Summary Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free‐living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close association with Legionella spp. in the aquatic environment. The putative role of biofilms and amoebae in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems and in controlling exposure to Legionella spp. and similar pathogens.

Klebsiella pneumoniae is a gram negative bacterium of significant clinical importance. This study examines the differential pulmonary host anti-bacterial responses towards two clinical isolates of K. pneumoniae. Intratracheal inoculation with 7×10 4 CFU of strain 43816 induced 100% mortality in C57BL/6J mice within 5 days post infection, whereas infection with 5×10 5 CFU of strain IA565 resulted in 100% survival. Infection with strain 43816 resulted in significant pulmonary and peripheral blood bacterial burden and induction of the chemokines MIP-2, KC and MCP-1 by 24 hours post infection. In contrast, IA565-infected mice displayed basal chemokine levels and no detectable bacteria by 24 hours post inoculation were isolated from lungs or peripheral blood. These data indicate an apparent lack of pathogenicity of strain IA565. Since little is known about Klebsiella-specific virulence genes, we have utilized PCR-based genomic DNA and cDNA suppressive subtractive hybridization and identified nine DNA sequences unique to a pathogenic strain of K. pneumoniae 43816. These sequences were highly homologous to enteric bacterial genes regulating iron uptake, fimbrial-mediated adhesion, energy production and conversion, transcriptional regulation, signal transduction, restriction endonuclease activity, and membrane transport.

Klebsiella pneumoniae is both an opportunistic pathogen and a commensal organism. We have previously reported that K. pneumoniae strain IA565 (KpIA565) is nonpathogenic in a murine model of acute pneumonia. In this study, KpIA565 was inoculated into wild-type mice and found to stably colonize and persist in the nasal cavity and gastrointestinal tract of mice for up to 3 weeks postinoculation. Intranasal inoculation of wild-type or germ-free mice with KpIA565 resulted in similar bacterial levels in the nasal cavity, suggesting KpIA565 nasal colonization is independent of normal nasal microbiota. In contrast, KpIA565 gastrointestinal tract colonization was significantly higher in germ-free mice than in wild-type mice, indicating that members of the endogenous microbiota regulate KpIA565 colonization. In the presence of non-specific dextran sodium sulfate-induced inflammation, KpIA565 gastrointestinal tract colonization was significantly higher when compared to non-DSS treated mice. Interestingly, KpIA565 colonization was unaffected by Citrobacter rodentium-induced gastrointestinal tract inflammation. However, gastrointestinal tract colonization with K. pneumoniae strain IA565 had no impact on the inflammatory histopathology in either colitis model. This study is the first to identify and describe mechanisms influencing the growth and behavior of a murine commensal strain of K. pneumoniae.

Klebsiella pneumoniae is a leading cause of gram-negative bacterial pneumonia, often resulting in bacteremia concurrent with the localized pulmonary infection. The beneficial role of tumor necrosis factor (TNF)-alpha during pulmonary infection has been well documented; however, consequences of TNF-alpha production during systemic bacterial infection are controversial. A murine model of K. pneumoniae was developed to address this important issue. Liver-associated TNF-alpha mRNA was induced within 30 min after intravenous bacterial inoculation and remained elevated through 6 h before returning to near-baseline at 24 h postinfection. Intravenous K. pneumoniae infection induced liver cellular injury that was completely ablated when mice were pretreated with a neutralizing anti-TNF-alpha antibody. Interestingly, this reduction in liver injury failed to translate into improved survival. Mice receiving anti-TNF-alpha continued to succumb to the infection even out to day 10 postinfection. Bacterial clearance after TNF-alpha neutralization was significantly impaired at later time points during infection. Correlating with impaired bacterial clearance was diminished production of liver-associated MIP-2, MIP-1alpha, MCP-1, and interferon-gamma. Further evidence of diminished antibacterial immune responses was noted when the activational status of splenic natural killer cells in anti-TNF-alpha-treated mice was examined 24 h postinfection. Natural killer cells displayed decreased CD69 expression. Combined, these data indicate that the beneficial effects of TNF-alpha during systemic K. pneumoniae infection outweigh the detrimental effects of TNF-alpha-mediated hepatocyte cellular injury. Anti-TNF-alpha therapy, although preventing liver injury during blood-borne bacterial infection, results in a dampened anti-bacterial host response, resulting in decreased bacterial clearance and overall survival.

Klebsiella pneumoniae is a leading cause of pneumonia due to gram-negative bacteria. A significant clinical complication of pulmonary infection with K. pneumoniae is peripheral blood dissemination, which results in a systemic infection coincident with the localized pulmonary infection. This study describes the critical importance of tumor necrosis factor (TNF) receptor 1 (TNFR1)-mediated signaling during K. pneumoniae bacteremia. TNFR1-deficient mice displayed a significantly increased mortality rate after intravenous inoculation. Unexpectedly, this increased mortality occurred in the absence of either increased bacterial burden or increased liver injury. However, excessive production of proinflammatory cytokines, including TNF-alpha , was observed in TNFR1-deficient mice, compared with that observed in infected C57BL/6 mice, which suggests that production was dysregulated in the absence of TNFR1 signaling. In contrast, other experiments examined the effect of immunotherapy with anti-TNF-alpha during K. pneumoniae bacteremia. Administration of a neutralizing anti-TNF-alpha antibody completely ablated K. pneumoniae-induced liver injury. This reduction in liver injury failed to translate into an improved survival rate, because mice died of the infection as late as 10 days after infection. Bacterial clearance after neutralization of TNF-alpha was significantly impaired at later time points during infection. Diminished production of liver-associated cytokines and chemokines correlated with impaired bacterial clearance, which suggests that antibacterial immune responses were dampened. These data indicate that the antibacterial host response is dysregulated in mice lacking TNFR1 or TNF-alpha bioactivity.