Increase In Bacterial Burden Research Articles

Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice.

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1β, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2-/- mice were predominantly populated with CD11bintLy6GintCXCR2loCD62Llo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2-/- mice. In contrast, neutralizing IL-1α and IL-1β, which are previously known factors responsible for TB pathogenesis in Nox2-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition.

Comparison of genomic DNA sequencing to anerobic cultures to detect efficacy of hydrogen peroxide at reducing cutibacterium acnes bacterial burden during primary shoulder arthroplasty

BackgroundThe purpose of this investigation is to compare Cutibacterium acnes diagnosis using culturing vs. genomic DNA sequencing (NextGen) at various timepoints and locations during primary total shoulder arthroplasty. Additionally, we intend to compare the effects of hydrogen peroxide on standard culture technique results with genomic DNA sequencing. MethodsA prospective diagnostic study of 40 patients undergoing primary total shoulder arthroplasty was performed. Intraoperatively, 4 tissue samples were collected per patient: subdermal skin edge following initial skin incision, subdermal skin edge following hydrogen peroxide soak for 5 minutes, glenohumeral joint capsule, and subdermal skin edge prior to wound closure. Each tissue specimen was collected twice (one for culture analysis and the other for genomic (NextGen) DNA sequencing analysis) for a total of 8 specimens per patient and 320 tissue specimens in total. All anaerobic cultures were held for 14 days. Each culture plate was divided into quadrants and the amount of growth was quantified. Tissue samples were collected for genomic sequencing DNA analysis. Genomic sequencing results provided relative percentage of bacteria for each specimen detected. ResultsThere were 18/40 males (45%) and no postoperative complications. Average age was 72.2 ± 11.8. Overall, 18% (29/160) of standard anaerobic cultures were positive for C. Acnes and 26% (942/160) were positive with genomic (Nextgen) DNA sequencing. When comparing the NextGen results with anaerobic standard cultures, there was a calculated negative predictive value of 85.6% and positive predictive value of 28.6%. Sensitivity of the NextGen was 41.4% and specificity was 77.1%. Bacterial culture rates did not significantly change from the beginning to the end of surgery as demonstrated on standard culturing and the NextGen analysis (P > .05). After treatment with hydrogen peroxide, the standard culturing technique showed no significant difference between the samples; however, there was a significant increase in bacterial burden (12.4%) noted with NextGen analysis (P = .0147). ConclusionNextGen culturing is a novel technique to help identify shoulder prosthetic joint infections. These results show that, NextGen is better at identifying the absence of infection, but has a high false positive rate indicative of its ability to identify contaminants as compared to standard anaerobic culturing methods. The increase in bacterial burden after peroxide treatment noted with NextGen could also be secondary to the test’s increased ability to identify both living and dead bacterial pathogens.

Tacrolimus Pharmacotherapy: Infectious Complications and Toxicity in Organ Transplant Recipients; An Updated Review.

After allogeneic organ transplantation, in order to reduce the risk of rejection, tacrolimus is given. In fact, infection is reported as one of the most common side effects of tacrolimus that might be associated with graft failure. This study aims to review the association between the occurrence of infections due to toxicity following the administration of tacrolimus in organ transplant recipients. Scientific literature on the pharmacotherapy of tacrolimus after organ transplantation, infections, and neurotoxicity were searched using PUBMED.Gov (https://pubmed.ncbi.nlm.nih.gov/), Web of Science, and Scopus (n=108). All articles were screened, and the data associated with the topic of interest was extracted. The primary outcome was infection and neurotoxicity. Total area under the curve exposure, the ratio of parent drug/metabolites of tacrolimus was reported to be correlated with aggressive events such as infection episodes. A trough/dose ratio may demonstrate the net state of immunosuppression and drug-related events. The most frequent infectious complication of tacrolimus after organ transplantation was reported as urinary tract infections (UTIs). Virulent strains of recombinant Listeria monocytogenes, in addition to an increase in bacterial burden in the liver and spleen tissues, were reported in experimental animal studies. Patient survival was significantly lower in recipients with UTIs in the first post-transplant month. A higher degree of immunosuppression was associated with recurrent UTIs and drug-resistant organisms. By inhibiting the cerebral immune system, tacrolimus could cause neurodegeneration. Transplant type, gut dysmotility, acute or chronic condition before transplant surgery, use of azole, antifungal, hematocrit, tacrolimus methods of detection, the total area under the curve, and duration of hospital stay could define the risk of infection through the first month of transplant surgery. In addition, neurological and infectious complications could be associated with the higher amounts of tacrolimus trough levels (C0). Polypharmacy based on tacrolimus, antiviral, and antifungal drugs, in addition to neurotoxicity, could increase the risk of opportunistic infections such as cytomegalovirus within the first year of organ transplantation.

BATF2 enhances proinflammatory cytokine responses in macrophages and improves early host defense against pulmonary Klebsiella pneumoniae infection.

Basic leucine zipper transcription factor ATF-like 2 (BATF2) is a transcription factor that is emerging as an important regulator of the innate immune system. BATF2 is among the top upregulated genes in human alveolar macrophages treated with LPS, but the signaling pathways that induce BATF2 expression in response to Gram-negative stimuli are incompletely understood. In addition, the role of BATF2 in the host response to pulmonary infection with a Gram-negative pathogen like Klebsiella pneumoniae (Kp) is not known. We show that induction of Batf2 gene expression in macrophages in response to Kp in vitro requires TRIF and type I interferon (IFN) signaling, but not MyD88 signaling. Analysis of the impact of BATF2 deficiency on macrophage effector functions in vitro showed that BATF2 does not directly impact macrophage phagocytic uptake and intracellular killing of Kp. However, BATF2 markedly enhanced macrophage proinflammatory gene expression and Kp-induced cytokine responses. In vivo, Batf2 gene expression was elevated in lung tissue of wild-type (WT) mice 24 h after pulmonary Kp infection, and Kp-infected BATF2-deficient (Batf2-/-) mice displayed an increase in bacterial burden in the lung, spleen, and liver compared with WT mice. WT and Batf2-/- mice showed similar recruitment of leukocytes following infection, but in line with in vitro observations, proinflammatory cytokine levels in the alveolar space were reduced in Batf2-/- mice. Altogether, these results suggest that BATF2 enhances proinflammatory cytokine responses in macrophages in response to Kp and contributes to the early host defense against pulmonary Kp infection.NEW & NOTEWORTHY This study investigates the signaling pathways that mediate induction of BATF2 expression downstream of TLR4 and also the impact of BATF2 on the host defense against pulmonary Kp infection. We demonstrate that Kp-induced upregulation of BATF2 in macrophages requires TRIF and type I IFN signaling. We also show that BATF2 enhances Kp-induced macrophage cytokine responses and that BATF2 contributes to the early host defense against pulmonary Kp infection.

The effect of decontamination using quaternary ammonium chloride on the bacterial burden of hospital privacy curtains.

This quasi-experimental study aimed to identify the effect of decontamination using quaternary ammonium chloride (QAC) on bacterial burden on hospital privacy curtains. The objects were the high-touch edges of 66 polyester curtains in inpatient wards. The decontamination was performed daily (n = 22), twice-weekly (n = 22), or not performed (n = 22) for 28 days. The bacterial burden on the curtains was measured based on the number of bacteria, the proportion of curtains with >2.5 colony-forming unit/cm2 , and the proportion of curtains with multidrug-resistant organisms (MDROs). As a result, the daily or twice-weekly decontamination groups showed a significantly lower increase in bacterial burden than the no-decontamination group overall and at all four posttest times. On day 28, daily decontamination showed a lower increase in the number of bacteria (p < 0.001) and proportions of curtains with >2.5 colony form units/cm2 (p < 0.001) than the no-decontamination condition, and in the number of curtains with MDROs than twice-weekly decontamination. In conclusion, decontamination of curtains using QAC helps reduce bacterial burden, and daily decontamination is recommended up to 28 days after installation.

Nitric oxide is a host cue for Salmonella Typhimurium systemic infection in mice

Nitric oxide (NO) is produced as an innate immune response against microbial infections. Salmonella Typhimurium (S. Typhimurium), the major causative pathogen of human gastroenteritis, induces more severe systemic disease in mice. However, host factors contributing to the difference in species-related virulence are unknown. Here, we report that host NO production promotes S. Typhimurium replication in mouse macrophages at the early infection stage by activating Salmonella pathogenicity island-2 (SPI-2). The NO signaling-induced SPI-2 activation is mediated by Fnr and PhoP/Q two-component system. NO significantly induced fnr transcription, while Fnr directly activated phoP/Q transcription. Mouse infection assays revealed a NO-dependent increase in bacterial burden in systemic organs during the initial days of infection, indicating an early contribution of host NO to virulence. This study reveals a host signaling-mediated virulence activation pathway in S. Typhimurium that contributes significantly to its systemic infection in mice, providing further insights into Salmonella pathogenesis and host–pathogen interaction.

1722. In vivo Activity of Imipenem/XNW4107 Human-Simulated Regimen against Serine Carbapenemase-Producing Acinetobacter baumannii and Pseudomonas aeruginosa in the Neutropenic Murine Thigh Infection Model

Abstract Background Imipenem (IPM)/XNW4107 is a novel β-lactam/β-lactamase inhibitor with in vitro activity against serine carbapenemase-producing Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales. Herein, we evaluated the in vivo activity of an IPM/XNW4107 human-simulated regimen (HSR) against clinical OXA-23- and OXA-24-producing A. baumannii as well as KPC- and GES-producing P. aeruginosa using a neutropenic murine thigh infection model. Methods Seven A. baumannii and 4 P. aeruginosa isolates were included. IPM and IPM/XNW4107 MICs (XNW4107 fixed at 8 mg/L) were tested in triplicate by broth microdilution. One thigh of neutropenic ICR mice (6 mice per group) was inoculated with ∼107 CFU/mL bacterial suspensions. HSR that mimicked the clinical exposures of IPM 500 mg q6h alone or in combination with XNW4107 250 mg q6h each as 1 h infusion were developed in the murine model. In efficacy studies, two hours after inoculation, placebo, IPM 500 mg q6h 1 h infusion HSR, or IPM/XNW4107 500/250 mg q6h 1 h infusion HSR were administered subcutaneously. Efficacy was measured as the change in log10CFU/thigh at 24 h compared with 0 h controls. Results Isolates were IPM resistant (MICs 16 - &amp;gt; 64 mg/L). IPM/XNW4107 A. baumannii and P. aeruginosa MIC ranges were 1-16 and 1- 8 mg/L, respectively. Across all examined isolates, 0 h mean ± SD bacterial burden was 5.86 ± 0.32 log10 CFU/thigh. The 24 h increase in bacterial burden was 2.68 ± 0.91 log10 CFU/thigh in the sham controls. IPM HSR monotherapy groups showed mean increase in bacterial burden of 2.34 ± 0.95 log10 CFU/thigh. Bacterial kill with IPM/XNW4107 500/250 mg q6h 1 h infusion HSR ranged from -0.46 ± 1.69 to -3.77 ± 0.15 and -2.33 ± 0.25 to -3.76 ± 0.57 among A. baumannii and P. aeruginosa isolates, respectively. IPM/XNW4107 500/250 mg q6h 1 h infusion HSR produced &amp;gt; 1-log kill against 6/7 examined A. baumannii with the exception of A. baumannii 160 (IPM/XNW4107 MIC 16 mg/L) and 4/4 P. aeruginosa as well as &amp;gt; 2-log kill against 4/7 A. baumannii and 4/4 P. aeruginosa. Conclusion IPM/XNW4107 500/250 mg q6h 1 h infusion HSR showed potent in vivo activity against serine carbapenemase-producing A. baumannii and P. aeruginosa. These data support the consideration of IPM/XNW4107 for the treatment of serious infections due to these organisms in clinical trials. Disclosures Haitao Yuan, PhD, Evopoint Biosciences Co., Ltd: Stocks/Bonds Xiao Liu, PhD, Evopoint Biosciences Co., Ltd: Stocks/Bonds Xi Chen, PhD, Evopoint Biosciences Co., Ltd: Stocks/Bonds Yuchuan Wu, PhD, Evopoint Biosciences Co., Ltd: Stocks/Bonds David P. Nicolau, PharmD, Shionogi: Grant/Research Support Kamilia Abdelraouf, PhD, Evopoint Biosciences Co., Ltd: Grant/Research Support|Venatorx Pharmaceuticals, Inc.: Grant/Research Support.

Toll-like receptor 4 signaling-mediated responses are critically engaged in optimal host protection against highly virulent Mycobacterium tuberculosis K infection

ABSTRACT Toll-like receptors (TLRs) play critical roles in the innate recognition of Mycobacterium tuberculosis (Mtb) by host immune cells. However, controversy has arisen regarding the role of TLR4 in determining the outcomes of Mtb infection. To address this controversy, the function of TLR4 in the induction of an optimal protective immune response against the highly virulent Mtb K-infection was comparatively investigated in C3 H/HeJ (TLR4-deficient mutant) and C3 H/HeN (TLR4-competent wild-type) mice. Interestingly, following Mtb infection, C3 H/HeJ mice showed a more severe disease phenotype than C3 H/HeN mice, exhibiting reduced weight and a marked increase in bacterial burden along with necrotic lung inflammation. Analysis of the immune cell composition revealed significantly increased neutrophils in the lung and significant production of IL-10 accompanied by the impairment of the protective Th1 response in C3 H/HeJ mice. Reducing the neutrophil numbers by treating C3 H/HeJ mice with an anti-Ly6 G monoclonal antibody (mAb) and blocking IL-10 signaling with an anti-IL-10 receptor mAb reduced the excessive lung inflammation and bacterial burden in C3 H/HeJ mice. Therefore, abundant IL-10 signaling and neutrophils have detrimental effects in TLR4-deficient mice during Mtb infection. However, the blockade of IL-10 signaling produced an increase in the CD11bhiLy6 Ghi neutrophil population, but the phenotypes of these neutrophils were different from those of the CD11bintLy6 Gint neutrophils from mice with controlled infections. Collectively, these results show that TLR4 positively contributes to the generation of an optimal protective immunity against Mtb infection. Furthermore, investigating the TLR4-mediated response will provide insight for the development of effective control measures against tuberculosis.

Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.

A mouse model of pulmonary Mycobacteroides abscessus infection

There is no preclinical mouse model to investigate pulmonary Mycobacteroides abscessus (formerly Mycobacterium abscessus) infection in an immunocompetent mouse strain, especially in the context of antibiotic testing and regimen development. We developed a mouse model of pulmonary M. abscessus infection using the aerosolized route of infection that leads to an increase in bacterial burden post- implantation and develops pathology as a result. In this mouse model, treatment with corticosteroid allows for initial proliferation and sustained M. abscessus pulmonary infection and permits evaluation of efficacies of antibiotics. Administration of corticosteroids that permitted higher levels of bacterial burden in the lungs were more likely to have pathology. Treatment of mice with antibiotics administered intranasally or subcutaneously significantly reduced lung M. abscessus burden. In addition to the reference strain, independent clinical isolates of M. abscessus also readily establish infection and proliferate in the lungs of mice in this model.

Expression of human TLR4/MD-2 directs an early innate immune response associated with modest increase in bacterial burden during Coxiella burnetii infection

Abstract Human and mouse TLR4 molecules respond differently to hypo-acylated LPS. The LPS of Coxiella burnetii is hypo-acylated, heavily glycosylated and known to cause a minimal response by human cells. We hypothesized that mice expressing hTLR4 molecules would be more susceptible to C. burnetii infection. Our results suggested that transgenic mice expressing hTLR4 and the human MD-2 adaptor protein (hTLR4/MD-2) generally respond similarly to wild type mice, contrary to our hypothesis. Small differences in bacterial burdens in spleens and/or lungs of infected mice were noted and lung pathology was increased in hTLR4/MD2 compared to wild type mice. Surprisingly, bone marrow chimera experiments indicated that hTLR4/MD-2 expression on non-hematopoietic cells, rather than the target cells for C. burnetii infection, accounted for increased bacterial burden. Very early during infection, cytokines involved in myeloid cell recruitment were detected in the plasma of hTLR4/MD2 mice, but not in wild type mice. This restricted cytokine response was accompanied by early neutrophil recruitment to the lung in hTLR4/MD2 mice. These data suggest that hTLR4/MD-2 may alter early responses to C. burnetii infection. Our results suggest that during C. burnetii infection, the species of origin for TLR4 may be less important than with other pathogens with hypoacylated LPS.

Mycobacterium avium Infection in a C3HeB/FeJ Mouse Model.

Infections caused by Mycobacterium avium complex (MAC) species are increasing worldwide, resulting in a serious public health problem. Patients with MAC lung disease face an arduous journey of a prolonged multidrug regimen that is often poorly tolerated and associated with relatively poor outcome. Identification of new animal models that demonstrate a similar pulmonary pathology as humans infected with MAC has the potential to significantly advance our understanding of nontuberculosis mycobacteria (NTM) pathogenesis as well as provide a tractable model for screening candidate compounds for therapy. One new mouse model is the C3HeB/FeJ which is similar to MAC patients in that these mice can form foci of necrosis in granulomas. In this study, we evaluated the ability of C3HeB/FeJ mice exposure to an aerosol infection of a rough strain of MAC 2285 to produce a progressive infection resulting in small necrotic foci during granuloma formation. C3HeB/FeJ mice were infected with MAC and demonstrated a progressive lung infection resulting in an increase in bacterial burden peaking around day 40, developed micronecrosis in granulomas and was associated with increased influx of CD4+ Th1, Th17, and Treg lymphocytes into the lungs. However, during chronic infection around day 50, the bacterial burden plateaued and was associated with the reduced influx of CD4+ Th1, Th17 cells, and increased numbers of Treg lymphocytes and necrotic foci during granuloma formation. These results suggest the C3HeB/FeJ MAC infection mouse model will be an important model to evaluate immune pathogenesis and compound efficacy.

2388. Efficacy of Humanized Cefiderocol Exposures Over 72 Hours Against a Diverse Group of Gram-Negative Isolates in the Neutropenic Murine Thigh Infection Model

BackgroundPrevious pharmacodynamic (PD) assessments conducted over a 24 hours dosing period have revealed that cefiderocol humanized exposures produced predictable bacterial kill against MDR Gram-negatives with MICs ≤4 mg/L. Our current aim was to evaluate the sustainability of cefiderocol in vivo activity over 72 hours against MDR pathogens. A. baumannii (AB, n = 4), P. aeruginosa (PSA, n = 2), K. pneumoniae (KP, n = 4) and E. coli (EC, n = 2) displaying cefiderocol MICs of 0.5–16 mg/L were used in the neutropenic murine thigh model.MethodsMice received either humanized exposures of cefiderocol equivalent to the clinical dose [2g q8h 3h inf.] or cefepime (FEP) reflective of a 2g q8h 3h inf or vehicle. Efficacy was determined as the change in log CFU at 24, 48 and 72h compared with 0 hours controls. MICs were determined on organisms recovered from both the control and treatment animals.ResultsIn AB, PSA and Enterobacteriaceae (EB) displaying MICs 0.5–8 (n = 9), infected mice given cefiderocol showed reductions of 0.5–3 log CFU at 72 hours. The killing profile observed among these 9 isolates followed a similar trend, demonstrating an initial reduction in bacterial burden at 24 hours which was sustained at 48 hours and 72 hours. As expected based on the PD profile of cefiderocol, no killing was seen with the AB isolate (MIC = 16). While cefiderocol exposure resulted in the killing of the FEP-resistant phenotype of the EB, mice receiving FEP displayed growth similar to controls. Infection with the remaining 2 organisms (EC 462, MIC = 1; KP 531, MIC = 4) resulted in a cumulative increase in bacterial burden over the study duration resulting in 1–2 logs growth following cefiderocol exposure over 72 hours. Retest MICs revealed an increase (≥2 dilutions) compared with control in only 1 animal (1/54 samples or 1.8%) observed in EC 462 at 72 hours. Additional samples from this group (2/3) remained unchanged throughout the study duration. Importantly, the retest MIC for this sample did not exceed the MIC of 4 mg/L.ConclusionThese data show that for isolates demonstrating kill at 24 hours, cefiderocol efficacy was unchanged over the 72 hours treatment period. Despite the MDR profile of the pathogens tested their phenotypic profile remained largely unchanged and adaptive resistance during therapy was not observed.Disclosures M. Tsuji, Shionogi & Co., Ltd.: Employee, Salary. Y. Yamano, Shionogi & Co., Ltd.: Employee, Salary.

TSLP Protects Corneas From Pseudomonas aeruginosa Infection by Regulating Dendritic Cells and IL-23-IL-17 Pathway.

PurposeWe sought to determine the role of epithelium-produced thymic stromal lymphopoietin (TSLP) and its underlying mechanisms in corneal innate immune defense against Pseudomonas (P.) aeruginosa keratitis.MethodsThe expression of TSLP and TSLPR in cultured human corneal epithelial cells (HCECs) and mouse corneas was determined by PCR, Western, and/or ELISA. Cellular localization of TSLP receptor (TSLPR) was determined by whole mount confocal microscopy. TSLP-TSLPR signaling was downregulated by neutralizing antibodies and/or small interfering (si)RNA; their effects on the severity of P. aeruginosa–keratitis and cytokine expression were assessed using clinical scoring, bacterial counting, PMN infiltration, and real-time PCR. The role of dendritic cells (DCs) in corneal innate immunity was determined by local DC depletion using CD11c-DTR mice.ResultsP. aeruginosa–infection induced the expression of TSLP and TSLPR in both cultured primary HCECs and in C57BL/6 mouse corneas. While TSLP was mostly expressed by epithelial cells, CD11c-positive cells were positive for TSLPR. Targeting TSLP or TSLPR with neutralizing antibodies or TSLPR with siRNA resulted in more severe keratitis, attributable to an increase in bacterial burden and PMN infiltration. TSLPR neutralization significantly suppressed infection-induced TSLP and interleukin (IL)-17C expression and augmented the expression of IL-23 and IL-17A. Local depletion of DCs markedly increased the severity of keratitis and exhibited no effects on TSLP and IL-23 expression while suppressing IL-17A and C expression in P. aeruginosa–infected corneas.ConclusionsThe epithelium-expressed TSLP plays a protective role in P. aeruginosa keratitis through targeting of DCs and in an IL-23/IL-17 signaling pathway-related manner.

Systematic Analysis of Two-Component Systems in Citrobacter rodentium Reveals Positive and Negative Roles in Virulence.

Citrobacter rodentium is a murine pathogen used to model intestinal infections caused by the human diarrheal pathogens enterohemorrhagic and enteropathogenic Escherichia coli During infection, bacteria use two-component systems (TCSs) to detect changing environmental cues within the host, allowing for rapid adaptation by altering the expression of specific genes. In this study, 26 TCSs were identified in C. rodentium, and quantitative PCR (qPCR) analysis showed that they are all expressed during murine infection. These TCSs were individually deleted, and the in vitro and in vivo effects were analyzed to determine the functional consequences. In vitro analyses only revealed minor differences, and surprisingly, type III secretion (T3S) was only affected in the ΔarcA strain. Murine infections identified 7 mutants with either attenuated or increased virulence. In agreement with the in vitro T3S assay, the ΔarcA strain was attenuated and defective in colonization and cell adherence. The ΔrcsB strain was among the most highly attenuated strains. The decrease in virulence of this strain may be associated with changes to the cell surface, as Congo red binding was altered, and qPCR revealed that expression of the wcaA gene, which has been implicated in colanic acid production in other bacteria, was drastically downregulated. The ΔuvrY strain exhibited increased virulence compared to the wild type, which was associated with a significant increase in bacterial burden within the mesenteric lymph nodes. The systematic analysis of virulence-associated TCSs and investigation of their functions during infection may open new avenues for drug development.

Detection of Vibrio spp., Salmonella spp., and Shigella spp. among the frozen food samples employing enrichment culture technique

Freezing has long been an established method for food preservation. Freezing temperature may act as a stress factor for microbial cells, transforming the cells into injured or dormant state. Upon inoculation, these debilitated cells cannot grow on solid media and hence produce false negative results. Foods contaminated with injured cells of pathogenic bacterial strains are of potential health risk. Employing enrichment cultivation technique, present study attempted to detect such injured, dormant or viable but non culturable (VBNC) cells in different frozen food samples, collected from local markets and super-shops of Dhaka metropolis. Compared to the conventional cultivation means, the enrichment procedure revealed a significant increase in bacterial burden as well as increase in the pathogenic load. A maximum of 3 log increase in case of total bacterial load while 4 log, 5 log and 2 log increase in case of Vibrio spp., Salmonella spp. and Shigella spp., consecutively were observed. These findings clearly demonstrated the presence of injured cells in frozen foods which could be lethal under normal condition thereby posing public health risk.Stamford Journal of Microbiology, Vol.5(1) 2015: 26-29

PcpA Promotes Higher Levels of Infection and Modulates Recruitment of Myeloid-Derived Suppressor Cells during Pneumococcal Pneumonia.

We used two different infection models to investigate the kinetics of the PcpA-dependent pneumococcal disease in mice. In a bacteremic pneumonia model, we observed a PcpA-dependent increase in bacterial burden in the lungs, blood, liver, bronchoalveolar lavage, and spleens of mice at 24 h postinfection. This PcpA-dependent effect on bacterial burden appeared earlier (within 12 h) in the focal pneumonia model, which lacks bacteremia or sepsis. Histological changes show that the ability of pneumococci to make PcpA was associated with unresolved inflammation in both models of infection. Using our bacteremic pneumonia model we further investigated the effects of PcpA on recruitment of innate immune regulatory cells. The presence of PcpA was associated with increased IL-6 levels, suppressed production of TRAIL, and reduced infiltration of polymorphonuclear cells. The ability of pneumococci to make PcpA negatively modulated both the infiltration and apoptosis of macrophages and the recruitment of myeloid-derived suppressor-like cells. The latter have been shown to facilitate the clearance and control of bacterial pneumonia. Taken together, the ability to make PcpA was strongly associated with increased bacterial burden, inflammation, and negative regulation of innate immune cell recruitment to the lung tissue during bacteremic pneumonia.

IL-6 regulates pro- and anti-inflammatory cytokine production and mortality of Mycobacterium tuberculosis infected type 2 diabetic mice (INC2P.418)

Abstract In this study, to determine whether diabetic (DM) mice are susceptible to Mycobacterium tuberculosis (Mtb) infection, we developed experimentally induced diabetes in wild type C57BL/6 mice with streptozotocin &amp; nicotinamide. We then infected non-DM and DM mice with Mtb H37Rv by aerosol. Around 50% of Mtb infected DM (p=0.05) and 25% of uninfected DM (p=0.07) mice died in 5 to 6 months compared to no deaths in infected control mice. Six months after Mtb infection, there was a statistically significant but marginal increase in bacterial burden in the lungs of Mtb infected DM mice compared to non-DM infected mice (3.1 ± 0.4 x106 vs 0.9 ± 0.1 x 106, p=0.001) suggesting increased mortality was not due to increased bacterial burden. Real time PCR analysis of Mtb infected DM lungs indicated significantly increased pro- and anti-inflammatory cytokine gene expression compared to uninfected DM and infected control mice, including increased levels of IL-6 mRNA. Anti-IL-6 antibody treatment of Mtb infected DM mice enhanced the survival (100% vs 60% survival in isotype control antibody treated, p=0.02) and reduced pro- and anti-inflammatory cytokine production. Dendritic cells (DC), but not other immune cells were the major source for IL-6 in Mtb infected DM mice. NK cells of Mtb infected DM mice further enhanced IL-6 production by autologous DCs. Our results suggest NK-DC interaction enhance IL-6 production which drives the immune pathology and mortality of Mtb infected diabetic mice

Staphylococcus aureusInfection in Humanized Mice: A New Model to Study Pathogenicity Associated With Human Immune Response

Staphylococcus aureus is a common pathogen among humans worldwide, with an increasing prevalence of multidrug resistance. The understanding of virulence factors inducing pathogenicity is still incomplete, and thus far the transfer of results from animal studies into successful clinical trials has been difficult. In this study, we established an S. aureus infection model in mice engrafted with a human immune system, compared it with infected wild-type and nonhumanized mice, and investigated pathogenesis in these models. Staphylococcus aureus infection was aggravated in humanized mice, compared with wild-type or nonengrafted mice. The humanized mice displayed a significantly reduced survival percentage, increased weight loss, and a more-rapid increase in bacterial burden. In addition, S. aureus infection induced T-cell activation, apoptosis, and Fas receptor expression in humanized but not wild-type mice. Our findings demonstrate the different pathogenetic mechanisms in wild-type and humanized mice and the possible benefit of including humanized mice in future studies involving S. aureus as a prior step to human clinical trials.

Antibacterial and pro-resolving mechanisms in lung diseases: role of C-type lectin receptors (INM2P.430)

Abstract Lower respiratory tract infection with bacteria can lead to sepsis development, a complex immune disorder with systemic hyperinflammation. There are currently no effective therapies for sepsis. To understand the function of specific pulmonary components in regulation of immune responses in this immune disorder, we utilize an acute pulmonary bacterial infection model of Klebsiella pneumoniae (KPn). Nosocomial infections with this opportunistic pathogen account for 5-20% of Gram-negative sepsis cases. C-type lectin receptors (CLRs), expressed mainly by myeloid cells, can shape immune responses in diverse pathological conditions. However, their role in development of pneumonic sepsis is unknown. Our preliminary analysis of a panel of CLRs showed an upregulated expression of two CLRs, Clec4d and Clec4e in the lungs of KPn infected mice. While the wild-type mice were able to resolve a sublethal pneumonic KPn infection, the Clec4d-/- and Clec4e-/- mice displayed increased susceptibility with a progressive increase in bacterial burden, hyperinflammatory response and massive accumulation of neutrophils in lungs. Importantly, we show that coordinated function of Clec4d and Clec4e in neutrophils during bacterial pneumonia, and in a chronic lung disease, leads to control of bacterial growth and hyperinflammation by regulating phagocytosis and efferocytosis (clearance of dead cells). Our studies provide novel insights into regulation of inflammatory derangements in immune disorders.