Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat, Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.

Klebsiella pneumoniae is a nosocomial pathogen and an important propagator of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Like other Gram-negative bacteria, they secrete outer membrane vesicles (OMVs) that distribute virulence and resistance factors. Here, we subjected a K. pneumoniae-XDR to subinhibitory concentrations of meropenem, amikacin, polymyxin B, and a combination of these agents to evaluate changes in the protein cargo of OMVs through liquid chromatography–tandem mass spectrometry (LC-MS/MS). Genome sequencing of the clinical isolate K. pneumoniae strain HCD1 (KpHCD1) revealed the presence of 41 resistance genes and 159 virulence factors. We identified 64 proteins in KpHCD1-OMVs modulated with different antibiotic treatments involved in processing genetic information, environmental information, cell envelope formation, energy metabolism, and drug resistance. The OMV proteome expression profile suggests that OMVs may be associated with pathogenicity, survival, stress response, and resistance dissemination.

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3⁺ Escherichia coli strain. The fosA3⁺ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3⁺ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3⁺ bacteria.