Current Clostridium difficile Strains May Be Less Susceptible to Metronidazole

Chris Berrie, MA, MPhil, PhD

April 23, 2008

April 23, 2008 (Barcelona) — Significant differences in metronidazole (MTZ) minimum inhibitory concentrations (MICs) between recent vs historic strains of Clostridium difficile reveal the emergence of the bacillus' reduced susceptibility to MTZ. Furthermore, the identification of ribotype subgroups that are more closely associated with reduced susceptibility to MTZ provides indications for clonal spread.

A new study was presented here at the 18th European Congress of Clinical Microbiology and Infectious Diseases on behalf of the C difficile Research Group by Simon Baines, PhD, from the Institute of Molecular and Cellular Biology in the Old Medical School of the University of Leeds in the United Kingdom.

The standard antimicrobial therapy for treating C difficile infection is generally MTZ or vancomycin (VAN). Indeed, as Dr. Baines said during his presentation, "There has been little change in this over the past 2 decades, although there are numerous other antimicrobials currently in development." During the last 10 years, there have also been sporadic reports of C difficile with reduced susceptibility to both MTZ and VAN.

Dr. Baines also initially noted the need to consider ribotype-specific data when performing susceptibility studies, as certain polymerase chain reaction (PCR) ribotypes might be less susceptible to certain antibiotics than other PCR ribotypes, with it now being common practice to ribotype panels of strains before susceptibility testing.

This study arose from the laboratory's updating of some of their C difficile strains. Dr. Bains said, "We noted that there were several strains that apparently had reduced susceptibility to [MTZ]." Investigating this further, the researchers decided to look at 4 groups of strains: their top 3 occurring PCR ribotypes of 001 (n = 87), 027 (n = 48), and 106 (n = 81), plus 10 of their most common other ribotypes (n = 57), with all these strains being single-patient isolates.

The initial screening was carried out using spiral gradient endpoint (SGE) analysis, with MTZ alone, and with an American Type Culture Collection control strain plus their own strain with a known reduced susceptibility to MTZ. C difficile MTZ MICs of 6 mg/L or more went forward for further analysis using agar incorporation methodologies.

The first of these techniques was the Leeds method, which had been the laboratory's standard method for 8 years. "We have demonstrated more reproducible MICs with this method, and better growth, and less spoor formation," added Dr. Baines.

The MICs were also determined according to Clinical and Laboratory Standards Institute guidelines and E-tests. Furthermore, any strains that were identified with potentially reduced susceptibility to MTZ were subjected to multilocus variable tandem repeat analysis (MLVA) to investigate the genetic relatedness of these strains.

In the initial SGE screening, with the MIC50 for ribotype 001 (4.16 mg/L) being from 4- to 20-fold higher than for the other ribotype groups, it did indeed have a significantly higher geometric mean over ribotypes 106, 027, and the others (3.51 vs 1.11 vs 0.90 vs 0.32; P < .001).

This potentially reduced susceptibility to MTZ was seen for 24% of the researchers' ribotype 001 C difficile. Dr. Bains also noted that 1 of their ribotype 001 strains was in actual fact an unknown ribotype that was 1 band different from ribotype 001. Of note, none of their ribotype 106 or 127 strains were identified as showing reduced MTZ susceptibility at this cut-off.

To put these changed susceptibilities into context, the researchers located historic 72 ribotype 001 strains stored from 1995 to 2001 and compared these directly. Indeed, these historic strains showed MIC50 to MTZ of 1.0 mg/L, with the recent ribotype 001 again showing a MIC50of 4 mg/L and a significantly greater geometric mean (5.94 vs 1.03; P < .001).

For the MLVA analysis, the researchers included their 22 isolates with potentially reduced MTZ susceptibility, along with 6 fully susceptible ribotypes for comparison; this initially identified 3 subgroups of highly related strains, with 18 of the 21 recent strains belonging to 1 of these 3 subgroups. "Only 2 of the 6 susceptible strains were actually related to one another," Dr. Baines added.

An interesting finding, but not the main focus of the present study, was that they also observed methodological variations. First, their MTZ E-tests were difficult to interpret because of variable results, although not for VAN. Further, both agar and broth formulation affected the MTZ MICs, although not for VAN again. Finally, the Clinical and Laboratory Standards Institute MICs were approximately 2 doubling dilutions lower than for the Leeds method.

Thus, Dr. Baines stated that, "We feel that we have observed an emergence of reduced susceptibility in PCR ribotype 001 predominantly at our institution." The clinical significance of this reduced MIC susceptibility remains to be determined, as does the evidence of clonal spread with the MLVA, which Dr. Baines noted can actually discriminate PCR ribotype 027 into 23 groups. Similarly, the mechanisms behind this decreased MTZ susceptibility remain to be investigated further.

The cochairman of the session, Ian R. Poxton, PhD, from the Department of Medical Microbiology at the University of Edinburgh Medical School, Edinburgh, United Kingdom, had relatively little comment to make at this time, he told Medscape Infectious Diseases. On the one hand, he felt that the potential problems with the E-test did remain "a bit of a mystery," whereas for the changes in susceptibility of C difficile to MTZ, he added, "I think that it is telling us what we know...and although the differences are very slight, it does seem to be moving up."

Dr. Baines and Dr. Poxton have disclosed no relevant financial relationships.

18th European Congress of Clinical Microbiology and Infectious Diseases: Abstract O463. Presented April 22, 2008.

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