Identification of Klebsiella pneumoniae genes uniquely expressed in a strain virulent using a murine model of bacterial pneumonia
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)
- et al.
Friedlander's pneumonia. A report of 11 cases and appraisal of antibiotic therapy
Dis Chest
(1968) - et al.
Rapidly fatal outcome of bacteremic Klebsiella pneumoniae pneumonia in alcoholics
Chest
(1995) - et al.
Identification and characterization of KvgAS, a two-component system in Klebsiella pneumoniae CG43
FEMS Microbiol Lett
(2003) - et al.
A rapid and simple microfluorometric phagocytosis assay
J Immunol Methods
(1993) - et al.
Bacteriology of hospital-acquired pneumonia
Arch Intern Med
(1986) - et al.
Factors associated with antimicrobial resistance among clinical isolates of Klebsiella pneumoniae: 1-year survey in a French university hospital
Eur J Clin Microbiol Infect Dis
(2004) - et al.
Klebsiella bacteraemia: community versus nosocomial infection
QJM
(1996) Inactivation of antibiotics and the dissemination of resistance genes
Science
(1994)- et al.
Nosocomial outbreak of Klebsiella infection resistant to late-generation cephalosporins
Ann Intern Med
(1993) - et al.
Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia
J Immunol
(1995)