligonucleotides T7 (5'-GTAATACGACTCACTATAG-3')(46) and RAPD-#3 (5'-GTAGACCCGT-3'; Amersham Pharmacia Biotech,Uppsala, Sweden) were used to prime the RAPD reactions.

Statistical methods. The significance of differences in susceptibility patterns wasanalyzed by the chi-square or the two-tailed Fisher exact test.A P value of <0.05 was considered statistically significant.

RESULTS

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Results

The incidence of CDAD per 1,000 admissions at our institutionincreased from 2.1 in 1993 to 8.9 in 2000. The most common underlyingdiseases of our patients were as follows: 294 were human immunodeficiencyvirus (HIV)-positive patients (16.5%), 139 were organ transplantpatients (7.8%), 106 were oncology patients (5.9%), and 1,248had a miscellany of other diseases (69.8%)

Susceptibility to metronidazole. The metronidazole MIC at which 50% of isolates tested are inhibited(MIC₅₀) and the MIC₉₀ for the 415 strains assayed were 0.5 and4 µg/ml, respectively (range, 0.016 to >32 µg/ml).

A total of 26 strains of C. difficile (6.3% of all strains tested)were resistant to metronidazole. The distribution of resistantstrains throughout the study period is shown in Table 1. Therate of resistance ranged from 2.8 to 12.5%. The distributionof the MICs for the nonsusceptible strains is shown in Table2.

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TABLE 1. Chronological distribution of nonsusceptible C. difficile isolates

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TABLE 2. Distribution of metronidazole and vancomycin MICs for nonsusceptible C. difficile isolates

The rate of resistance was not uniform among the groups withdifferent underlying conditions (Table 3) and was significantlyhigher among the isolates from HIV-infected patients (12.6%)than among the isolates from the other patient groups (P <0.001).

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TABLE 3. Number of isolates of C. difficile and percent resistance to metronidazole and vancomycin, by patient risk group

Susceptibility to vancomycin. The vancomycin MIC₅₀ and MIC₉₀ for the 415 assayed strains were1 and 2 µg/ml, respectively (range, 0.016 to 16 µg/ml).

Strains not susceptible to vancomycin were first detected inour laboratory in 1996, although none of our isolates showedfull resistance to this agent. The evolution of vancomycin-intermediateisolates throughout the study period is summarized in Table1. Overall, 3.1% of all our isolates of C. difficile were notsusceptible to vancomycin. The distribution of the MICs forthe nonsusceptible strains is shown in Table 2.

The distribution among the different groups of patients of strainsnot susceptible to vancomycin is shown in Table 3. The proportionsranged from 0 to 4.2%, although the differences were not statisticallysignificant.

A high degree of genetic diversity was detected among the isolatesstudied by typing analysis (Fig. 1).

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FIG. 1. RAPD typing patterns of a representative subset of isolates. (A) Primer T7 (low level of discrimination); (B) primer RAPD-#3 (high level of discrimination). Lanes 1 and 15, molecular weight markers; lane 2, negative control; lanes 3 and 14, collection strain (C. difficile ATCC 9689); lanes 4 to 13, clinical C. difficile isolates.

DISCUSSION

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Discussion

C. difficile is the most frequently identified causal agentof hospital-acquired diarrhea. Advanced age, severe underlyingdisease, recent surgery, and, much more importantly, the prioruse of antimicrobial therapy are among the predisposing conditionsrelated to CDAD (10, 17, 28, 29, 33, 45, 48). Populations athigher risk also include those undergoing organ transplantationand HIV-infected patients [3, 5, 6, 11, 26, 30, 51; P. Muñoz,T. Peláez, J. Palomo, R. Muñoz, M. J. Ruiz Serrano,and E. Bouza, abstract from the 7th ECCMID 1997, Clin. Microbiol.Infect. 3(Suppl. 2):218, 1997].

The basis for adequate therapy relies on the rapid diagnosisof either toxin A or toxin B, or both, directly from fecal samplesor from strains of C. difficile isolates obtained from fecesby a negative direct test (a "second-look" assay for cytotoxicity)(3, 4, 12, 13, 15).

Metronidazole is the drug of choice for the treatment of firstepisodes of CDAD. It is effective when it is administered byeither the oral or the parenteral route, has a low potentialfor selection of VRE, and is inexpensive. Nowadays, vancomycinis considered a second-line drug, mainly due to its potentialfor the selection of VRE, its efficacy only with oral administration,and its high cost. Both drugs are considered to have equivalentefficacies, and both are associated with infection recurrencerates of 15 to 35% (7-9, 14, 18, 19, 21, 31, 39, 47, 50).

C. difficile is considered susceptible to metronidazole in mostinstitutions, and tests to assess the in vitro activity of metronidazoleagainst C. difficile isolates are rarely performed in most centers(20, 23, 34, 36, 50). It is well known, however, that CDAD maydevelop in patients during metronidazole therapy (43) and thatisolates of C. difficile resistant to metronidazole in vitrohad already been reported anecdotally in 1981 (37), althoughthese facts are not necessarily related.

In 1994, our group reported a 6% rate of resistance to metronidazoleamong 78 isolates of C. difficile (Peláez et al., 38thICAAC). In another study (T. Peláez, L. Alcalá,L. Martínez-Sánchez, P. Muñoz, J. M. García-Lechuz,M. Rodríguez-Créixems, and E. Bouza, Abstr. 38thIntersci. Conf. Antimicrob. Agents Chemother., abstr. E-173,p. 219, 1998), we found that 9% of strains were resistant (MICs, $>=$ 32 µg/ml). Other recent reports from different investigatorshave also warned about this problem. In 1997, high-level metronidazoleresistance was demonstrated in C. difficile isolates obtainedfrom horses [S. S. Jang, L. M. Hansen, J. E. Breher, D. A. Riley,K. G. Magdesian, J. E. Madigan, Y. J. Tang, J. Silva, Jr., andD. C. Hirsh, abstract from the 35th Annual Meeting of InfectiousDiseases Society of America 1997, Clin. Infect. Dis. 25(Suppl.2):S266-S267, 1997], and Wong et al. (49) later reported ona clinical isolate of C. difficile, from among 100 isolatesstudied, for which the metronidazole MIC was >64 µg/ml.In our study, the highest rate of metronidazole resistance wasobserved in HIV-infected patients. This fact may be explained,among other reasons, by a theoretically higher probability ofexposure to the antimicrobial agent on previous occasions.

To our knowledge, isolates of C. difficile that were not susceptibleto vancomycin were reported only three times, from Poland in1991 (16), although the study was performed by a disk diffusionmethod. Our data also showed that 13 strains (3.1%) had decreasedsusceptibility to vancomycin.

The first isolates of C. difficile not susceptible to vancomycinappeared in our institution in 1996, and since then we havehad a low but persistent number of resistant isolates everyyear. This should be another reason to relegate vancomycin toa second position as the drug of choice for the treatment ofCDAD. The risk of development of colonization with VRE and VREsuperinfections in patients treated with oral vancomycin isalready known (39, 40, 42).

The present paper demonstrates that this situation is far fromanecdotal in our institution and that this problem was endemicduring the study period. It could be argued that the situationin our institution may represent the epidemic spread of a singleclone of a resistant strain, but molecular studies involvingisolates from our institution have already shown that this isnot the case (3). The typing study performed with some of theisolates as part of the present study demonstrates that a highdegree of clonal heterogeneity was present. This fact was evenobserved with primer T7, described as an oligonucleotide witha low discriminatory ability (46), which is useful for the establishmentof groups with high degrees of similarity for further analysis.Primer RAPD-#3 was able to show a high degree of clonal heterogeneitywith a greater discriminatory ability.

It is also worth discussing at this point the fact that MICbreakpoints are usually set for isolates causing systemic infectionsand that they are based upon antimicrobial levels in blood.In this case, levels in the colon seem to be much more interestingthan the levels in the circulation. For metronidazole and vancomycin,levels in the colon might even be high enough to kill "resistant"strains, although this issue has not been explored.

Our study tries to draw attention to a new problem in the emergenceof antimicrobial resistance. Microbiology laboratories shouldnot continue considering C. difficile a pathogen that cannotdevelop resistance to metronidazole and/or vancomycin. The immediatechallenges in this area, in our opinion, are to demonstratethe extension of the problem in other institutions, to outlinethe predisposing factors for CDAD caused by strains which arenot susceptible to vancomycin and/or metronidazole, and to clarifythe impact of nonsusceptible strains on clinical therapy andtheir potential role in relapses. It is also necessary to demonstratethe mechanism of resistance and to look for new alternativesfor the treatment and prevention of CDAD.

ACKNOWLEDGMENTS

We thank Thomas O'Boyle for help with the correction of theEnglish version of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Servicio de Microbiologia Clínica y Enfermedades Infecciosas, Hospital General Universitario "Gregorio Marañon," C/Dr. Esquerdo, 46, 28007, Madrid, Spain. Phone: 34-91-5868793. Fax: 34-91-504 49 06. E-mail: ebouza{at}microb.net.

REFERENCES

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