Characterizing staphylococcus aureus properties in patient biopsies to test current concepts of antibiotic persistence

Staphylococcal infections vary from superficial skin infections to life threatening bacteraemia. They are often associated with prolonged hospital stays leading to high morbidity and mortality. Deep-seated musculoskeletal and blood stream infections are difficult to eradicate with antimicrobial therapy even if the causative S. aureus strain is susceptible in laboratory assays. Various mechanisms explain survival of antibiotic-exposed S. aureus but their relevance in humans is lacking, primarily due to difficulty associated with processing and analysing heterogeneous human tissues. In this dissertation, we developed a microscopy based method to visualize single S. aureus in millimetre sized human tissues in a time and data efficient manner. Specifically, we used combination of novel fluorescently conjugated vancomycin, immuno-staining and adaptive high resolution microscopy reliant on trained neural networks. Further, we used single cell microfluidic assays to observe clinically relevant antibiotic derived killing in vitro. We quantified 12,552 S. aureus and its respective niche in biopsies from musculoskeletal infected patients with different antibiotic regimens. We found ~80% S. aureus intracellularly and ~20% extracellularly. Intracellular S. aureus adopted monocyte-macrophage intermediate cells (CD14+CD16+CD68+) as their primary niche whereas macrophages, neutrophils and other eukaryotic cells contained ~30% S. aureus. All S. aureus, irrespective of their location, resided in singlets or in clusters of up to 10 cells. Large clusters, indicative of biofilm formation, were rare and typically undetectable in tissues. These data suggest location-independent inadequacy of conditions prevalent in tissues for sustaining massive S. aureus growth. Antibiotic therapy did not reduce extracellular S. aureus burden or select for particular intracellular niches but significantly increased individual cellular loads. These data fail to support specific stress tolerant niches as crucial permissive sites for observed antibiotic treatment failure. Instead, antibiotic survival seemed to be a S. aureus population-wide property in vivo. Collectively, human data also supports presence of S. aureus in a state of low toxicity, characterized by limited proliferation. This state allows for evasion from antibiotic and host derived killing of both intra- and extracellular subsets. To test this idea, we used microfluidic assay with slow growing S. aureus exposed to a clinically relevant antibiotic – flucloxacillin. In contrast to colony forming units (CFU) based assays, we observed substantial bactericidal effects after antibiotic was removed and growth resumed. This single-cell observation highlighted the complexity associated with understanding antibiotic effects, even when a single aspect of human relevance is focused upon. Thus, human-centric studies are necessary for understanding relevant bacterial phenotypes that account for diseases. In vitro assays recapitulating multiple aspects of in-patient conditions might serve as better predictors of treatment outcome and provide a basis for identifying more effective control strategies

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

International audienceGram-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

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 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 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

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

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