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During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased pyoverdine and pyochelin siderophore gene expression, causing macrophage ferroptosis and lysis. We show that iron-bound pyochelin was sufficient to cause macrophage ferroptosis and lysis, however, apo-pyochelin, iron-bound pyoverdine, or apo-pyoverdine were insufficient to kill macrophages. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

Denture-related stomatitis caused by Candida spp. affects elderly individuals using partial/total prosthesis, provoking several discomforts including burning sensation and altered taste. Herein, we have studied 52 denture-wearing individuals (>60 years-old), attended at the dentistry clinic of UNIVALE, aiming to isolate Candida spp. directly from the stomatitis lesions and to evaluate their potential to produce virulence attributes. A low prevalence of denture-related stomatitis was reported in these patients (4/52; 7.7%). Candida albicans was isolated in the 4 selected patients, with the ability to form biofilm over a polystyrene surface and to produce aspartic protease, esterase and hemolysin. However, neither phospholipase nor caseinase activities were detected. Planktonic-growing yeasts were susceptible to amphotericin B and caspofungin, while the susceptibility to azoles (fluconazol, itraconazole and voriconazole) varied depending on either the isolate or antifungal. Relevantly, biofilm-forming C. albicans cells exhibited resistance to all studied antifungals. So, new effective drugs against resistant C. albicans isolates causing denture-related stomatitis are urgently required.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104–107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

Biofilm formation is the preferred mode of growth lifestyle for many microorganisms, including bacterial and fungal human pathogens. Biofilm is a strong and dynamic structure that confers a broad range of advantages to its members, such as adhesion/cohesion capabilities, mechanical properties, nutritional sources, metabolite exchange platform, cellular communication, protection and resistance to drugs (e.g., antimicrobials, antiseptics, and disinfectants), environmental stresses (e.g., dehydration and ultraviolet light), host immune attacks (e.g., antibodies, complement system, antimicrobial peptides, and phagocytes), and shear forces. Microbial biofilms cause problems in the hospital environment, generating high healthcare costs and prolonged patient stay, which can result in further secondary microbial infections and various health complications. Consequently, both public and private investments must be made to ensure better patient management, as well as to find novel therapeutic strategies to circumvent the resistance and resilience profiles arising from biofilm-associated microbial infections. In this work, we present a general overview of microbial biofilm formation and its relevance within the biomedical context.