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

Cfr is a radical S-adenosyl-l-methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three cfr-like genes implicated in antibiotic resistance have been described, two of which, cfr(B) and cfr(C), have been sporadically detected in Clostridium difficile However, the methylase activity of Cfr(C) has not been confirmed. We found cfr(B), cfr(C), and a cfr-like gene that shows only 51 to 58% protein sequence identity to Cfr and Cfr-like enzymes in clinical C. difficile isolates recovered across nearly a decade in Mexico, Honduras, Costa Rica, and Chile. This new resistance gene was termed cfr(E). In agreement with the anticipated function of the cfr-like genes detected, all isolates exhibited high MIC values for several ribosome-targeting antibiotics. In addition, in vitro assays confirmed that Cfr(C) and Cfr(E) methylate Escherichia coli and, to a lesser extent, C. difficile 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 poxtA, optrA, and pleuromutilin resistance genes. Moreover, these cfr-like genes were found in Tn6218-like transposons or integrative and conjugative elements (ICE) that could facilitate their transfer. These results indicate selection of potentially mobile cfr-like genes in C. difficile 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).

Diversity of multidrug-resistant epidemic Clostridium difficile NAP1/RT027/ST01 strains in tertiary hospitals from Honduras.

In recent years, reports of NAP1/RT027/ST01 epidemic strains of Clostridium difficile producing outbreaks of healthcare-associated diarrhea have increased in America and Europe. We cultivated multidrug-resistant NAP1/RT027/ST01 strains from the FQR2 linage from TcdA/TcdB+ stool samples obtained from patients in two Honduran hospitals. The PFGE macrorestriction patterns of two of the isolates were new. These bacteria were toxigenic and induced with different magnitude classical cytopathic effects on HeLa cells. Besides their resistance to twelve antibiotics, including to clindamycin, fluoroquinolones, linezolid and tigecycline. In this regard, they show the gyrA mutation that typifies epidemic C. difficile genotypes and carry cfr-like genes in different molecular contexts, respectively. These results confirm the spread of multidrug-resistant NAP1/RT027/ST01 strains in Central America with potential idiosyncratic adaptations.

Prolonged dysbiosis and altered immunity under nutritional intervention in a physiological mouse model of severe acute malnutrition.

Severe acute malnutrition (SAM) is a multifactorial disease affecting millions of children worldwide. It is associated with changes in intestinal physiology, microbiota, and mucosal immunity, emphasizing the need for multidisciplinary studies to unravel its full pathogenesis. We established an experimental model in which weanling mice fed a high-deficiency diet mimic key anthropometric and physiological features of SAM in children. This diet alters the intestinal microbiota (less segmented filamentous bacteria, spatial proximity to epithelium), metabolism (decreased butyrate), and immune cell populations (depletion of LysoDC in Peyer's patches and intestinal Th17 cells). A nutritional intervention leads to a fast zoometric and intestinal physiology recovery but to an incomplete restoration of the intestinal microbiota, metabolism, and immune system. Altogether, we provide a preclinical model of SAM and have identified key markers to target with future interventions during the education of the immune system to improve SAM whole defects.

Peyer's patch phagocytes acquire specific transcriptional programs that influence their maturation and activation profiles.

Peyer's patches (PPs) are secondary lymphoid organs in contact with the external environment via the intestinal lumen, thus combining antigen sampling and immune response initiation sites. Therefore, they provide a unique opportunity to study the entire process of phagocyte differentiation and activation in vivo. Here, we deciphered the transcriptional and spatial landscape of PP phagocyte populations from their emergence in the tissue to their final maturation state at homeostasis and under stimulation. Activation of monocyte-derived Lysozyme-expressing dendritic cells (LysoDCs) differs from that of macrophages by their upregulation of conventional DC (cDC) signature genes such as Ccr7 and downregulation of typical monocyte-derived cell genes such as Cx3cr1. We identified gene sets that distinguish PP cDCs from the villus ones and from LysoDCs. We also identified key immature, early, intermediate, and late maturation markers of PP phagocytes. Finally, exploiting the ability of the PP interfollicular region to host both villous and subepithelial dome emigrated cDCs, we showed that the type of stimulus, the subset, but also the initial location of cDCs shape their activation profile and thus direct the immune response. Our study highlights the importance of targeting the right phagocyte subset at the right place and time to manipulate the immune response.