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The inoculum effect (i.e., reduction in antimicrobial activity at large starting inoculum) is a phenomenon described for various pathogens. Given that limited data exist regarding inoculum effect of Acinetobacter baumannii, we evaluated killing of A. baumannii by polymyxin B, a last-resort antibiotic, at several starting inocula and developed a pharmacokinetic-pharmacodynamic (PKPD) model to capture this phenomenon. In vitro static time-kill experiments were performed using polymyxin B at concentrations ranging from 0.125 to 128 mg/L against a clinical A. baumannii isolate at four starting inocula from 10(5) to 10(8) CFU/mL. Samples were collected up to 30 h to quantify the viable bacterial burden and were simultaneously modeled in the NONMEM software program. The expression of polymyxin B resistance genes (lpxACD, pmrCAB, and wzc), and genetic modifications were studied by RT-qPCR and DNA sequencing experiments, respectively. The PKPD model included a single homogeneous bacterial population with adaptive resistance. Polymyxin B effect was modeled as a sigmoidal E(max) model and the inoculum effect as an increase of polymyxin B EC(50) with increasing starting inoculum using a power function. Polymyxin B displayed a reduced activity as the starting inoculum increased: a 20-fold increase of polymyxin B EC(50) was observed between the lowest and the highest inoculum. No effects of polymyxin B and inoculum size were observed on the studied genes. The proposed PKPD model successfully described and predicted the pronounced in vitro inoculum effect of A. baumannii on polymyxin B activity. These results should be further validated using other bacteria/antibiotic combinations and in vivo models.

Resistance to colistin, one of the antibiotics of last resort against multidrug-resistant Gram-negative bacteria, is increasingly reported. Notably, MCR plasmids discovered in 2015 have now been reported worldwide in humans. To keep this antibiotic of last resort efficient, a way to tackle this mechanism seems essential. Terpene alcohols such as farnesol have been shown to improve the efficacy of some antibiotics. However, their high lipophilicity makes them difficult to use. This problem can be solved by encapsulating them in water-dispersible lipid nanoparticles (LNPs). The aim of this study was to discover, using checkerboard tests and time-kill curve experiments, an association between colistin and farnesol or geraniol loaded in LNPs, which would improve the efficacy of colistin against E. coli and, in particular, MCR-1 transconjugants. Then, the effect of the combination on E. coli inner membrane permeabilisation was evaluated using propidium iodide (PI) uptake and compared to human red blood cells plasma membrane permeabilisation. Both terpene alcohols were able to restore the susceptibility of E. coli J53 MCR-1 to colistin with the same efficacy (Emax = 16, i.e., colistin MIC was decreased from 8 to 0.5 mg/L). However, with an EC50 of 2.69 mg/L, farnesol was more potent than geraniol (EC50 = 39.49 mg/L). Time-kill studies showed a bactericidal effect on MCR-1 transconjugant 6 h after incubation, with no regrowth up to 30 h in the presence of 1 mg/L colistin (1/8 MIC) and 60 mg/L or 200 mg/L farnesol or geraniol, respectively. Colistin alone was more potent in increasing PI uptake rate in the susceptible strain (EC50 = 0.86 ± 0.08 mg/L) than in the MCR-1 one (EC50 = 7.38 ± 0.85 mg/L). Against the MCR-1 strain, farnesol-loaded LNP at 60 mg/L enhanced the colistin-induced inner membrane permeabilization effect up to 5-fold and also increased its potency as shown by the decrease in its EC50 from 7.38 ± 0.85 mg/L to 2.69 ± 0.25 mg/L. Importantly, no hemolysis was observed for LNPs loaded with farnesol or geraniol, alone or in combination with colistin, at the concentrations showing the maximum decrease in colistin MICs. The results presented here indicate that farnesol-loaded LNPs should be studied as combination therapy with colistin to prevent the development of resistance to this antibiotic of last resort.

International audience; Due to the low level of resistance observed with daptomycin, this antibiotic has an important place in the treatment of severe Gram-positive infections. It is the first-in-class of the group of calcium-dependent, membrane-binding lipopeptides, and is a cyclic peptide constituted of 13 amino acids and an n-decanoyl fatty acid chain. The antibacterial action of daptomycin requires its complexation with calcium. Daptomycin is not absorbed from the gastrointestinal tract and needs to be administered parenterally. The distribution of daptomycin is limited (volume of distribution of 0.1 L/kg in healthy volunteers) due to its negative charge at physiological pH and its high binding to plasma proteins (about 90%). Its elimination is mainly renal, with about 50% of the dose excreted unchanged in the urine, justifying dosage adjustment for patients with renal insufficiency. The pharmacokinetics of daptomycin are altered under certain pathophysiological conditions, resulting in high interindividual variability. As a result, therapeutic drug monitoring of daptomycin may be of interest for certain patients, such as intensive care unit patients, patients with renal or hepatic insufficiency, dialysis patients, obese patients, or children. A target for the ratio of the area under the curve to the minimum inhibitory concentration > 666 is usually recommended for clinical efficacy, whereas in order to limit the risk of undesirable muscular effects the residual concentration should not exceed 24.3 mg/L.

Experiments were conducted with polymyxin B and two Klebsiella pneumonia isogenic strains (the wild type, KP_WT, and its transconjugant carrying the mobile colistin resistance gene, KP_MCR-1) to demonstrate that conducting two consecutive time-kill experiments (sequential TK) represents a simple approach to discriminate between pharmacokinetics/pharmacodynamics models with two heterogeneous subpopulations or adaptive resistance.

International audience; Mycobacterium abscessus is responsible for difficult-to-treat chronic pulmonary infections in humans. Current regimens, including parenteral administrations of cefoxitin (FOX) in combination with amikacin and clarithromycin, raise compliance problems and are frequently associated with high failure and development of resistance. Aerosol delivery of FOX could be an interesting alternative. FOX was administered to healthy rats by intravenous bolus or intratracheal nebulization, and concentrations were determined in plasma and epithelial lining fluid (ELF) by liquid chromatography-tandem mass spectrometry. After intrapulmonary administration, the FOX area under the curve within ELF was 1,147 times higher than that in plasma, indicating that this route of administration offers a biopharmaceutical advantage over intravenous administration. FOX antimicrobial activity was investigated using time-kill curves combined with a pharmacokinetic/pharmacodynamic (PK/PD) type modeling approach in order to account for its in vitro instability that precludes precise determination of MIC. Time-kill data were adequately described by a model including in vitro degradation, a sensitive (S) and a resistant (R) bacteria subpopulation, logistic growth, and a maximal inhibition-type growth inhibition effect of FOX. Median inhibitory concentrations were estimated at 16.2 and 252 mg/liter for the S and R subpopulations, respectively. These findings suggest that parenteral FOX dosing regimens used in patients for the treatment of M. abscessus are not sufficient to reduce the bacterial burden and that FOX nebulization offers a potential advantage that needs to be further investigated.

Inflammasome-induced pyroptosis comprises a key cell-autonomous immune process against intracellular bacteria, namely the generation of dying cell structures. These so-called pore-induced intracellular traps (PITs) entrap and weaken intracellular microbes. However, the immune importance of pyroptosis against extracellular pathogens remains unclear. Here, we report that Type-3 secretion system (T3SS)-expressingPseudomonas aeruginosa(P. aeruginosa) escaped PIT immunity by inducing a NLRC4 inflammasome-dependent macrophage pyroptosis response in the extracellular environment. To the contrary, phagocytosis ofSalmonellaTyphimurium promoted NLRC4-dependent PIT formation and the subsequent bacterial caging. Remarkably, T3SS-deficientPseudomonaswere efficiently sequestered within PIT-dependent caging, which favored exposure to neutrophils. Conversely, both NLRC4 and caspase-11 deficient mice presented increased susceptibility to T3SS-deficientP. aeruginosachallenge, but not to T3SS-expressingP. aeruginosa.Overall, our results uncovered thatP. aeruginosauses its T3SS to overcome inflammasome-triggered pyroptosis, which is primarily effective against intracellular invaders.ImportanceAlthough innate immune components confer host protection against infections, the opportunistic bacterial pathogenPseudomonas aeruginosa(P. aeruginosa) exploits the inflammatory reaction to thrive. Specifically the NLRC4 inflammasome, a crucial immune complex, triggers an Interleukin (IL)-1β and -18 deleterious host response toP. aeruginosa. Here, we provide evidence that, in addition to IL-1 cytokines,P. aeruginosaalso exploits the NLRC4 inflammasome-induced pro-inflammatory cell death, namely pyroptosis, to avoid efficient uptake and killing by macrophages. Therefore, our study reveals that pyroptosis-driven immune effectiveness mainly depends onP. aeruginosalocalization. This paves the way toward our comprehension of the mechanistic requirements for pyroptosis effectiveness upon microbial infections and may initiate targeted approaches in order to ameliorate the innate immune functions to infections.Graphical abstractMacrophages infected with T3SS-expressingP. aeruginosadie in a NLRC4-dependent manner, which allows bacterial escape from PIT-mediated cell-autonomous immunity and neutrophil efferocytosis. However, T3SS-deficientP. aeruginosais detected by both NLRC4 and caspase-11 inflammasomes, which promotes bacterial trapping and subsequent efferocytosis ofP. aeruginosa-containing-PITs by neutrophils.

International audience

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.

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.

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.

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 infection by Staphylococcus aureus and Pseudomonas aeruginosa , using a pharmacodynamic approach allowing the determination of maximal relative efficacies ( E max values) and bacteriostatic concentrations ( C s values) on the basis of Hill equations of the 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/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. E max 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. C s 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.

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) log 10 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.

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 [ C s ] and maximal relative efficacies [ E max ]; 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 E max exceeded the limit of detection (−4.5 log 10 CFU compared to the initial inoculum). Intracellularly, C s values remained unchanged for β-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas E max values were markedly reduced (less negative), reaching −3 log 10 CFU for fluoroquinolones and only −1 to −1.5 log 10 CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher C s ) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the E max 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.

Background. Avibactam reverses ceftazidime resistance in Pseudomonas aeruginosa expressing many extended spectrum beta-lactamases when tested in broth. We examined whether similar reversal is observed against P. aeruginosa intracellular forms. Methods. Strains: reference strains; two engineered strains with basal non-inducible expression of AmpC and their isogenic mutants with stably-derepressed AmpC; clinical isolates with complete, partial or no resistance reversion with avibactam. Pharmacodynamic model: 24h-concentration-response to ceftazidime (0.01 to 200 mg/L alone or with avibactam [4 mg/L]) of bacteria in broth or bacteria phagocytozed by THP-1 monocytes, with calculation of ceftazidime relative potency (Cs: concentration yielding a static effect) and maximal relative effect (Emax: cfu decrease at infinitely large antibiotic concentrations [efficacy in the model]) using Hill's equation. Cellular content of avibactam: quantification by LC-MS-MS. Results. For both extracellular and intracellular bacteria, ceftazidime Cs was always close to its MIC. For ceftazidime-resistant strains, avibactam addition shifted ceftazidime Cs to values close to the MIC of the combination in broth. Emax was systematically below the detection limit (-5 log10) for extracellular bacteria, but limited to -1.3 log10 for intracellular bacteria (except for 2 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. Avibactam potential for inhibition of beta-lactamases is not different 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 beta-lactamases.

Macrolides show high minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa when tested in recommended media (cation-adjusted Muller-Hinton broth [CA-MHB]). Nevertheless, azithromycin is successfully used in cystic fibrosis patients, supposedly because of "nonantibiotic effects."CA-MHB and Roswell Park Memorial Institute (RPMI) 1640 medium (used for growing eukaryotic cells) were compared for measuring azithromycin MICs (with or without Phe-Arg-β-naphthylamide [PAβN], an efflux inhibitor), [(14)C]-clarithromycin accumulation, azithromycin-induced protein synthesis inhibition, oprM (encoding the outer-membrane protein coupled with MexAB and MexXY efflux systems) expression, outer-membrane permeability (tested with 1-N-phenylnaphthylamine and nitrocefin), and synergy (determined by checkerboard assay) between azithromycin and outer-membrane disrupting agents. Key experiments were repeated with CA-MHB supplemented with serum, mouse bronchoalveolar lavage fluid, other macrolides, and other gram-negative bacteria.Azithromycin MICs were ≥128 mg/L in CA-MHB, compared with 1-16 mg/L in RPMI 1640 medium, CA-MHB supplemented with serum, or bronchoalveolar lavage fluid (repeated for RPMI 1640 medium with clarithromycin, other macrolides, and other gram-negative bacteria). [(14)C]-clarithromycin accumulation was 2.2-fold higher in RPMI 1640 medium, compared with CA-MHB. Inhibition of95% of protein synthesis was obtained with azithromycin at 16 mg/L in RPMI 1640 medium, compared with512 mg/L in CA-MHB. Strains not expressing oprM showed an MIC of 4 mg/L in CA-MHB. PAβN decreased MICs in CA-MHB but not in RPMI 1640 medium. Real-time polymerase chain reaction showed downregulation of oprM by azithromycin in RPMI 1640 medium. Outer-membrane permeability was 3-4.5 times higher in RPMI 1640 medium or bronchoalveolar lavage fluid, compared with CA-MHB. Azithromycin combined with outer-membrane disrupting agents were synergistic in CA-MHB but indifferent in RPMI 1640 medium.Macrolides show antimicrobial activity against P. aeruginosa in eukaryotic media through increased uptake and reduced efflux. These data may help explain the clinical efficacy of macrolides against pseudomonal infections.