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We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

Background Rapid and effective screening of carbapenemase-producing Enterobacteriaceae (CPE) appears essential for adequate patient management and rapid implementation of infection control measures. Most of these screening techniques require a minimum of 24 h of culture. Molecular assays are an exception since these can be achieved within 1 h, but are expensive and usually require specialized facilities and trained personnel. In this context, lateral immunochromatography performed directly from rectal swab samples could represent a cost-effective alternative with a reduced turnaround time. Objectives In this study, we assessed the performance of the OKN K-SeT test (Coris BioConcept, Gembloux, Belgium) for the rapid detection of OXA-48, KPC and NDM CPE directly from rectal swab samples. Methods A total of 149 residual rectal swabs, routinely screened for CPE through selective culture and confirmed by PCR, were tested with a defined protocol consisting of a 2.5 h incubation of the swab in an enrichment medium containing meropenem followed by OKN K-SeT testing after centrifugation. Results This method displayed an overall sensitivity of 96% and a specificity of 100% with a limit of detection ranging between 104 and 105 cfu/mL. Conclusions Whereas this assay appears highly specific, it displays a reduced sensitivity compared with the standard procedure encompassing a culture step. Nonetheless, this rapid method allows an accelerated identification of most CPE carriers at a lower cost and, accordingly, the implementation of early appropriate management procedures.

OBJECTIVES: The increasing rate of carbapenem resistance in Gram-negative bacteria is a major public health problem and rapid detection is essential for infection management. We evaluated the performances of the MBT STAR®-Carba IVD assay (Bruker Daltonics) to detect carbapenemase-producing organisms (CPO) from bacterial colonies and directly from positive blood culture bottles with MALDI-TOF MS. METHODS: We analyzed 130 strains with a reduced susceptibility to at least one carbapenem including 109 CPO (6 KPC, 27 NDM, 21 VIM, 1 IMP, 41 OXA-48-like, 8 OXA-23, 2 OXA-24/-40, and 2 OXA-58) and 21 non-CPO. The assay on colonies was performed with all 130 strains while the assay on spiked blood cultures was performed with 45 strains. Samples were prepared with the MBT STAR®-CARBA IVD kit and imipenem hydrolysis by the potential carbapenemase was analyzed with the MBT STAR®-BL module (Bruker Daltonics) on MALDI-TOF MS. RESULTS: Performed on colonies, the assay detected all carbapenemase-producing Enterobacteriaceae (n = 78), Pseudomonas spp. (n = 19) and Acinetobacter spp. (n = 12). All 21 tested non-CPO remained negative resulting in sensitivity and specificity of 100%. Performed on positive blood cultures, the assay detected all carbapenemase-producing Enterobacteriaceae (n = 23) and Pseudomonas spp. (n = 4) but missed 9/12 carbapenemase-producing Acinetobacter spp. However, a prolonged imipenem-incubation time of the strain pellet improved carbapenemase detection. Non-CPO from positive blood culture bottles remained negative (n = 5) with the assay with the exception of one Klebsiella pneumoniae isolate. CONCLUSION: The MBT STAR®-Carba IVD assay is a highly reliable method for the detection of carbapenemase activity in Gram-negative bacteria. However, time-consuming sample preparation steps and reagent costs need to be considered before implementation in a routine clinical microbiology laboratory.

We recently described the novel anti-persister compound 1-[(2,4-dichlorophenethyl)amino]-3-phenoxypropan-2-ol (SPI009), capable of directly killing persister cells of the Gram-negative pathogen Pseudomonas aeruginosa. This compound also shows antibacterial effects against non-persister cells, suggesting that SPI009 could be used as an adjuvant for antibacterial combination therapy. Here, we demonstrate the broad-spectrum activity of SPI009, combined with different classes of antibiotics, against the clinically relevant ESKAPE pathogens Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, Enterococcus faecium and Burkholderia cenocepacia and Escherichia coli. Importantly, SPI009 re-enabled killing of antibiotic-resistant strains and effectively lowered the required antibiotic concentrations. The clinical potential was further confirmed in biofilm models of P. aeruginosa and S. aureus where SPI009 exhibited effective biofilm inhibition and eradication. Caenorhabditis elegans infected with P. aeruginosa also showed a significant improvement in survival when SPI009 was added to conventional antibiotic treatment. Overall, we demonstrate that SPI009, initially discovered as an anti-persister molecule in P. aeruginosa, possesses broad-spectrum activity and is highly suitable for the development of antibacterial combination therapies in the fight against chronic infections. ispartof: Frontiers in Microbiology vol:8 issue:DEC pages:2585- ispartof: location:Switzerland status: published

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).

The injectisome type three secretion system (T3SS) is a major virulence factor in Pseudomonas aeruginosa. This bacterium is responsible for severe infections in immunosuppressed or cystic fibrosis patients and has become resistant to many antibiotics. Inhibitors of T3SS may therefore constitute an innovative therapeutic target. After a brief description of the T3SS and its regulation, this review presents strategies to inhibit T3SS-mediated toxicity and describes the main families of existing inhibitors. Over the past few years, 12 classes of small-molecule inhibitors and two types of antibody have been discovered and evaluated in vitro for their capacity to inhibit T3SS expression or function, and to protect host cells from T3SS-mediated cytotoxicity. While only one small molecule has been tested in vivo, a bifunctional antibody targeting both the translocation apparatus of the T3SS and a surface polysaccharide is currently in Phase II clinical trials.

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.

International audience; Long-term exposure to pharmacological agents can select for cells that overexpress efflux transporters. We previously showed that mouse J774 macrophages cultivated for a prolonged period of time with toxic concentrations of the fluoroquinolone ciprofloxacin overexpress the efflux transporter Mrp4 and display a reduced accumulation of this antibiotic, but no change in the accumulation of moxifloxacin, a closely related molecule (Antimicrob. Agents Chemother. [2006] 50, 1689-1695 and [2009] 53, 2410-2416). Because of this striking difference between the two fluoroquinolones, we have now examined the modifications in the expression of ABC efflux transporters induced by the prolonged exposure of J774 macrophages to high concentrations of moxifloxacin. The resulting cell line showed (i) no difference in the accumulation of moxifloxacin but an increased accumulation and decreased efflux of ciprofloxacin; (ii) an overexpression of the multidrug transporters Abcb1a (P-gp), Abcc2 (Mrp2) and Abcg2 (Bcrp1), and a decreased expression of Abcc4 (Mrp4). While P-gp and Bcrp1 were functional, they did not modify the cellular accumulation of fluoroquinolones. The data show that exposing cells to high concentrations of a drug that is not affected by active efflux can trigger a pleiotropic response leading to a modulation in the expression of several transporters. These changes, however, are not sufficient to protect cells against the toxicity that fluoroquinolones may exert at large concentrations. They could also cause unanticipated drug interactions in vivo, should the drug exposure grossly exceed what is anticipated from its current registered use.