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Introduction: Streptococcus pneumoniae is the most common cause of community-acquired pneumonia (CAP) in children. Recently, the rapid emergence of antibiotic-resistant pneumococci has posed enormous challenges for CAP treatment and public health. This study aims to provide clinicians with updated data about the antimicrobial susceptibility of S. pneumoniae and improve the treatment guidelines in CAP in children. Methods: This cross-sectional and in-vitro study was conducted at three hospitals in Quang Nam province and Da Nang city in Vietnam. Pneumococcal strains were isolated from nasopharyngeal aspirate samples of 360 CAP in-patients under five years of age. The susceptibility of clinically used antibiotics was investigated using the disk diffusion test and the E-test for identifying the MIC. Multi-drug resistant (MDR) S. pneumoniae isolates were also determined. Results: 21.9% of CAP children patients were pneumococcal carriage. The susceptibility testing demonstrated that all 56 tested pneumococcal isolates were resistant to erythromycin and azithromycin, whereas none of the isolates developed the resistance to levofloxacin, vancomycin, and linezolid. The clindamycin- and cotrimoxazole-resistant pneumococci rates were 96.4% and 87.5%, respectively. For β-lactam antibiotics, the resistance proportions of pneumococcal isolates to penicillin G, co-amoxiclav, amoxicillin, cefotaxime, and ceftriaxone were 3.6%,1.8%, 1.8%, 1.8%, and 3.6%, respectively. 83.9% of the tested isolates were MDR S. pneumoniae. Conclusion: Our data support using penicillin G, amoxicillin, co-amoxiclav, or cefotaxime as the first-line therapy for uncomplicated-pneumococci-induced CAP in Quang Nam – Da Nang, while vancomycin, linezolid, and levofloxacin should be used as alternatives or in MDR cases.

The reduction in antimicrobial activity at high bacterial counts is a microbiological phenomenon known as the inoculum effect (IE). In a previous in vitro study, a significant IE was observed for polymyxin B (PMB) against a clinical isolate of Acinetobacter baumannii, and well described by a new pharmacokinetic-pharmacodynamic model. Few in vivo studies have investigated the impact of inoculum size on survival or antibiotic efficacy. Therefore, our objective was to confirm the influence of inoculum size of this A. baumannii clinical isolate on PMB in vivo effect over time. Pharmacokinetics and pharmacodynamics of PMB after a single subcutaneous administration (1, 15 and 40 mg/kg) were studied in a neutropenic murine thigh infection model. The impact of A. baumannii inoculum size (105, 106 and 107 CFU/thigh) on PMB efficacy was also evaluated. In vivo PMB PK was well described by a two-compartment model including saturable absorption from the subcutaneous injection site and linear elimination. The previous in vitro PD model was modified to adequately describe the decrease of PMB efficacy with increased inoculum size in infected mice. The IE was modeled as a decrease of 32% in the in vivo PMB bactericidal effect when the starting inoculum increases from 105 to 107 CFU/thigh. Although not as important as previously characterized in vitro an IE was confirmed in vivo.

The reduction in antimicrobial activity at high bacterial counts is a microbiological phenomenon known as the inoculum effect (IE). In a previous

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; Gram-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.

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.

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.

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).

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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.

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.

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.