<i>cfr</i> (B), <i>cfr</i> (C), and a New <i>cfr</i> -Like Gene, <i>cfr</i> (E), in Clostridium difficile Strains Recovered across Latin America

Abstract

Cfr is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three <i>cfr</i>-like genes implicated in antibiotic resistance have been described, two of which, <i>cfr</i>(B) and <i>cfr</i>(C), have been sporadically detected in <i>Clostridium difficile</i> However, the methylase activity of Cfr(C) has not been confirmed. We found <i>cfr</i>(B), <i>cfr</i>(C), and a <i>cfr</i>-like gene that shows only 51 to 58% protein sequence identity to Cfr and Cfr-like enzymes in clinical <i>C. difficile</i> isolates recovered across nearly a decade in Mexico, Honduras, Costa Rica, and Chile. This new resistance gene was termed <i>cfr</i>(E). In agreement with the anticipated function of the <i>cfr</i>-like genes detected, all isolates exhibited high MIC values for several ribosome-targeting antibiotics. In addition, <i>in vitro</i> assays confirmed that Cfr(C) and Cfr(E) methylate <i>Escherichia coli</i> and, to a lesser extent, <i>C. difficile</i> 23S rRNA fragments at the expected positions. The analyzed isolates do not have mutations in 23S rRNA genes or genes encoding the ribosomal proteins L3 and L4 and lack <i>poxtA</i>, <i>optrA</i>, and pleuromutilin resistance genes. Moreover, these <i>cfr</i>-like genes were found in Tn<i>6218</i>-like transposons or integrative and conjugative elements (ICE) that could facilitate their transfer. These results indicate selection of potentially mobile <i>cfr</i>-like genes in <i>C. difficile</i> from Latin America and provide the first assessment of the methylation activity of Cfr(C) and Cfr(E), which belong to a cluster of Cfr-like proteins that does not include the functionally characterized enzymes Cfr, Cfr(B), and Cfr(D).