Clostridium difficile Isolates with High Linezolid MICs Harbor the Multiresistance Gene cfr

Abstract

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC 50 and MIC 90 of linezolid were 0.75 g/ml and 1.5 g/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 g/ml to 16 g/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variablenumber tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance. Linezolid is the first antibiotic in the oxazolidinone group and has been used for more than 10 years to treat Gram-positive infections (1). Resistance to linezolid in Gram-positive bacteria is very uncommon and has been reported mainly in clinical isolates of coagulase-negative staphylococcus, Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium, as well as in nonhuman isolates of other Gram-positive bacteria and Gram-negative bacteria (2). Resistance is due to the presence of 1 or more of 3 mechanisms: point mutations in the 23S rRNA gene (central loop of domain V, mainly G2576T), mutations/deletions in ribosomal proteins L3 (rplC gene) and L4 (rplD gene), and methylation of position A2503 of the 23S rRNA gene mediated by an rRNA methyltransferase encoded by the multiresistance gene cfr (chloramphenicol-florfenicol resistance), which is harbored mainly in transferable plasmids (3). This last mechanism also has been associated with resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics (4).Clostridium difficile is the main cause of antibiotic-associated diarrhea. Although linezolid is not used for the treatment of C. difficile infection (5, 6), some observations suggest that patients treated with linezolid for ventilator-associated pneumonia could be protected against this infection (7).Resistance to linezolid occasionally has bee...

Diversity and Evolution in the Genome of Clostridium difficile

“…Recently, Marin et al (124) reported reduced susceptibility to linezolid (MIC, 6 to 16 mg/liter) in 9/891 clinical isolates of C. difficile. These 9 strains belonged to RT001, RT017, and RT078, and they all possessed the cfr gene, which showed 100% sequence identity with a fragment of Tn6218 (124).…”

Different Resistance Mechanisms for Cadazolid and Linezolid in Clostridium difficile Found by Whole-Genome Sequencing Analysis

“…Two other LZD-resistant clinical isolates, A-1290 and A-1291, did not have any mutations in L3 but contained a Val88Ile variation in ribosomal protein L20 (rplT; CD630_06870), just as was seen with resistant isolates A-1410 and A-1412. No mutations were found in any of the copies for the 23S rRNA PTC loop of domain V that correspond to the ones described in LZD-resistant S. aureus isolates (14), and no mutations were found in the topoisomerase II genes (DNA gyrase) in any of the clones selected with CDZ.…”

“…Marín et al had identified a cfr-like gene in the majority of clinical isolates with elevated LZD MICs (14). Sequencing of the amplified cfr fragments showed 2 different profiles with a 2-bp difference (GenBank sequence accession numbers KM359438 and KM359439).…”

“…Similarly to S. aureus findings, a 23S rRNA methylating gene (cfr) has been detected in a study in 7 of 9 C. difficile clinical isolates with elevated MICs of LZD (14). However, in contrast to S. aureus, no mutations were found in the 23S rRNA genes (14).…”

“…Similarly to S. aureus findings, a 23S rRNA methylating gene (cfr) has been detected in a study in 7 of 9 C. difficile clinical isolates with elevated MICs of LZD (14). However, in contrast to S. aureus, no mutations were found in the 23S rRNA genes (14). Finally, LZD resistance in a porcine Clostridium perfringens strain has recently been associated with a glycine-to-aspartate mutation at position 71 in ribosomal protein L4 (rplD) (15).…”