Characterization of the Achromobacter xylosoxidans Type VI Secretion System and Its Implication in Cystic Fibrosis.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine. A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management.

Antibiotic-related gut dysbiosis induces lung immunodepression and worsens lung infection in mice.

BACKGROUND: Gut dysbiosis due to the adverse effects of antibiotics affects outcomes of lung infection. Previous murine models relied on significant depletion of both gut and lung microbiota, rendering the analysis of immune gut-lung cross-talk difficult. Here, we study the effects of antibiotic-induced gut dysbiosis without lung dysbiosis on lung immunity and the consequences on acute P. aeruginosa lung infection. METHODS: C57BL6 mice received 7 days oral vancomycin-colistin, followed by normal regimen or fecal microbial transplant or Fms-related tyrosine kinase 3 ligand (Flt3-Ligand) over 2 days, and then intra-nasal P. aeruginosa strain PAO1. Gut and lung microbiota were studied by next-generation sequencing, and lung infection outcomes were studied at 24 h. Effects of vancomycin-colistin on underlying immunity and bone marrow progenitors were studied in uninfected mice by flow cytometry in the lung, spleen, and bone marrow. RESULTS: Vancomycin-colistin administration induces widespread cellular immunosuppression in both the lung and spleen, decreases circulating hematopoietic cytokine Flt3-Ligand, and depresses dendritic cell bone marrow progenitors leading to worsening of P. aeruginosa lung infection outcomes (bacterial loads, lung injury, and survival). Reversal of these effects by fecal microbial transplant shows that these alterations are related to gut dysbiosis. Recombinant Flt3-Ligand reverses the effects of antibiotics on subsequent lung infection. CONCLUSIONS: These results show that gut dysbiosis strongly impairs monocyte/dendritic progenitors and lung immunity, worsening outcomes of P. aeruginosa lung infection. Treatment with a fecal microbial transplant or immune stimulation by Flt3-Ligand both restore lung cellular responses to and outcomes of P. aeruginosa following antibiotic-induced gut dysbiosis.

Severe preschool asthmatics have altered cytokine and anti-viral responses during exacerbation.

BACKGROUND: Preschool asthma/recurrent wheeze is a heterogeneous condition. Different clinical phenotypes have been described, including episodic viral wheeze (EVW), severe intermittent wheeze (SIW), and multiple-trigger wheeze (MTW). OBJECTIVE: To compare clinical, viral, and inflammatory/immune profiling at exacerbation between MTW, SIW, and EVW phenotypes. METHODS: Multicenter, prospective, observational cohort (VIRASTHMA-2). Children (1-5 years) with preschool asthma were enrolled during hospitalization for a severe exacerbation. History and anamnestic data, plasma, and nasal samples were collected at exacerbation (T1) and at steady state, 8 weeks later (T2), and sputum samples were collected at T1. RESULTS: A total of 147 children were enrolled, 37 (25%) had SIW, 18 (12.2%) EVW, and 92 (63%) MTW. They were atopic (47%), exposed to mold (22%) and cigarette smoke (50%), and prone to exacerbations (≥2 in the previous year in 70%). At exacerbation, at least one virus was isolated in 94% and rhinovirus in 75%, with no difference between phenotypes. Children with MTW and SIW phenotypes displayed lower plasma concentrations of IFN-γ (P = .002), IL-5 (P = .020), TNF-α (P = .038), IL-10 (P = .002), IFN-ß (P = .036), and CXCL10 (P = .006) and lower levels of IFN-γ (P = .047) in sputum at exacerbation than children with EVW. At T2, they also displayed lower plasma levels of IFN-γ (P = .045) and CXCL10 (P = .013). CONCLUSION: Among preschool asthmatic children, MTW and SIW, prone to exacerbations, display lower systemic levels of Th1, Th2 cytokines, pro- and anti-inflammatory cytokines, and antiviral responses during severe virus-induced exacerbation.

Differential risk of severe infection in febrile neutropenia among children with blood cancer or solid tumor.

OBJECTIVE: To describe and analyze the differences between infections in children with febrile neutropenia (FN) treated for solid tumor or blood cancer. METHODS: A prospective study included all episodes of FN in children from April 2007 to April 2016 in 2-pediatric cancer centers in France. Medical history, clinical and laboratory data available at admission and final microbiological data were collected. The proportion of FN, severe infection, categories of microorganisms and outcomes were compared between the two groups. The presumed gateway of the infection was a posteriori considered and evaluated. RESULTS: We analyzed 1197 FN episodes (mean age: 8 years). 66% of the FN episodes occurred in children with blood cancer. Severe infections were identified in 23.4% of episodes overall. The rate of severe infection (28.4% vs. 10.4%), types of microorganisms and the need for a management in intensive care unit (2.6% vs. 0.5%) was significantly different between children with blood cancer and solid tumor. Digestive or respiratory presumed gateway of the infections was less frequent for patients with solid tumor. CONCLUSION: Given these important microbiological and clinical differences, it may be appropriate to consider differently the risk of severe infection in these two populations and therefore the management of FN.

The human NAIP-NLRC4-inflammasome senses the Pseudomonas aeruginosa T3SS inner-rod protein.

While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIP-NLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1ß maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components.

Vaginal Mucosal Homeostatic Response May Determine Pregnancy Outcome in Women With Bacterial Vaginosis: A Pilot Study.

Bacterial vaginosis (BV) is considered as a trigger for an inflammatory response that could promote adverse pregnancy outcome (APO). We hypothesized that BV-related inflammation could be counterbalanced by anti-inflammatory and mucosal homeostatic responses that could participate in pregnancy outcomes.A total of 402 vaginal self-samples from pregnant women in their first trimester were screened by Nugent score. In this population, we enrolled 23 pregnant women with BV but without APO, 5 pregnant women with BV and developing APO, 21 pregnant women with intermediate flora, and 28 random control samples from pregnant women without BV or APO.BV without APO in pregnant women was associated with 28-fold interleukin-8, 5-fold interleukin-10, and 40-fold interleukin-22 increases in expression compared to controls. BV associated with APO in pregnant women shared 4-fold increase in tumor necrosis factor, 100-fold decrease in interleukin-10, and no variation in interleukin-22 expressions compared to controls. Next-generation sequencing of vaginal microbiota revealed a shift from obligate anaerobic bacteria dominance in BV without APO pregnant women to Lactobacillus dominance microbiota in BV with APO.Our results show that the anti-inflammatory and mucosal homeostatic responses to BV may determine outcome of pregnancy in the setting of BV possibly through effects on the vaginal microbiota.

PscI is a type III secretion needle anchoring protein with in vitro polymerization capacities.

The export of bacterial toxins across the bacterial envelope requires the assembly of complex, membrane-embedded protein architectures. Pseudomonas aeruginosa employs type III secretion (T3S) injectisome to translocate exotoxins directly into the cytoplasm of a target eukaryotic cell. This multi-protein channel crosses two bacterial membranes and extends further as a needle through which the proteins travel. We show in this work that PscI, proposed to form the T3S system (T3SS) inner rod, possesses intrinsic properties to polymerize into flexible and regularly twisted fibrils and activates IL-1ß production in mouse bone marrow macrophages in vitro. We also found that point mutations within C-terminal amphipathic helix of PscI alter needle assembly in vitro and T3SS function in cell infection assays, suggesting that this region is essential for an efficient needle assembly. The overexpression of PscF partially compensates for the absence of the inner rod in PscI-deficient mutant by forming a secretion-proficient injectisome. All together, we propose that the polymerized PscI in P. aeruginosa optimizes the injectisome function by anchoring the needle within the envelope-embedded complex of the T3S secretome and - contrary to its counterpart in Salmonella - is not involved in substrate switching.

Antiadhesive properties of glycoclusters against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa lung infections are a major cause of death in cystic fibrosis and hospitalized patients. Treating these infections is becoming difficult due to the emergence of conventional antimicrobial multiresistance. While monosaccharides have proved beneficial against such bacterial lung infection, the design of several multivalent glycosylated macromolecules has been shown to be also beneficial on biofilm dispersion. In this study, calix[4]arene-based glycoclusters functionalized with galactosides or fucosides have been synthesized. The characterization of their inhibitory properties on Pseudomonas aeruginosa aggregation, biofilm formation, adhesion on epithelial cells, and destruction of alveolar tissues were performed. The antiadhesive properties of the designed glycoclusters were demonstrated through several in vitro bioassays. An in vivo mouse model of lung infection provided an almost complete protection against Pseudomonas aeruginosa with the designed glycoclusters.

Pseudomonas aeruginosa type-3 secretion system dampens host defense by exploiting the NLRC4-coupled inflammasome.

RATIONALE: Pseudomonas aeruginosa, a major problem pathogen responsible for severe infections in critically ill patients, triggers, through a functional type-3 secretion system (T3SS), the activation of an intracellular cytosolic sensor of innate immunity, NLRC4. Although the NLRC4-inflammasome-dependent response contributes to increased clearance of intracellular pathogens, it seems that NLRC4 inflammasome activation decreases the clearance of P. aeruginosa, a mainly extracellular pathogen. OBJECTIVES: We sought to determine the underlying mechanisms of this effect of the activation of NLRC4 by P. aeruginosa. METHODS: We established acute lung injury in wild-type and Nlrc4(-/-) mice using sublethal intranasal inocula of P. aeruginosa strain CHA expressing or not a functional T3SS. We studied 96-hour survival, lung injury, bacterial clearance from the lungs, cytokine secretion in bronchoalveolar lavage, lung antimicrobial peptide expression by quantitative polymerase chain reaction, and flow cytometry analysis of lung cells. MEASUREMENTS AND MAIN RESULTS: Nlrc4(-/-) mice showed enhanced bacterial clearance and decreased lung injury contributing to increased survival against extracellular P. aeruginosa strain expressing a functional T3SS. The mechanism involved decreased NLRC4-inflammasome-driven IL-18 secretion attenuating lung injury caused by excessive neutrophil recruitment. Additionally, in the lungs of Nlrc4(-/-) mice secretion of IL-17 by innate immune cells was increased and responsible for increased expression of lung epithelial antimicrobial peptides. Furthermore, IL-18 secretion was found to repress IL-17 and IL-17-driven lung antimicrobial peptide expression. CONCLUSIONS: We report a new role of the T3SS apparatus itself, independently of exotoxin translocation. Through NLRC4 inflammasome activation, the T3SS promotes IL-18 secretion, which dampens a beneficial IL-17-mediated antimicrobial host response.

Candida albicans airway exposure primes the lung innate immune response against Pseudomonas aeruginosa infection through innate lymphoid cell recruitment and interleukin-22-associated mucosal response.

Pseudomonas aeruginosa and Candida albicans are two pathogens frequently encountered in the intensive care unit microbial community. We have demonstrated that C. albicans airway exposure protected against P. aeruginosa-induced lung injury. The goal of the present study was to characterize the cellular and molecular mechanisms associated with C. albicans-induced protection. Airway exposure by C. albicans led to the recruitment and activation of natural killer cells, innate lymphoid cells (ILCs), macrophages, and dendritic cells. This recruitment was associated with the secretion of interleukin-22 (IL-22), whose neutralization abolished C. albicans-induced protection. We identified, by flow cytometry, ILCs as the only cellular source of IL-22. Depletion of ILCs by anti-CD90.2 antibodies was associated with a decreased IL-22 secretion and impaired survival after P. aeruginosa challenge. Our results demonstrate that the production of IL-22, mainly by ILCs, is a major and inducible step in protection against P. aeruginosa-induced lung injury. This cytokine may represent a clinical target in Pseudomonas aeruginosa-induced lung injury.

Optimization of continuous infusion of piperacillin-tazobactam in children with fever and neutropenia.

The study through Monte Carlo simulation of ß-lactam pharmacokinetic/pharmacodynamic target attainment and determination of subsequent serum concentrations of piperacillin-tazobactam administered through continuous infusion to children treated for fever and neutropenia shows that 400 mg/kg/day has the highest probability of target attainment against Pseudomonas aeurginosa in our oncology ward compared with the standard regimen of 300 mg/kg/day.

Defensins couple dysbiosis to primary immunodeficiency in Crohn's disease.

Antimicrobial peptides, including defensins, are essential effectors in host defence and in the maintenance of immune homeostasis. Clinical studies have linked the defective expression of both α- and ß-defensin to the reduced killing of certain microorganisms by the intestinal mucosa of patients suffering from ileal and colonic Crohn's disease (CD), respectively. Only recently have the events leading to defective expression of defensins in CD been further elucidated, and are discussed herein. These events may account for CD-associated alterations in the microbiome and may subsequently precipitate the development of granulomatous inflammatory lesions in genetically-predisposed patients. We also address how these discoveries may pave the way for the development of a molecular medicine aimed at restoring gut barrier function in CD.

Autocrine induction of invasive and metastatic phenotypes by the MIF-CXCR4 axis in drug-resistant human colon cancer cells.

Metastasis and drug resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil, methotrexate, doxorubicin, or oxaliplatin. Drug-resistant invasive cells were compared with noninvasive cells using cDNA microarray, quantitative reverse transcription-PCR, flow cytometry, immunoblots, and ELISA. Functional and cellular signaling analyses were undertaken using pharmacologic inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-Fluorouracil- and methotrexate-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behavior in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration-inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, monoclonal antibody 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 was associated with the upregulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2alpha and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from patients with colon cancer treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis could potentially counteract the emergence of the invasive metastatic behavior in clonal derivatives of drug-resistant colon cancer cells.

Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

Innate immunity in Crohn's disease: the reverse side of the medal.

The etiology of Crohn's disease (CD) remains poorly understood. Both mice and human studies suspected a perverse link between the microbiota and the lining of the gut mucosa. There is now emerging evidence that suggests that such a pathologic condition might result from an overly aggressive immune response to microbial antigens in genetically predisposed individuals. However, the multiple pathophysiologic processing steps linking environmental exposure to the clinical expression of CD are, for the most part, unknown. Herein, we review evidences reflecting a general causing defect of the innate immune function of the intestinal mucosa of CD patients, which might lead to a sustained microbial-induced inflammatory response. Changing the paradigms of CD pathophysiology might lead to entirely new therapeutic approaches aiming to boost the innate immune response.

NODs in defence: from vulnerable antimicrobial peptides to chronic inflammation.

Defensins and cathelicidins are prevalent and essential gastrointestinal cationic antimicrobial peptides (CAPs). However, these defensive peptides are not infallible because certain enteropathogens can overcome their protective function. Furthermore, impaired defensin synthesis has been linked to the occurrence of Crohn's disease (CD), a chronic inflammatory bowel disease. Recently, defective bacterial sensing through NOD1 and NOD2 has been related to reduced defensin production, CD predisposition and susceptibility to enteric infection. Hence, we propose that microbial sensors at the gut interface monitor the levels of these effector peptides, which might function as "danger" signals to confer tolerance and alert immunocytes. Further work is required to clarify how gastrointestinal CAPs are regulated and to assess their role in maintaining epithelial homeostasis and triggering adaptive immunity.

An 18-case outbreak of drug-resistant Pseudomonas aeruginosa bacteriemia in hematology patients.

We retrospectively identified an outbreak of 18 episodes of P. aeruginosa bacteriemia in 17 patients with hematologic malignancies in 2004. All strains were ticarcillin I/R, 77% ciprofloxacin I/R, 72% ceftazidime I/R, 72% amikacin I/R and 50% imipenem I/R. The outbreak was multiclonal. Colistin was employed for documented therapy in ten cases including seven in which it was the only active drug. Outcomes were resolution of infection in 12 out of 18 episodes (67%), and death in six cases, five (25%) of which were attributable to the infection. Colistin was useful even in highly resistant strains and the efficacy of antibacterial therapy was similar (57%)in bacteriemia due to strains only susceptible to colistin.