Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

Pathogenicity and virulence of Clostridioides difficile.

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen.