Intracellular persistence of Staphylococcus aureus : from observations to mechanisms

Staphylococcus aureus is an etiological agent involved in a multitude of infectious diseases, ranging from minor skin infections to more serious diseases. Bacterial persisters are subpopulations that adopt a transient phenotype characterized by a non-growing state and a tolerance to lethal concentrations of antibiotics. When the antibiotic treatment is discontinued, persisters give rise to a population that is, again, mostly drug susceptible. Their rare and transient nature has long hampered their experimental study, but there is now rising evidence on their implication in relapses of chronic infections. A switch to a persister phenotype has been suggested to occur inside eukaryotic cells for only very few intracellular bacteria, and the mechanisms leading to their formation are still poorly understood. Given the capacity of S. aureus to promote persistent or recurrent infections, we demonstrated that antibiotics could induce persisters within host cells and thus explain the impossibility of eradicating intracellular reservoirs. Our data further indicate that permissive host cells can host alternatively persistent bacterial forms upon antibiotic exposure, and replicative forms upon antibiotic removal and could therefore constitute a viable reservoir and a source of dissemination. Further transcriptomic analysis contributed to a better understanding of the mechanism of persistence formation. Persisters remained metabolically active and redirect energy-consuming processes to the benefit of multiple stress responses that contribute to the maintenance of vital processes, notably cell wall, DNA and transcription. In non-permissive cells, host cell oxidative stress modulates dormancy of S. aureus persisters, which may then reach different levels of dormancy but remain infectious, unveiling complex strategies developed by S. aureus to cope with the host and antibiotic treatments.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

Cellular pharmacokinetics and intracellular activity of the novel peptide deformylase inhibitor GSK1322322 against Staphylococcus aureus laboratory and clinical strains with various resistance phenotypes. Studies with human THP-1 monocytes and J774 murine macrophages

GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity, we used an established model of human THP-1 monocytes and tested one fully susceptible (ATCC25923) and 8 clinical S. aureus strains with resistance to oxacillin, vancomycin,daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release and subcellular distribution (cell fractionation) of [14C]-GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (Emax) of 0.5-1 log10 CFU decrease over the original inoculum within 24 h and a static concentration (Cs) close to its MIC in broth. Influx and efflux were very fast (< 5 min to equilibrium) and accumulation was about 4-fold, with no or minimal effect of the broad-spectrum eukaryotic efflux transporters inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell soluble fraction, dissociated from the main subcellular organelles and from bacteria (in infected cells). This study shows that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. It also suggests that GSK1322322 has the ability to freely diffuse in and out eukaryotic cells as well as within cells subcellular compartments

Host Cell Oxidative Stress Induces Dormant Staphylococcus aureus Persisters

Persisters are transiently nongrowing and antibiotic-tolerant phenotypic variants identified in major human pathogens, including intracellular Staphylococcus aureus. Due to their capacity to regrow once the environmental stress is relieved and to promote resistance, persisters possibly contribute to therapeutic failures. While persistence and its related quiescence have been mostly studied under starvation, little is known within host cell environments. Here, we examined how the level of reactive oxygen species (ROS) in different host cells affects dormancy depth of intracellular S. aureus. Using single-cell approaches, we found that host ROS induce variable dormant states in S. aureus persisters, displaying heterogeneous and increased lag times for resuscitation in liquid medium. Dormant persisters displayed decreased translation and energy metabolism, but remained infectious, exiting from dormancy and resuming growth when reinoculated in low-oxidative-stress cells. In high-oxidative-stress cells, ROS-induced ATP depletion was associated with the formation of visible dark foci similar to those induced by the protein aggregation inducer CCCP (carbonyl cyanide m-chlorophenylhydrazone) and with the recruitment of the DnaK-ClpB chaperone system involved in the clearance of protein aggregates. ATP depletion led to higher fractions of dormant persisters than ROS, due to a counterbalancing effect of ROS-induced translational repression, suggesting a pivotal role of translation in the dormant phenotype. Consistently, protein synthesis inhibition limited dormancy to levels similar to those observed in low-oxidative-stress cells. This study supports the hypothesis that intracellular S. aureus persisters can reach heterogeneous dormancy depths and highlights the link between ROS, ATP depletion, dark focus formation, and subsequent dormancy state

RX-P873, a novel protein synthesis inhibitor, accumulates in human THP-1 monocytes and is active against intracellular infections by Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria.

The pyrrolocytosine RX-P873, a new broad-spectrum antibiotic in preclinical development, inhibits protein synthesis at the translation step. The aims of this work were to study RX-P873's ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infections by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies [Emax] and bacteriostatic concentrations [Cs] based on Hill equations of concentration-response curves. RX-P873's apparent concentration in human THP-1 monocytes was about 6-fold higher than the extracellular one. In broth, MICs ranged from 0.125 to 0.5 mg/L (S. aureus) and 2 to 8 mg/L (P. aeruginosa), with no significant shift in these values against strains resistant to currently-used antibiotics. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as CFU decrease from the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log and 5 log in broth, respectively, and 0.7 log and 2.7 log in infected THP-1 cells, respectively, after 24 h. Cs values remained close to the MIC in all cases, making RX-P873 more potent than antibiotics to which the strains were resistant (moxifloxacin, vancomycin, daptomycin for S. aureus; ciprofloxacin, ceftazidime for P. aeruginosa). Kill curves in broth showed that RX-P873 was more rapidly bactericidal against P. aeruginosa than against S. aureus. Taken together, these data suggest that RX-P873 may constitute a useful alternative for infections involving intracellular bacteria, especially Gram-negative species

Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor, afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells.

Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio 1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log after incubation in broth for 30 h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30 h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S. aureus and remains fully active against strains resistant to these comparators

Intracellular activity of antibiotics against Coxiella burnetii in a model of activated human THP-1 cells

We evaluated antibiotic activity against the intracellular bacterium Coxiella burnetii using an activated THP-1 cell model of infection. At clinically relevant concentrations, the intracellular bacterial load was reduced 300-fold by levofloxacin and finafloxacin, 40-fold by doxycycline, and 4-fold by ciprofloxacin and was unaffected by azithromycin. Acidification of the culture medium reduced antibiotic activity, with the exceptions of doxycycline (no change) and finafloxacin (slight improvement). This model may be used to select antibiotics to be evaluated in vivo

Activity of moxifloxacin against biofilms formed by clinical isolates of Staphylococcus aureus differing by their resistant or persister character to fluoroquinolones

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC

The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (≤10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ≥ 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (Emax) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin Emax was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics

In Vitro Models for the Study of the Intracellular Activity of Antibiotics

Intracellular bacteria are poorly responsive to antibiotic treatment. Pharmacological studies are thus needed to determine the antibiotics which are the most potent or effective against intracellular bacteria as well as to explore the reasons for poor bacterial responsiveness. An in vitro pharmacodynamic model is described, consisting of (1) phagocytosis of preopsonized bacteria by eukaryotic cells, (2) elimination of noninternalized bacteria with gentamicin, (3) incubation of infected cells with antibiotics, and (4) determination of surviving bacteria by viable cell counting and normalization of the counts based on sample protein content. The use of strains expressing fluorescent proteins under the control of an inducible promoter allows to follow intracellular bacterial division at the individual level and therefore to monitor bacterial persisters that do not multiply anymore