Quesada-Gomez, C. | Search bioRxiv

bioRxiv2020

Clostridium perfringenosum sp. nov., a closely related species toClostridium perfringens and its virulence factors, isolated from a humansoft tissue infection

Two Gram-positive, anaerobic bacteria, designated 27733 and 27737, were isolated from a soft tissue infection from a human patient. They were preliminarily identified as Clostridium perfringens through a series of phenotypic tests, including Gram-staining, determination of lipase and hemolytic activities, MALDI-ToF profiling, and a commercial biochemical identification system. In line with these results, genomes obtained for both isolates were ~3.56 Mbp in size, showed a DNA G+C content of ~28.4%, and contained C. perfringens ribosomal markers (i.e. 16S rRNA gene identity >99.0% to C. perfringens ATCC13124T). A closer examination of these sequences; however, revealed low average Nucleotide Identity (~87%) and digital DNA-DNA Hybridization (~35%) values between isolates 27733/27737 and C. perfringens ATCC13124T, as well as substantial differences in gene content to multiple C. perfringens strains, indicating that they represent a novel species within the genus Clostridium. Congruently, Bayesian dating analyses placed the divergence of this new species and C. perfringens from its common ancestor hundreds of thousands of years ago. Isolates 27733/27737 are not genomically identical (34-197 SNPs apart) and carry genes for C. perfringens-like toxins (<94% nucleotide sequence identity), including plc (alpha toxin), pfoA (perfringolysin O, theta-toxin), nagHIJKL (hyalorudinase, mu-toxin), nanHIJ (exo-alpha sialidase), and cloSI (alpha-clostripain). They do not have known antibiotic resistance genes but were catalogued as resistant to clindamycin through phenotypic tests. On the basis of the presented evidence, and due to its resemblance and potential confusion with C. perfringens, we herein propose the species C. perfringenosum sp. nov. and strain 27733 as its type strain.

microbiology↗

bioRxiv2019

cfrB,cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

Cfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

microbiology↗