Julien M. Buyck

Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes.

ABSTRACT Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [Cs] and maximal relative efficacies [Emax]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine β-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their Emax exceeded the limit of detection (−4.5 log10 CFU compared to the initial inoculum). Intracellularly, Cs values remained unchanged for β-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas Emax values were markedly reduced (less negative), reaching −3 log10 CFU for fluoroquinolones and only −1 to −1.5 log10 CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher Cs) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the Emax may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections.

New Amphiphilic Neamine Derivatives Active against Resistant Pseudomonas aeruginosa and Their Interactions with Lipopolysaccharides

ABSTRACT The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3′,6-di-O-[(2″-naphthyl)propyl]neamine (3′,6-di2NP), 3′,6-di-O-[(2″-naphthyl)butyl]neamine (3′,6-di2NB), and 3′,6-di-O-nonylneamine (3′,6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691–7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 μg/ml), The most active one (3′,6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3′,6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa.

Activities of Antibiotic Combinations against Resistant Strains of Pseudomonas aeruginosa in a Model of Infected THP-1 Monocytes

ABSTRACT Antibiotic combinations are often used for treating Pseudomonas aeruginosa infections but their efficacy toward intracellular bacteria has not been investigated so far. We have studied combinations of representatives of the main antipseudomonal classes (ciprofloxacin, meropenem, tobramycin, and colistin) against intracellular P. aeruginosa in a model of THP-1 monocytes in comparison with bacteria growing in broth, using the reference strain PAO1 and two clinical isolates (resistant to ciprofloxacin and meropenem, respectively). Interaction between drugs was assessed by checkerboard titration (extracellular model only), by kill curves, and by using the fractional maximal effect (FME) method, which allows studying the effects of combinations when dose-effect relationships are not linear. For drugs used alone, simple sigmoidal functions could be fitted to all concentration-effect relationships (extracellular and intracellular bacteria), with static concentrations close to (ciprofloxacin, colistin, and meropenem) or slightly higher than (tobramycin) the MIC and with maximal efficacy reaching the limit of detection in broth but only a 1 to 1.5 (colistin, meropenem, and tobramycin) to 2 to 3 (ciprofloxacin) log10 CFU decrease intracellularly. Extracellularly, all combinations proved additive by checkerboard titration but synergistic using the FME method and more bactericidal in kill curve assays. Intracellularly, all combinations proved additive only based on both FME and kill curve assays. Thus, although combinations appeared to modestly improve antibiotic activity against intracellular P. aeruginosa, they do not allow eradication of these persistent forms of infections. Combinations including ciprofloxacin were the most active (even against the ciprofloxacin-resistant strain), which is probably related to the fact this drug was the most effective alone intracellularly.

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 which antibiotics are 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 pre-opsonized bacteria by eukaryotic cells; (2) elimination of non-internalized 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.

Correlation between cytotoxicity induced by Pseudomonas aeruginosa clinical isolates from acute infections and IL-1β secretion in a model of human THP-1 monocytes

Type III secretion system (T3SS) in Pseudomonas aeruginosa is associated with poor clinical outcome in acute infections. T3SS allows for injection of bacterial exotoxins (e.g. ExoU or ExoS) into the host cell, causing cytotoxicity. It also activates the cytosolic NLRC4 inflammasome, activating caspase-1, inducing cytotoxicity and release of mature IL-1β, which impairs bacterial clearance. In addition, flagellum-mediated motility has been suggested to also modulate inflammasome response and IL-1β release. Yet the capacity of clinical isolates to induce IL-1β release and its relation with cytotoxicity have never been investigated. Using 20 clinical isolates from acute infections with variable T3SS expression levels and human monocytes, our aim was to correlate IL-1β release with toxin expression, flagellar motility and cytotoxicity. ExoU-producing isolates caused massive cell death but minimal release of IL-1β, while those expressing T3SS but not ExoU (i.e. expressing ExoS or no toxins) induced caspase-1 activation and IL-1β release, the level of which was correlated with cytotoxicity. Both effects were prevented by a specific caspase-1 inhibitor. Flagellar motility was not correlated with cytotoxicity or IL-1β release. No apoptosis was detected. Thus, T3SS cytotoxicity is accompanied by a modification in cytokine balance for P. aeruginosa clinical isolates that do not express ExoU.

Inhibition of the Injectisome and Flagellar Type III Secretion Systems by INP1855 Impairs Pseudomonas aeruginosa Pathogenicity and Inflammasome Activation

With the rise of multidrug resistance, Pseudomonas aeruginosa infections require alternative therapeutics. The injectisome (iT3SS) and flagellar (fT3SS) type III secretion systems are 2 virulence factors associated with poor clinical outcomes. iT3SS translocates toxins, rod, needle, or regulator proteins, and flagellin into the host cell cytoplasm and causes cytotoxicity and NLRC4-dependent inflammasome activation, which induces interleukin 1β (IL-1β) release and reduces interleukin 17 (IL-17) production and bacterial clearance. fT3SS ensures bacterial motility, attachment to the host cells, and triggers inflammation. INP1855 is an iT3SS inhibitor identified by in vitro screening, using Yersinia pseudotuberculosis Using a mouse model of P. aeruginosa pulmonary infection, we show that INP1855 improves survival after infection with an iT3SS-positive strain, reduces bacterial pathogenicity and dissemination and IL-1β secretion, and increases IL-17 secretion. INP1855 also modified the cytokine balance in mice infected with an iT3SS-negative, fT3SS-positive strain. In vitro, INP1855 impaired iT3SS and fT3SS functionality, as evidenced by a reduction in secretory activity and flagellar motility and an increase in adenosine triphosphate levels. As a result, INP1855 decreased cytotoxicity mediated by toxins and by inflammasome activation induced by both laboratory strains and clinical isolates. We conclude that INP1855 acts by dual inhibition of iT3SS and fT3SS and represents a promising therapeutic approach.

Pharmacodynamics of ceftazidime/avibactam against extracellular and intracellular forms of Pseudomonas aeruginosa

Objectives When tested in broth, avibactam reverses ceftazidime resistance in many Pseudomonas aeruginosa that express ESBLs. We examined whether similar reversal is observed against intracellular forms of P. aeruginosa . Methods Strains: reference strains; two engineered strains with basal non-inducible expression of AmpC and their isogenic mutants with stably derepressed AmpC; and clinical isolates with complete, partial or no resistance to reversion with avibactam. Pharmacodynamic model: 24 h concentration-response to ceftazidime [0.01-200 mg/L alone or with avibactam (4 mg/L)] of bacteria in broth or bacteria phagocytosed by THP-1 monocytes, with calculation of ceftazidime relative potency ( C s : concentration yielding a static effect) and maximal relative effect [ E max : cfu decrease at infinitely large antibiotic concentrations (efficacy in the model)] using the Hill equation. Cellular content of avibactam: quantification by LC-MS/MS. Results For both extracellular and intracellular bacteria, ceftazidime C s was always close to its MIC. For ceftazidime-resistant strains, avibactam addition shifted ceftazidime C s to values close to the MIC of the combination in broth. E max was systematically below the detection limit (-5 log 10 ) for extracellular bacteria, but limited to -1.3 log 10 for intracellular bacteria (except for two isolates) with no effect of avibactam. The cellular concentration of avibactam reflected extracellular concentration and was not influenced by ceftazidime (0-160 mg/L). Conclusions The potential for avibactam to inhibit β-lactamases does not differ for extracellular and intracellular forms of P. aeruginosa , denoting an unhindered access to its target in both situations. The loss of maximal relative efficacy of ceftazidime against intracellular P. aeruginosa was unrelated to resistance via avibactam-inhibitable β-lactamases.

Salicylidene Acylhydrazides and Hydroxyquinolines Act as Inhibitors of Type Three Secretion Systems in Pseudomonas aeruginosa by Distinct Mechanisms

ABSTRACT Type 3 secretion systems (T3SSs) are major virulence factors in Gram-negative bacteria. Pseudomonas aeruginosa expresses two T3SSs, namely, an injectisome (iT3SS) translocating effector proteins in the host cell cytosol and a flagellum (fT3SS) ensuring bacterial motility. Inhibiting these systems is an appealing therapeutic strategy for acute infections. This study examines the protective effects of the salicylidene acylhydrazide INP0341 and of the hydroxyquinoline INP1750 (previously described as T3SS inhibitors in other species) toward cytotoxic effects of P. aeruginosain vitro. Both compounds reduced cell necrosis and inflammasome activation induced by reference strains or clinical isolates expressing T3SS toxins or only the translocation apparatus. INP0341 inhibited iT3SS transcriptional activation, including in strains with constitutive iT3SS expression, and reduced the total expression of toxins, suggesting it targets iT3SS gene transcription. INP1750 inhibited toxin secretion and flagellar motility and impaired the activity of the YscN ATPase from Yersinia pseudotuberculosis (homologous to the ATPase present in the basal body of P. aeruginosa iT3SS and fT3SS), suggesting that it rather targets a T3SS core constituent with high homology among iT3SS and fT3SS. This mode of action is similar to that previously described for INP1855, another hydroxyquinoline, against P. aeruginosa. Thus, although acting by different mechanisms, INP0341 and INP1750 appear as useful inhibitors of the virulence of P. aeruginosa. Hydroxyquinolines may have a broader spectrum of activity by the fact they act upon two virulence factors (iT3SS and fT3SS).

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.

ABSTRACT 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-P873s ability to accumulate in eukaryotic cells, together with its activity against extracellular and intracellular forms of infection by Staphylococcus aureus and Pseudomonas aeruginosa, using a pharmacodynamic approach allowing the determination of maximal relative efficacies (Emax values) and bacteriostatic concentrations (Cs values) on the basis of Hill equations of the concentration-response curves. RX-P873s 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/liter (S. aureus) and 2 to 8 mg/liter (P. aeruginosa), with no significant shift in these values against strains resistant to currently used antibiotics being noted. In concentration-dependent experiments, the pharmacodynamic profile of RX-P873 was not influenced by the resistance phenotype of the strains. Emax values (expressed as the decrease in the number of CFU from that in the initial inoculum) against S. aureus and P. aeruginosa reached more than 4 log units and 5 log units in broth, respectively, and 0.7 log unit and 2.7 log units 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, and daptomycin for S. aureus; ciprofloxacin and 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.