Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa.

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Molecular reprogramming and phenotype switching in Staphylococcus aureus lead to high antibiotic persistence and affect therapy success.

Staphylococcus aureus causes invasive infections and easily acquires antibiotic resistance. Even antibiotic-susceptible S. aureus can survive antibiotic therapy and persist, requiring prolonged treatment and surgical interventions. These so-called persisters display an arrested-growth phenotype, tolerate high antibiotic concentrations, and are associated with chronic and recurrent infections. To characterize these persisters, we assessed S. aureus recovered directly from a patient suffering from a persistent infection. We show that host-mediated stress, including acidic pH, abscess environment, and antibiotic exposure promoted persister formation in vitro and in vivo. Multiomics analysis identified molecular changes in S. aureus in response to acid stress leading to an overall virulent population. However, further analysis of a persister-enriched population revealed major molecular reprogramming in persisters, including down-regulation of virulence and cell division and up-regulation of ribosomal proteins, nucleotide-, and amino acid-metabolic pathways, suggesting their requirement to fuel and maintain the persister phenotype and highlighting that persisters are not completely metabolically inactive. Additionally, decreased aconitase activity and ATP levels and accumulation of insoluble proteins involved in transcription, translation, and energy production correlated with persistence in S. aureus, underpinning the molecular mechanisms that drive the persister phenotype. Upon regrowth, these persisters regained their virulence potential and metabolically active phenotype, including reduction of insoluble proteins, exhibiting a reversible state, crucial for recurrent infections. We further show that a targeted antipersister combination therapy using retinoid derivatives and antibiotics significantly reduced lag-phase heterogeneity and persisters in a murine infection model. Our results provide molecular insights into persisters and help explain why persistent S. aureus infections are so difficult to treat.

A Far-Red Fluorescent Probe to Visualize Gram-Positive Bacteria in Patient Samples.

Gram-positive bacteria, in particular Staphylococcus aureus (S. aureus), are the leading bacterial cause of death in high-income countries and can cause invasive infections at various body sites. These infections are associated with prolonged hospital stays, a large economic burden, considerable treatment failure, and high mortality rates. So far, there is only limited knowledge about the specific locations where S. aureus resides in the human body during various infections. Hence, the visualization of S. aureus holds significant importance in microbiological research. Herein, we report the development and validation of a far-red fluorescent probe to detect Gram-positive bacteria, with a focus on staphylococci, in human biopsies from deep-seated infections. This probe displays strong fluorescence and low background in human tissues, outperforming current tools for S. aureus detection. Several applications are demonstrated, including fixed- and live-cell imaging, flow cytometry, and super-resolution bacterial imaging.

A prevalent and culturable microbiota links ecological balance to clinical stability of the human lung after transplantation.

There is accumulating evidence that the lower airway microbiota impacts lung health. However, the link between microbial community composition and lung homeostasis remains elusive. We combine amplicon sequencing and bacterial culturing to characterize the viable bacterial community in 234 longitudinal bronchoalveolar lavage samples from 64 lung transplant recipients and establish links to viral loads, host gene expression, lung function, and transplant health. We find that the lung microbiota post-transplant can be categorized into four distinct compositional states, 'pneumotypes'. The predominant 'balanced' pneumotype is characterized by a diverse bacterial community with moderate viral loads, and host gene expression profiles suggesting immune tolerance. The other three pneumotypes are characterized by being either microbiota-depleted, or dominated by potential pathogens, and are linked to increased immune activity, lower respiratory function, and increased risks of infection and rejection. Collectively, our findings establish a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant.