Antibiotics are widely used as growth promoters in animal husbandry; among them, the tetracyclines are a chemical group of relevance, due to their wide use in agriculture, surpassing in quantities applied almost every other antibiotic family. Seeing the considerable amounts of tetracyclines used worldwide, monitoring of these antibiotics is paramount. Advances must be made in the analysis of antibiotics to assess correct usage and dosage of tetracyclines in food and feedstuffs and possible residues in pertinent environmental samples. The tetracyclines are still considered a clinically relevant group of antibiotics, though dissemination of tolerance and resistance determinants have limited their use. This review focuses on four different aspects: (i) tetracyclines, usage, dosages, and regulatory issues that govern their food-related application, with particular attention to the prohibitions and restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union, (ii) analytical methods for tetracyclines, determination, and residues thereof in feedstuffs and related matrices with an emphasis on the most relevant and novel techniques, including both screening and confirmatory methods, (iii) tetracycline resistance and tetracycline-resistant bacteria in feedstuff, and (iv) environmental and health risks accompanying the use of tetracyclines in animal nutrition. In the last two cases, we discuss the more relevant undesirable effects that tetracyclines exert over bacterial communities and nontarget species including unwanted effects in farmers.

Clostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, and pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI–III. The emergence of these three novel genomospecies predates clades C1–5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work may impact the diagnosis of CDI.

Multilocus Sequence Typing (MLST) is a precise microbial typing approach at the intra-species level for epidemiologic and evolutionary purposes. It operates by assigning a sequence type (ST) identifier to each specimen, based on a combination of alleles of multiple housekeeping genes included in a defined scheme. The use of MLST has multiplied due to the availability of large numbers of genomic sequences and epidemiologic data in public repositories. However, data processing speed has become problematic due to the massive size of modern datasets. Here, we present FastMLST, a tool that is designed to perform PubMLST searches using BLASTn and a divide-and-conquer approach that processes each genome assembly in parallel. The output offered by FastMLST includes a table with the ST, allelic profile, and clonal complex or clade (when available), detected for a query, as well as a multi-FASTA file or a series of FASTA files with the concatenated or single allele sequences detected, respectively. FastMLST was validated with 91 different species, with a wide range of guanine-cytosine content (%GC), genome sizes, and fragmentation levels, and a speed test was performed on 3 datasets with varying genome sizes. Compared with other tools such as mlst, CGE/MLST, MLSTar, and PubMLST, FastMLST takes advantage of multiple processors to simultaneously type up to 28 000 genomes in less than 10 minutes, reducing processing times by at least 3-fold with 100% concordance to PubMLST, if contaminated genomes are excluded from the analysis. The source code, installation instructions, and documentation of FastMLST are available at https://github.com/EnzoAndree/FastMLST

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.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAP _CR1 . Whole-genome sequencing and ribotyping indicated that the NAP _CR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAP _CR1 strains are resistant to fluoroquinolones due to a mutation in gyrA , and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAP _CR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAP _CR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAP _CR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

The present widespread use of antimicrobials in crop farming is based upon their successful application in human medicine. However, recent evidence suggests that the massive anthropogenic release of antimicrobials into the biosphere has selected for resistant bacteria and facilitated the transfer of resistance genes among them. This work deals with the examination of iceberg lettuce collected at 10 farms from two regions in Costa Rica. Farmers from nine sampling sites regularly apply commercial formulations containing gentamicin, oxytetracycline, streptomycin, or a combination of them without being able to indicate how often and how much of these products have been sprayed onto the crops. One organic farm was also investigated for comparative purposes. Oxytetracycline- and gentamicin-resistant bacteria were abundantly detected using selective enrichment cultures. Furthermore, colony mixtures from selective plates were characterized by chemotaxonomical and molecular fingerprinting methods. Both types of resistant communities accounted for a significant fraction of all culturable bacteria and included several resistance genes as well as factors for their potential horizontal transfer. Given the fact that lettuce is eaten raw, it may contribute to the dissemination of antimicrobial-resistant bacteria and/or their resistance genes from the environment to the microbial biota of the human intestine.