Elsevier

Microbial Pathogenesis

Volume 42, Issue 4, April 2007, Pages 148-155
Microbial Pathogenesis

Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia

https://doi.org/10.1016/j.micpath.2007.01.001Get rights and content

Abstract

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 7×104 CFU of strain 43816 induced 100% mortality in C57BL/6J mice within 5 days post infection, whereas infection with 5×105 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 h post infection. In contrast, IA565-infected mice displayed basal chemokine levels and no detectable bacteria by 24 h 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 the 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.

Introduction

Klebsiella pneumoniae is a gram-negative opportunistic bacterial pathogen primarily infecting immunocompromised individuals who are hospitalized and suffer from severe underlying diseases [1], [2]. K. pneumoniae can cause a range of infections, from mild urinary tract infections to severe septicemia and bacterial pneumonia with mortality rates that may exceed 50% [1], [3]. Pulmonary K. pneumoniae infections are often complicated by multilobular involvement, formation of lung abscesses, and dissemination of bacteria from within the pulmonary airspace into the bloodstream [4], [5]; all of which are accompanied by the characteristic rapidly progressive clinical course. The recent emergence of antibiotic resistant K. pneumoniae strains [5], [6], [7] emphasizes the importance of determining the mechanisms in which K. pneumoniae interacts with the host, bacterial factors that contribute to the disease course, and the role the immune host defense plays in the eventual outcome of the bacterial infection.

Animal models have proven useful in determining host:pathogen interactions during K. pneumoniae infection. Utilizing a murine model of K. pneumoniae pneumonia, IFN-γ, IL-10, IL-12, and TNFα have all been shown to play an important role in mediating lung antibacterial host responses during K. pneumoniae infection [8], [9], [10], [11], [12]. The majority of these studies utilized a single clinical isolate of K. pneumoniae, 43816, prompting the question of whether or not measurable immune responses can be observed employing different K. pneumoniae strains. In this study, K. pneumoniae clinical isolates 43816 and IA565 were used in a murine model of bacterial pneumonia. Our results show that strain IA565 is rapidly cleared from the lungs and failed to induce any animal mortality, in sharp contrast to strain 43816.

While significant advances have been made in the understanding of host responses following infection with K. pneumoniae, very little is known about bacterial virulence factors that can contribute to in vivo pathogenicity. However, both 43816 and IA565 strains express the prototypic virulence factors associated with K. pneumoniae pathogenicity; being capsule, lipopolysaccharide, and type 1 and 3 fimbriae. Of these three, perhaps capsule is the best studied virulence factor. There are over 70 serological types of capsular antigens associated with Klebsiella pneumoniae. Strain 43816 is classified as having a K2 serotype and strain IA565 has a K15 capsular serotype. K. pneumoniae strains displaying in vivo pathogenicity in murine studies most often express capsular serotypes K1 and K2, however this is not an absolute correlation [13]. Capsule switch mutants have been constructed using strains expressing K2 and K21a that have indicated that the genetic background of virulent strains, independent of the capsule serotype, confers significant in vivo murine pathogenicity [14], [15]. These studies concluded that pathogenesis of K. pneumoniae was multifactorial and that capsule can only partially account for in vivo murine virulence.

Since our two strains of K. pneumoniae differ significantly in their ability to cause disease using a murine model of pneumonia, this raised the question of whether or not there are any other Klebsiella-specific genes responsible for its virulence uniquely contained within the genome of strain 43816. We utilized PCR-based suppressive subtractive hybridization to identify putative virulence genes in K. pneumoniae and have identified 9 DNA sequences unique to our pathogenic K. pneumoniae strain 43816.

Section snippets

Increased mortality in 43816 infected mice following pulmonary K. pneumoniae infection

To determine the in vivo pathogenicity of strains IA565 and 43816, mice were infected and overall survival was determined over a 7 day course of infection. Strain 43816 has been previously reported to be a virulent strain of K. pneumoniae [16], [17]. Intratracheal inoculation of 7×104 CFU of strain 43816 into C57BL/6J mice induced mortality within 2–3 days post infection and resulted in 100% mortality by day 5 post infection (Fig. 1). Inoculation of animals with this same dose of strain IA565

Discussion

The use of K. pneumoniae clinical isolates in various animal models has provided valuable information regarding strain dependence in producing respiratory tract infections [24], [25], [26]. In this study, clinical isolates IA565 and 43816 were used to establish an acute pulmonary infection in C57BL/6J mice that closely resembles that of human bacterial pneumonia. K. pneumoniae 43816 induced 100% mortality at doses of 7×104 CFU whereas inoculation of 5×105 CFU of IA565 failed to induce any

Animals

C57BL/6J wild-type mice were purchased from The Jackson Laboratory and housed in specific pathogen-free conditions within the animal care facility at the University of Michigan until the day of sacrifice. All experimental animal procedures were approved by the University Committee on Use and Care of Animals at the University of Michigan.

K. pneumoniae inoculation

K. pneumoniae strain 43816 is a clinical isolate with a K2 serotype [25] (ATCC, Rockville, MD). Strain IA565 is a clinical isolate from the University of Iowa

Acknowledgments

This work was supported in part by grants AI49448 (TAM) and AI050011 (SC) from the National Institutes of Health and a Career Investigator Award from the American Lung Association (TAM).

References (43)

  • L.L. Laichalk et al.

    Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia

    Infect Immun

    (1996)
  • T.A. Moore et al.

    Divergent role of gamma interferon in a murine model of pulmonary versus systemic Klebsiella pneumoniae infection

    Infect Immun

    (2002)
  • T.J. Standiford et al.

    Expression and regulation of chemokines in acute bacterial pneumonia

    Biol Signals

    (1996)
  • T.J. Standiford et al.

    Cytokines in host defense against pneumonia

    J Invest Med

    (1997)
  • K. Mizuta et al.

    Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types

    Infect Immun

    (1983)
  • I. Ofek et al.

    Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a

    Infect Immun

    (1993)
  • K. Kabha et al.

    Relationships among capsular structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae

    Infect Immun

    (1995)
  • J.C. Deng et al.

    Krieg AM and Standiford TJ CpG oligodeoxynucleotides stimulate protective innate immunity against pulmonary Klebsiella infection

    J Immunol

    (2004)
  • T.A. Moore et al.

    Gamma delta-T cells are critical for survival and early proinflammatory cytokine gene expression during murine Klebsiella pneumonia

    J Immunol

    (2000)
  • S.A. Fulton et al.

    Neutrophil-mediated mycobacteriocidal immunity in the lung during Mycobacterium bovis BCG infection in C57BL/6 mice

    Infect Immun

    (2002)
  • S. Knapp et al.

    Alveolar macrophages have a protective antiinflammatory role during murine pneumococcal pneumonia

    Am J Respir Crit Care Med

    (2003)
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