Re-evaluation of cefepime or piperacillin/tazobactam to decrease use of carbapenems in ESBL-producing Enterobacterales urinary tract infections (REDUCE-UTI).

Objectives: To re-examine the use of non-carbapenems (NCBPs), specifically piperacillin/tazobactam and cefepime, for ESBL-producing Enterobacterales (ESBL-E) urinary tract infections (UTIs). Patients: Retrospective cohort study of adults hospitalized between January 2016 and June 2020 with pyuria on urinalysis, a urine culture positive for ESBL-E treated with a study antibiotic (meropenem, ertapenem, cefepime or piperacillin/tazobactam) and did not meet criteria for study exclusion. Methods: To compare carbapenems (CBPs) with cefepime or piperacillin/tazobactam for the treatment of ESBL-E UTI. The primary outcome was clinical cure, defined as complete resolution of signs and symptoms of infection. Secondary outcomes included in-hospital mortality, recurrence within 30 days and resistance emergence within 30 days. Results: One-hundred and thirty-three patients were included, based on definitive therapy received; 69 (51.9%) received CBP and 64 (48.1%) received NCBP therapy. Of the total patient population, 17 (12.8%) were admitted to the ICU, 84 (63.1%) had a complicated UTI and 64 (48.1%) had pyelonephritis. There was no difference in clinical cure between the CBP and NCBP groups (95.7% versus 96.9%, P = 0.999). Additionally, no differences in secondary outcomes were observed. Conclusions: When compared with CBPs, cefepime and piperacillin/tazobactam resulted in similar clinical cure, in-hospital mortality, recurrence and resistance emergence in the treatment of ESBL-E UTI.

Re-evaluation of cefepime or piperacillin-tazobactam to decrease use of carbapenems in extended-spectrum beta-lactamase-producing Enterobacterales bloodstream infections (REDUCE-BSI).

Objective: To re-examine the use of noncarbapenems (NCBPs), specifically piperacillin-tazobactam (PTZ) and cefepime (FEP), for extended-spectrum beta-lactamase-producing Enterobacterales bloodstream infections (ESBL-E BSIs). Design: Retrospective cohort study. Setting: Tertiary-care, academic medical center. Patients: The study included patients hospitalized between May 2016 and May 2019 with a positive blood culture for ESBL-E. Patients were excluded if they received treatment with antibiotics other than meropenem, ertapenem, PTZ, or FEP. Patients were also excluded if they were aged <18 years, received antibiotics for <24 hours, were treated for polymicrobial BSI, or received concomitant antibiotic therapy for a separate gram-negative infection. Methods: We compared CBPs with FEP or PTZ for the treatment of ESBL-E BSI. The primary outcome was in-hospital mortality. Secondary outcomes included clinical cure, microbiologic cure, infection recurrence, and resistance development. Results: Data from 114 patients were collected and analyzed; 74 (65%) patients received carbapenem (CBP) therapy and 40 (35%) patients received a NCBP (30 received FEP and 10 received PTZ). The overall in-hospital mortality was 6% (N = 7), with a higher death rate in the CBP arm than in the N-CBP arm, (8% vs 3%; P = .42). No difference in mortality was detected between subgroups with Pitt bacteremia score ≥4, those requiring ICU admission, those whose infections were cause by a nongenitourinary source or causative organism (ie, 76 had Escherichia coli and 38 had Klebsiella spp). We detected no differences in secondary outcomes between the groups. Conclusion: Compared to CBPs, FEP and PTZ did not result in greater mortality or decreased clinical efficacy for the treatment of ESBL-E BSI caused by susceptible organisms.

High pyocyanin production and non-motility of Pseudomonas aeruginosa isolates are correlated with septic shock or death in bacteremic patients.

Studies of the outcome of Pseudomonas aeruginosa bacteremia (Pab) have focused mainly on antibiotic appropriateness. However, P. aeruginosa possesses many virulence factors whose roles in outcomes have not been examined in humans, except for the type III secretion system (T3SS) toxins. The purpose of this study was to examine the role of virulence factors other than the T3SS toxins. Bacterial isolates were collected from 75 patients who suffered from Pa blood stream infections. Host factors such as neutropenia, immunosuppression, comorbidities, time to effective antibiotics, source of bacteremia, and presence of multidrug resistant (MDR) isolate were studied. The isolates were analyzed for the presence of toxin genes, proteolytic activity, swimming and twitching motility, and pyocyanin production. The data were analyzed to ascertain which virulence factors correlated with poor outcomes defined as septic shock or death (SS) within 7 days. Septic shock or death occurred in 25/75 patients. Univariate analysis identified age as a host factor that exerted a significant effect on these outcomes. Ineffective antibiotics administered during the first 24 hours of treatment or MDR P. aeruginosa did not influence the frequency of SS, nor did the presence of lasB, exoA, exoS exoU, plcH genes and proteolytic activity. However, 6/8 patients infected with non-motile isolates, developed SS, p = 0.014 and 5/6 isolates that produced large amounts of pyocyanin (>18ug/ml), were associated with SS, p = 0.014. Multivariate analysis indicated that the odds ratio (OR) for development of SS with a non-motile isolate was 6.8, with a 95% confidence interval (CI) (1.37, 51.5), p = 0.030 and with high pyocyanin producing isolates, an OR of 16.9, 95% CI = (2.27, 360), p = .017. This study evaluating the role of microbial factors that significantly effect outcomes following Pa bloodstream infection suggests that P. aeruginosa strains showing high pyocyanin production and the lack of motility independently increase the risk of SS.

Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets.

BACKGROUND: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process. METHODS: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa. RESULTS: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation. CONCLUSIONS: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.

Pseudomonas aeruginosa Lipoxygenase LoxA Contributes to Lung Infection by Altering the Host Immune Lipid Signaling.

Pseudomonas aeruginosa is an opportunistic bacteria and a major cause of nosocomial pneumonia. P. aeruginosa has many virulence factors contributing to its ability to colonize the host. LoxA is a lipoxygenase enzyme secreted by P. aeruginosa that oxidizes polyunsaturated fatty acids. Based on previous in vitro biochemical studies, several biological roles of LoxA have been hypothesized, including interference of the host lipid signaling, and modulation of bacterial invasion properties. However, the contribution of LoxA to P. aeruginosa lung pathogenesis per se remained unclear. In this study, we used complementary in vitro and in vivo approaches, clinical strains of P. aeruginosa as well as lipidomics technology to investigate the role of LoxA in lung infection. We found that several P. aeruginosa clinical isolates express LoxA. When secreted in the lungs, LoxA processes a wide range of host polyunsaturated fatty acids, which further results in the production of bioactive lipid mediators (including lipoxin A4). LoxA also inhibits the expression of major chemokines (e.g., MIPs and KC) and the recruitment of key leukocytes. Remarkably, LoxA promotes P. aeruginosa persistence in lungs tissues. Hence, our study suggests that LoxA-dependent interference of the host lipid pathways may contribute to P. aeruginosa lung pathogenesis.

Ampicillin for the treatment of complicated urinary tract infections caused by vancomycin-resistant Enterococcus spp (VRE): a single-center university hospital experience.

Vancomycin-resistant enterococci (VRE) are a common cause of urinary tract infections (UTIs) and are typically multidrug resistant, including ampicillin. This retrospective study evaluated outcomes of 84 adult patients hospitalized between January 2007 and December 2015 with ampicillin- and vancomycin-resistant enterococcus isolates causing UTI and treated with ampicillin. Treatment response was classified as clinical cure and microbiological eradication. Clinical cure was achieved in 88.1% (74/84) of patients. In patients with follow-up cultures, microbiological eradication was achieved in 86% (50/58) of patients. Cure rates were similar in patients with indwelling urinary catheters (n = 45) receiving catheter exchange/removal (90.47%; 19/21) versus catheter retention (87.5%; 21/24). Presence of co-morbidities, such as diabetes and chronic kidney disease, were not associated with increased risk of treatment failure. Immunocompromised patients achieved lower cure rates of 78.1% (25/32) compared with 94.2% (49/52) among those without immune impairment (P = 0.038). Presence of an underlying urinary tract abnormality was also associated with a lower cure rate of 71.4% (15/21) compared with 93.7% (59/63) in those without urinary tract abnormalities (P = 0.0135). Overall cure rates remained high in all groups providing good evidence to support ampicillin for the treatment of complicated UTI caused by ampicillin- and vancomycin-resistant enterococci.

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.

Computed Tomography (CT) Scanning Facilitates Early Identification of Neonatal Cystic Fibrosis Piglets.

BACKGROUND: Cystic Fibrosis (CF) is the most prevalent autosomal recessive disease in the Caucasian population. A cystic fibrosis transmembrane conductance regulator knockout (CFTR-/-) pig that displays most of the features of the human CF disease has been recently developed. However, CFTR-/- pigs presents a 100% prevalence of meconium ileus that leads to death in the first hours after birth, requiring a rapid diagnosis and surgical intervention to relieve intestinal obstruction. Identification of CFTR-/- piglets is usually performed by PCR genotyping, a procedure that lasts between 4 to 6 h. Here, we aimed to develop a procedure for rapid identification of CFTR-/- piglets that will allow placing them under intensive care soon after birth and immediately proceeding with the surgical correction. METHODS AND PRINCIPAL FINDINGS: Male and female CFTR+/- pigs were crossed and the progeny was examined by computed tomography (CT) scan to detect the presence of meconium ileus and facilitate a rapid post-natal surgical intervention. Genotype was confirmed by PCR. CT scan presented a 94.4% sensitivity to diagnose CFTR-/- piglets. Diagnosis by CT scan reduced the birth-to-surgery time from a minimum of 10 h down to a minimum of 2.5 h and increased the survival of CFTR-/- piglets to a maximum of 13 days post-surgery as opposed to just 66 h after later surgery. CONCLUSION: CT scan imaging of meconium ileus is an accurate method for rapid identification of CFTR-/- piglets. Early CT detection of meconium ileus may help to extend the lifespan of CFTR-/- piglets and, thus, improve experimental research on CF, still an incurable disease.

Flagellin concentrations in expectorations from cystic fibrosis patients.

BACKGROUND: The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation. METHODS: Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files. RESULTS: Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration. CONCLUSIONS: This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject.

Time to positivity of blood cultures supports antibiotic de-escalation at 48 hours.

BACKGROUND: Appropriate de-escalation of empirical antimicrobial therapy is a fundamental component of antimicrobial stewardship. Concern for the late detection of bloodstream pathogens may undermine early streamlining efforts and subject patients to protracted courses of nonessential therapy. OBJECTIVE: To quantify the prevalence of bacterial bloodstream infection (BSI) detection after more than 48 hours of culture incubation. We also assessed the impact of antimicrobial therapy delivered prior to blood sample collection. METHODS: We retrospectively evaluated time to blood culture positivity (TTP) in adult patients at an academic tertiary care hospital. Microbiology reports were reviewed to identify the TTP for the first positive blood culture bottle for each episode of BSI occurring from February 1, 2011, to July 31, 2011. Isolates were classified as true pathogens or contaminants. Blood culture results after 48 hours of incubation were compared with results after 120 hours of incubation. RESULTS: The median TTP of 416 monomicrobial BSIs and 210 contamination episodes was 13.7 and 24.4 hours, respectively (P < .001). The median TTPs in those who received and did not receive prior antibiotics were 17.0 and 12.8 hours, respectively (P < .001). By 48 hours, 98% of aerobic Gram-positive and Gram-negative BSIs were detected. Culture results at 48 hours were 97% sensitive and had a negative predictive value of 99.8%. CONCLUSION: Few true BSIs are detected after more than 48 hours of culture incubation. Clinicians may adjust empirical antibiotic coverage at this time with little risk for subsequent bacterial pathogen detection.

Poly-L-Lysine compacts DNA, kills bacteria, and improves protease inhibition in cystic fibrosis sputum.

RATIONALE: Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES: We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS: We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS: Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS: Poly-L-lysine may be an alternative to dornase-α to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.

Toll-like receptor 5 (TLR5), IL-1ß secretion, and asparagine endopeptidase are critical factors for alveolar macrophage phagocytosis and bacterial killing.

A deficit in early clearance of Pseudomonas aeruginosa (P. aeruginosa) is crucial in nosocomial pneumonia and in chronic lung infections. Few studies have addressed the role of Toll-like receptors (TLRs), which are early pathogen associated molecular pattern receptors, in pathogen uptake and clearance by alveolar macrophages (AMs). Here, we report that TLR5 engagement is crucial for bacterial clearance by AMs in vitro and in vivo because unflagellated P. aeruginosa or different mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. In addition, the clearance of PAK (a wild-type P. aeruginosa strain) by primary AMs was causally associated with increased IL-1ß release, which was dramatically reduced with PAK mutants or in WT PAK-infected primary TLR5(-/-) AMs, demonstrating the dependence of IL-1ß production on TLR5. We showed that this IL-1ß production was important in endosomal pH acidification and in inducing the killing of bacteria by AMs through asparagine endopeptidase (AEP), a key endosomal cysteine protease. In agreement, AMs from IL-1R1(-/-) and AEP(-/-) mice were unable to kill P. aeruginosa. Altogether, these findings demonstrate that TLR5 engagement plays a major role in P. aeruginosa internalization and in triggering IL-1ß formation.

Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling.

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/ß, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.

Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection.

BACKGROUND: The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research. METHODS: Using a model of pneumonia in Toll-like receptor (TLR) 2,4(-/-) mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology. RESULTS: Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS(+) strains killed mice within a day, and the T2SS(+) strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS(+) strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria. CONCLUSIONS: The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.

Sepsis induces early alterations in innate immunity that impact mortality to secondary infection.

Sepsis, the systemic inflammatory response to microbial infection, induces changes in both innate and adaptive immunity that presumably lead to increased susceptibility to secondary infections, multiorgan failure, and death. Using a model of murine polymicrobial sepsis whose severity approximates human sepsis, we examined outcomes and defined requirements for survival after secondary Pseudomonas aeruginosa pneumonia or disseminated Listeria monocytogenes infection. We demonstrate that early after sepsis neutrophil numbers and function are decreased, whereas monocyte recruitment through the CCR2/MCP-1 pathway and function are enhanced. Consequently, lethality to Pseudomonas pneumonia is increased early but not late after induction of sepsis. In contrast, lethality to listeriosis, whose eradication is dependent upon monocyte/macrophage phagocytosis, is actually decreased both early and late after sepsis. Adaptive immunity plays little role in these secondary infectious responses. This study demonstrates that sepsis promotes selective early, impaired innate immune responses, primarily in neutrophils, that lead to a pathogen-specific, increased susceptibility to secondary infections.

Pseudomonas aeruginosa LPS or flagellin are sufficient to activate TLR-dependent signaling in murine alveolar macrophages and airway epithelial cells.

BACKGROUND: The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 liters of air. Innate immunity is thus essential to defend the lungs against these pathogens. This defense is mediated in part through the recognition of specific microbial ligands by Toll-like receptors (TLR) of which there are at least 10 in humans. Pseudomonas aeruginosa is the main pathogen that infects the lungs of cystic fibrosis patients. Based on whole animal experiments, using TLR knockout mice, the control of this bacterium is believed to occur by the recognition of LPS and flagellin by TLRs 2,4 and 5, respectively. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we investigated in vitro the role of these same TLR and ligands, in alveolar macrophage (AM) and epithelial cell (EC) activation. Cellular responses to P. aeruginosa was evaluated by measuring KC, TNF-alpha, IL-6 and G-CSF secretion, four different markers of the innate immune response. AM and EC from WT and TLR2, 4, 5 and MyD88 knockout mice for were stimulated with the wild-type P. aeruginosa or with a mutant devoid of flagellin production. CONCLUSIONS/SIGNIFICANCE: The results clearly demonstrate that only two ligand/receptor pairs are necessary for the induction of KC, TNF-alpha, and IL-6 synthesis by P. aeruginosa-activated cells, i.e. TLR2,4/LPS and TLR5/flagellin. Either ligand/receptor pair is sufficient to sense the bacterium and to trigger cell activation, and when both are missing lung EC and AM are unable to produce such a response as were cells from MyD88(-/-) mice.