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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.

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.

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.

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. aeruginosa in 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).

International audience

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.

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 .

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.