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Metronidazole Susceptibility in Clostridium difficile Isolates Recovered from Cases of C. difficile -associated Disease Treatment Failures and Successes
Anaerobe (1999) 5, 201±204 Article No. anae.1999.0268
CLOSTRIDIUM DIFFICILE AND ITS ROLE IN INFECTION (ORAL PRESENTATION)
Metronidazole Susceptibility in Clostridium di¤cile Isolates Recovered from Cases of C. di¤cile-associated DiseaseTreatment Failures and Successes Jason L. Sanchez*, Dale N. Gerding, Mary M. Olson, and Stuart Johnson{ 1
Medical Service, VA Chicago Health Care System, Lakeside Division; 2 Infectious Disease Section, Department of Medicine, Northwestern University Medical School, Chicago, IL, 3 Surgical Service, Minneapolis VA Medical Center, Minneapolis, MN, U.S.A. Key Words: Clostridium difficile, Metronidazole resistance, C. difficile-associated disease
Despite high clinical efficacy, a small number of patients with Clostridium difficile-associated disease (CDAD) do not respond to treatment with metronidazole. We looked for evidence of metronidazole resistance in human C. difficile isolates from 632 patients with CDAD treated with metronidazole, 14(2%) of whom failed treatment. C. difficile isolates were available from 10 of the metronidazole-failure cases and were matched with isolates from 20 contemporary control CDAD patients who responded to treatment with metronidazole. The mean (+SD) MIC of metronidazolefailure-associated C. difficile isolates was similar to the mean (+SD) MIC of isolates from metronidazole-success cases (E-test; 0.23+0.21 vs 0.29+0.19 mg/mL, P = 0.4). Restriction endonuclease analysis typing revealed that no particular C. difficile strain was associated with metronidazole treatment failure. Clinical CDAD treatment failures with metronidazole could not be attributed to decreased susceptibility of the causative C. difficle isolate to metronidazole. # 1999 Academic Press
Introduction Oral metronidazole is both an effective and inexpensive agent in the treatment of Clostridium difficileassociated diarrhea (CDAD) [1]. Metronidazole is now considered as the drug of choice for this *Current address: Washington University School of Medicine, Campus Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110, U.S.A. {Corresponding author: Medical Service, VACHCS, Lakeside Division, 333 East Huron, Chicago, IL 60611, U.S.A. Tel.: +1 312 640 2193; Fax: +1 312 640 2313; E-mail: [email protected]. Supported by the U.S. Department of Veterans Affairs Research Service.
1075±9964/99/030201 + 04 $30.00/0
indication because of comparable efficacy to that of vancomycin and the concern of spread of glycopeptide resistance to other pathogens [2]. Despite high efficacy in the initial treatment of CDAD (495%), a small number of patients fail therapy with metronidazole. The reasons behind treatment failure with metronidazole have not been extensively studied. C. difficle isolates, in general, display high susceptibility to metronidazole in vitro [3], however, high level resistance to metronidazole (48 mg/mL) was recently reported in C. difficile isolates recovered from horses [4]. To investigate whether or not metronidazole resistance of C. difficile is a factor in human # 1999 Academic Press
202
J.L. Sanchez et al.
treatment failures, C. difficile isolates recovered from clinical treatment failures over a 10-year period at a single institution were tested for metronidazole susceptibility and subjected to REA typing.
Materials and Methods Patients and controls Clostridium difficile isolates were saved and clinical data recorded on all patients with CDAD over a 10year period (1982 to 1991) at the Minneapolis VA Medical Center [5]. CDAD was defined as the presence of diarrhea ( 6 watery stools in a 36-h period) in a patient with a positive stool culture or stool cytotoxin assay. Treatment failure was defined as persistent watery stools ( 4 stools/day) after 7 days of treatment with metronidazole or vancomycin. Metronidazole treatment regimens were typically 250 mg given orally four times daily or 500 mg three times daily. Of 632 patients with CDAD treated with metronidazole, 14(2%) were treatment failures. C. difficile isolates were available for analysis from 10 of the 14 cases. The stool specimen isolates were obtained prior to initiation of specific treatment for CDAD. Two controls were chosen for each treatment failure case from patients successfully treated with oral metronidazole and represent the first patient who presented chronologically before and after each treatment failure case. Metronidazole susceptibility testing E-test susceptibility testing was performed using prereduced Brucella-based blood agar (BBA) plates inoculated with C. difficile for confluent growth and overlaid with test strips impregnated with metronidazole in a continuous gradient (Biodisk, Solna, Sweden). The plates were then incubated in an anaerobic chamber at 378C for 24 h and the MIC
value read from the test strip at the intercept of the inhibition ellipse. Agar dilution susceptibility testing was performed using BBA plates prepared with metronidazole to achieve concentrations ranging from 0.05 to 4.0 mg/ mL [6]. Clostridium difficile isolates were inoculated into pre-reduced trypticase soy broth media and incubated overnight at 378C. The inoculum was adjusted to a 1.0 McFarland standard and transferred to the pre-reduced BBA-metronidazole plates using a replicator device. Plates were incubated at 378C for 48 h and examined for growth. Restriction endonuclease analysis (REA) REA typing using HindIII restriction digestion was performed as previously described [7]. DNA fragment banding patterns were visually compared to patterns of previously identified REA types from a large library collection of C. difficile isolates. Patterns which show 90% homology were placed in the same REA group designated by a letter.
Results All C. difficile isolates tested by E-test and agar dilution were susceptible to metronidazole. The highest MIC for any isolate was 0.75 mg/mL. The mean MIC of C. difficle isolates recovered from the metronidazole-failure cases was similar to that of the metronidazole-success cases (0.23+0.21 vs 0.29+0.19 mg/mL by E-test, P = 0.40, Student's t-test) (Table 1). The median MIC of the metronidazolefailure isolates appeared to be higher than that of the metronidazole-success isolates when tested by the agar dilution method (Table 1) but the MIC values for the controls were tightly clustered and this difference is of doubtful clinical significance. Results of E-testing showed a modest correlation with the agar dilution results (correlation coefficient =0.58).
Table 1. In vitro metronidazole susceptibility (MIC in mg/mL) of Clostridium difficile isolated associated with metronidazole treatment failure and treatment success of C. difficile diarrhea cases
E-test
Agar dilution
Mean +SD Median Range Geometric mean Median Range
*2-tailed Student's t-test (assuming equal variances). Mann±Whitney U-test.
{
Metronidazole treatment failure (n=10)
Metronidazole treatment success (n=20)
0.23 +0.21 0.19 0.064 to 0.75 0.32 0.38 0.19 to 0.5
0.29 +0.19 0.25 0.094 to 0.75 0.38 0.38 0.25 to 0.5
P = 0.40*
P = 0.05{
Metronidazole Susceptibility in Clostridium di¤cile Isolates
203
Figure 1. Percentage of Clostridium difficile isolates from metronidazole treatment-failure and treatment-success cases of C. difficile-associated diarrhea in each of the 13 restriction endonuclease analysis (REA) groups identified.
Clostridium difficile isolates from one patient were obtained prior to metronidazole therapy and after 10 days of treatment at which time his therapy was switched to vancomycin because of continuing diarrhea. The stool cytotoxin assay was positive on both occasions. The MICs of both isolates were similar by E-test (0.25 and 0.38 mg/mL) and identical by the agar dilution method (0.50 mg/mL). Thirteen different REA groups were present among the C. difficile isolates and these REA groups were randomly distributed between the treatment failure and success cases (Figure 1). REA group Y accounted for 45% of the treatment failure isolates and 20% of the treatment success isolates. Two isolates from each group had the identical REA type pattern, YIP. No particular C. difficile REA group or type was associated with metronidazole treatment failure.
Discussion Metronidazole resistance of the infecting C. difficile strain was not an explanation for treatment failure in this cohort of 632 CDAD cases identified through 10 years of active surveillance at one hospital. None of the C. difficile isolates from either treatment failures or their successfully treated controls were resistant to metronidazole. In the one treatment failure case with pre- and post-treatment C. difficile isolates available for testing, metronidazole resistance did not develop after 10 days of treatment. Moreover, relative susceptibility of C. difficile isolates did not distinguish between these two groups of patients. REA typing corroborated these findings in that no particular C. difficile strain was associated with metronidazole treatment failures.
The reasons behind metronidazole treatment failures in CDAD remain obscure and are probably multifactorial. Achievement of significant fecal drug levels has been a concern with metronidazole as it is well absorbed and difficult to detect in the stool of volunteers in the absence of diarrhea. However, bactericidal fecal concentrations are present in patients with CDAD and decline as the diarrhea improves [8]. In this study patient stool specimens were not available for analysis of metronidazole concentrations. Although metronidazole resistance was not found among clinical C. difficile isolates in this study, the recent report of high-level resistance in a C. difficile strain recovered from horses [4] warrants continued surveillance among human cases of CDAD. In conclusion, metronidazole is an efficacious agent for the treatment of CDAD and no cases of metronidazole resistance among human clinical strains have been clearly documented. For the small group of patients with CDAD who fail treatment with metronidazole, mechanisms other than resistance of C. difficile are operative. Acknowledgements The authors thank Connie R. Clabots for REA typing, Lance R. Peterson, MD for clinical laboratory support, Susan P. Sambol, BS and Michelle M. Merrigan, MS for laboratory assistance and David W. Hecht, MD for advice.
References 1. Gerding D.N., Johnson S., Peterson L.R., Mulligan M.E. and Silva J. (1995) Clostridium difficile-associated diarrhea and colitis. Infect Control Hosp Epidemiol 16: 459±477
204
J.L. Sanchez et al.
2. Hospital Infection Control Practices Advisory Committee (HICPAC) (1995) Recommendations for preventing the spread of vancomycin resistance. Infect Control Hosp Epidemiol 16: 105±113 3. Johnson S. and Gerding D.N. (1998) Clostridium difficile. IN: Yu V.L., Merigan T.C. and Barriere S. (eds) Antimicrobial Chemotherapy and Vaccines. Williams&Wilkins, Baltimore, MD 4. Jang S.S., Hansen L.M., Breher J.E. et al. (1997) Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 25(Suppl. 2): S266±267 5. Olson M.M., Shanholtzer C.J., Lee J.T.Jr. and Gerding D.N. (1994) Ten years of prospective Clostridium difficile-associated disease surveillance and treatment at the Minneapolis VA Medical Center, 1982±1991. Infect Control Hosp Epidemiol 15: 371±381
6. National Committee for Clinical Laboratory Standards (1997) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 4 Edn. Approved Standard. NCCLS Document No. M11-A4. National Committee for Clinical Laboratory Standards, Wayne, PA 7. Clabots C.R., Johnson S., Bettin K.M., Mathie P.A., Mulligan M.E., Schaberg D.R., Peterson L.R. and Gerding D.N. (1993) Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. J Clin Microbiol 31: 1870±1875 8. Bolton R.P. and Culshaw M.A. (1986) Fecal metronidazole concentrations during oral and intravenous therapy for antibiotic-associated colitis due to Clostridium difficile. Gut 27: 1169±1172
CLOSTRIDIUM DIFFICILE AND ITS ROLE IN INFECTION (ORAL PRESENTATION)
Metronidazole Susceptibility in Clostridium di¤cile Isolates Recovered from Cases of C. di¤cile-associated DiseaseTreatment Failures and Successes Jason L. Sanchez*, Dale N. Gerding, Mary M. Olson, and Stuart Johnson{ 1
Medical Service, VA Chicago Health Care System, Lakeside Division; 2 Infectious Disease Section, Department of Medicine, Northwestern University Medical School, Chicago, IL, 3 Surgical Service, Minneapolis VA Medical Center, Minneapolis, MN, U.S.A. Key Words: Clostridium difficile, Metronidazole resistance, C. difficile-associated disease
Despite high clinical efficacy, a small number of patients with Clostridium difficile-associated disease (CDAD) do not respond to treatment with metronidazole. We looked for evidence of metronidazole resistance in human C. difficile isolates from 632 patients with CDAD treated with metronidazole, 14(2%) of whom failed treatment. C. difficile isolates were available from 10 of the metronidazole-failure cases and were matched with isolates from 20 contemporary control CDAD patients who responded to treatment with metronidazole. The mean (+SD) MIC of metronidazolefailure-associated C. difficile isolates was similar to the mean (+SD) MIC of isolates from metronidazole-success cases (E-test; 0.23+0.21 vs 0.29+0.19 mg/mL, P = 0.4). Restriction endonuclease analysis typing revealed that no particular C. difficile strain was associated with metronidazole treatment failure. Clinical CDAD treatment failures with metronidazole could not be attributed to decreased susceptibility of the causative C. difficle isolate to metronidazole. # 1999 Academic Press
Introduction Oral metronidazole is both an effective and inexpensive agent in the treatment of Clostridium difficileassociated diarrhea (CDAD) [1]. Metronidazole is now considered as the drug of choice for this *Current address: Washington University School of Medicine, Campus Box 8051, 660 S. Euclid Ave., St. Louis, MO 63110, U.S.A. {Corresponding author: Medical Service, VACHCS, Lakeside Division, 333 East Huron, Chicago, IL 60611, U.S.A. Tel.: +1 312 640 2193; Fax: +1 312 640 2313; E-mail: [email protected]. Supported by the U.S. Department of Veterans Affairs Research Service.
1075±9964/99/030201 + 04 $30.00/0
indication because of comparable efficacy to that of vancomycin and the concern of spread of glycopeptide resistance to other pathogens [2]. Despite high efficacy in the initial treatment of CDAD (495%), a small number of patients fail therapy with metronidazole. The reasons behind treatment failure with metronidazole have not been extensively studied. C. difficle isolates, in general, display high susceptibility to metronidazole in vitro [3], however, high level resistance to metronidazole (48 mg/mL) was recently reported in C. difficile isolates recovered from horses [4]. To investigate whether or not metronidazole resistance of C. difficile is a factor in human # 1999 Academic Press
202
J.L. Sanchez et al.
treatment failures, C. difficile isolates recovered from clinical treatment failures over a 10-year period at a single institution were tested for metronidazole susceptibility and subjected to REA typing.
Materials and Methods Patients and controls Clostridium difficile isolates were saved and clinical data recorded on all patients with CDAD over a 10year period (1982 to 1991) at the Minneapolis VA Medical Center [5]. CDAD was defined as the presence of diarrhea ( 6 watery stools in a 36-h period) in a patient with a positive stool culture or stool cytotoxin assay. Treatment failure was defined as persistent watery stools ( 4 stools/day) after 7 days of treatment with metronidazole or vancomycin. Metronidazole treatment regimens were typically 250 mg given orally four times daily or 500 mg three times daily. Of 632 patients with CDAD treated with metronidazole, 14(2%) were treatment failures. C. difficile isolates were available for analysis from 10 of the 14 cases. The stool specimen isolates were obtained prior to initiation of specific treatment for CDAD. Two controls were chosen for each treatment failure case from patients successfully treated with oral metronidazole and represent the first patient who presented chronologically before and after each treatment failure case. Metronidazole susceptibility testing E-test susceptibility testing was performed using prereduced Brucella-based blood agar (BBA) plates inoculated with C. difficile for confluent growth and overlaid with test strips impregnated with metronidazole in a continuous gradient (Biodisk, Solna, Sweden). The plates were then incubated in an anaerobic chamber at 378C for 24 h and the MIC
value read from the test strip at the intercept of the inhibition ellipse. Agar dilution susceptibility testing was performed using BBA plates prepared with metronidazole to achieve concentrations ranging from 0.05 to 4.0 mg/ mL [6]. Clostridium difficile isolates were inoculated into pre-reduced trypticase soy broth media and incubated overnight at 378C. The inoculum was adjusted to a 1.0 McFarland standard and transferred to the pre-reduced BBA-metronidazole plates using a replicator device. Plates were incubated at 378C for 48 h and examined for growth. Restriction endonuclease analysis (REA) REA typing using HindIII restriction digestion was performed as previously described [7]. DNA fragment banding patterns were visually compared to patterns of previously identified REA types from a large library collection of C. difficile isolates. Patterns which show 90% homology were placed in the same REA group designated by a letter.
Results All C. difficile isolates tested by E-test and agar dilution were susceptible to metronidazole. The highest MIC for any isolate was 0.75 mg/mL. The mean MIC of C. difficle isolates recovered from the metronidazole-failure cases was similar to that of the metronidazole-success cases (0.23+0.21 vs 0.29+0.19 mg/mL by E-test, P = 0.40, Student's t-test) (Table 1). The median MIC of the metronidazolefailure isolates appeared to be higher than that of the metronidazole-success isolates when tested by the agar dilution method (Table 1) but the MIC values for the controls were tightly clustered and this difference is of doubtful clinical significance. Results of E-testing showed a modest correlation with the agar dilution results (correlation coefficient =0.58).
Table 1. In vitro metronidazole susceptibility (MIC in mg/mL) of Clostridium difficile isolated associated with metronidazole treatment failure and treatment success of C. difficile diarrhea cases
E-test
Agar dilution
Mean +SD Median Range Geometric mean Median Range
*2-tailed Student's t-test (assuming equal variances). Mann±Whitney U-test.
{
Metronidazole treatment failure (n=10)
Metronidazole treatment success (n=20)
0.23 +0.21 0.19 0.064 to 0.75 0.32 0.38 0.19 to 0.5
0.29 +0.19 0.25 0.094 to 0.75 0.38 0.38 0.25 to 0.5
P = 0.40*
P = 0.05{
Metronidazole Susceptibility in Clostridium di¤cile Isolates
203
Figure 1. Percentage of Clostridium difficile isolates from metronidazole treatment-failure and treatment-success cases of C. difficile-associated diarrhea in each of the 13 restriction endonuclease analysis (REA) groups identified.
Clostridium difficile isolates from one patient were obtained prior to metronidazole therapy and after 10 days of treatment at which time his therapy was switched to vancomycin because of continuing diarrhea. The stool cytotoxin assay was positive on both occasions. The MICs of both isolates were similar by E-test (0.25 and 0.38 mg/mL) and identical by the agar dilution method (0.50 mg/mL). Thirteen different REA groups were present among the C. difficile isolates and these REA groups were randomly distributed between the treatment failure and success cases (Figure 1). REA group Y accounted for 45% of the treatment failure isolates and 20% of the treatment success isolates. Two isolates from each group had the identical REA type pattern, YIP. No particular C. difficile REA group or type was associated with metronidazole treatment failure.
Discussion Metronidazole resistance of the infecting C. difficile strain was not an explanation for treatment failure in this cohort of 632 CDAD cases identified through 10 years of active surveillance at one hospital. None of the C. difficile isolates from either treatment failures or their successfully treated controls were resistant to metronidazole. In the one treatment failure case with pre- and post-treatment C. difficile isolates available for testing, metronidazole resistance did not develop after 10 days of treatment. Moreover, relative susceptibility of C. difficile isolates did not distinguish between these two groups of patients. REA typing corroborated these findings in that no particular C. difficile strain was associated with metronidazole treatment failures.
The reasons behind metronidazole treatment failures in CDAD remain obscure and are probably multifactorial. Achievement of significant fecal drug levels has been a concern with metronidazole as it is well absorbed and difficult to detect in the stool of volunteers in the absence of diarrhea. However, bactericidal fecal concentrations are present in patients with CDAD and decline as the diarrhea improves [8]. In this study patient stool specimens were not available for analysis of metronidazole concentrations. Although metronidazole resistance was not found among clinical C. difficile isolates in this study, the recent report of high-level resistance in a C. difficile strain recovered from horses [4] warrants continued surveillance among human cases of CDAD. In conclusion, metronidazole is an efficacious agent for the treatment of CDAD and no cases of metronidazole resistance among human clinical strains have been clearly documented. For the small group of patients with CDAD who fail treatment with metronidazole, mechanisms other than resistance of C. difficile are operative. Acknowledgements The authors thank Connie R. Clabots for REA typing, Lance R. Peterson, MD for clinical laboratory support, Susan P. Sambol, BS and Michelle M. Merrigan, MS for laboratory assistance and David W. Hecht, MD for advice.
References 1. Gerding D.N., Johnson S., Peterson L.R., Mulligan M.E. and Silva J. (1995) Clostridium difficile-associated diarrhea and colitis. Infect Control Hosp Epidemiol 16: 459±477
204
J.L. Sanchez et al.
2. Hospital Infection Control Practices Advisory Committee (HICPAC) (1995) Recommendations for preventing the spread of vancomycin resistance. Infect Control Hosp Epidemiol 16: 105±113 3. Johnson S. and Gerding D.N. (1998) Clostridium difficile. IN: Yu V.L., Merigan T.C. and Barriere S. (eds) Antimicrobial Chemotherapy and Vaccines. Williams&Wilkins, Baltimore, MD 4. Jang S.S., Hansen L.M., Breher J.E. et al. (1997) Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 25(Suppl. 2): S266±267 5. Olson M.M., Shanholtzer C.J., Lee J.T.Jr. and Gerding D.N. (1994) Ten years of prospective Clostridium difficile-associated disease surveillance and treatment at the Minneapolis VA Medical Center, 1982±1991. Infect Control Hosp Epidemiol 15: 371±381
6. National Committee for Clinical Laboratory Standards (1997) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 4 Edn. Approved Standard. NCCLS Document No. M11-A4. National Committee for Clinical Laboratory Standards, Wayne, PA 7. Clabots C.R., Johnson S., Bettin K.M., Mathie P.A., Mulligan M.E., Schaberg D.R., Peterson L.R. and Gerding D.N. (1993) Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems. J Clin Microbiol 31: 1870±1875 8. Bolton R.P. and Culshaw M.A. (1986) Fecal metronidazole concentrations during oral and intravenous therapy for antibiotic-associated colitis due to Clostridium difficile. Gut 27: 1169±1172