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Published in final edited form as: Microbes Infect. 2008 Aug 13;10(12-13):1283–1290. doi: 10.1016/j.micinf.2008.07.040

Host and microbiota factors that control Klebsiella pneumoniae mucosal colonization in mice

Helen Y Lau a,c, Gary B Huffnagle b, Thomas A Moore b,*
PMCID: PMC2633640  NIHMSID: NIHMS80753  PMID: 18762269

Abstract

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 post-inoculation. 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.

Keywords: Klebsiella pneumoniae, Colitis, Dextran Sulfate, Citrobacter rodentium, Gastrointestinal Tract, Nasal Cavity

1. Introduction

The commensal microbiota is present on body surfaces covered by epithelial cells including those exposed to the external environment. The most abundant microbiota is present on the mucosal surface of the gastrointestinal (GI)-tract and has been shown to play an important role in development of the mucosal immune system. Germ-free (GF) mice that lack commensal enteric bacteria have fewer numbers of intra-epithelial lymphocytes and IgA-producing cells, contain relatively small Peyer’s patches and lack germinal centers [1, 2]. Upon colonization with commensal bacteria, GF mice are indistinguishable from their conventionally reared counterparts [3–5].

The mucosal immune system is responsible for oral and mucosal tolerance to not only food antigens but commensal bacteria as well. A break in mucosal tolerance leading to inappropriate immune responses to commensal bacteria as well as microbial gut overgrowth is hypothesized to be an important contributing factor to inflammatory bowel disease (IBD). This is supported by studies that show spontaneous and induced murine IBD models do not develop pathology in GF conditions [6, 7]. Current models of IBD include treatment with mucosal-injuring agents (dextran sodium sulfate, DSS) [8, 9] and infection with the murine enteric pathogen Citrobacter rodentium [9, 10]. Probiotic treatment of mice has been shown to alleviate acute and chronic colitis. In the C. rodentium-induced colitis model, administration of Lactobacillus rhamnosus, L. acidophilus, or Bacillus subtilis spores has been shown to reduce C. rodentium growth and subsequent pathology [8, 11, 12]. Likewise, Escherichia coli strain Nissle 1917 diminished DSS-induced mucosal injury [13, 14].

Klebsiella pneumoniae is traditionally described as a clinically significant opportunistic bacterial pathogen that can infect immunocompromised individuals who are hospitalized and suffer from underlying diseases [15, 16]. In addition to being a significant clinical pathogen, K. pneumoniae is also a normal component of the upper respiratory and GI-tract microbiota of both humans and mice [17–19]. We have previously characterized two human clinical K. pneumoniae isolates (strains 43816 and IA565) that differ significantly in their in vivo pathogenicity in a murine model of acute bacterial pneumonia [20]. Strain 43816 is a commonly used K. pneumoniae isolate previously shown to be highly pathogenic in mouse models of pneumonia and bacteremia, inducing high levels of mortality [21, 22]. In contrast, strain IA565 is rapidly cleared from the lungs with no observed mortality [20].

In this study, K. pneumoniae strain IA565 (KpIA565) mucosal colonization in the upper respiratory and gastrointestinal tract of wild-type and GF mice was examined to determine the mechanisms responsible for colonization. Upper respiratory tract colonization was found to be independent of host endogenous microbiota. In contrast, extensive GI-tract colonization occurred in the absence of endogenous microbiota and this colonization was regulated by both the presence and microbial complexity of the endogenous microbiota. These data suggest that KpIA565 represents a commensal strain of K. pneumonia capable of colonizing murine mucosal surfaces.

2. Material and Methods

2.1 Animals

C57BL/6J wild type mice were purchased from The Jackson Laboratory and housed in specific pathogen-free (SPF) conditions within the animal care facility at the University of Michigan until the day of sacrifice. GF Swiss Webster mice were maintained in flexible plastic isolators in the GF Mouse Resource Laboratory in the Life Science Institute at the University of Michigan. The University Committee on Use and Care of Animals at the University of Michigan approved all experimental procedures.

2.2 Bacterial Strains

K. pneumoniae strain IA565, described elsewhere [20], and it’s streptomycin resistant derived strain, IA565S were grown in tryptic soy broth (Difco, Detroit, MI) and luria broth (LB) containing 50μg/mL streptomycin (Teknova, Hollister, CA), respectively, overnight at 37 °C. Citrobacter rodentium (ATCC 51459, Rockville, MD) was grown in LB media overnight at 37 °C. Bacterial concentration was determined by measuring the amount of absorbance at 600 nm and compared to a predetermined standard curve. Bacteria were then diluted to the desired concentration in sterile saline. For intranasal inoculation, mice were anesthetized with ketamine and xylazine and a 5–10 μL inoculum of bacteria was given into each nostril followed by a 5 μL flush with sterile saline in each nostril. For oral gavage of bacteria into the GI tract, mice were inoculated with a 24-gauge feeding needle attached to a 1 ml syringe. The syringe containing bacteria was mounted onto a Stepper repetitive pipette (Tridak, Brookfield, CT) to deliver an equal amount of inoculum (50 μL) to each mouse. Aliquots of the inoculum were analyzed for CFU to monitor the dose delivered.

In some experiments, animals were pretreated with the broad-spectrum antibiotic cefoperazone in their drinking water (500 μg/ml) for 4 days. Mice were then placed on antibiotic-free drinking water for 24 hours prior to K. pneumoniae inoculation.

2.3 Inducing murine colitis for models of inflammatory bowel disease

For the C. rodentium model of murine colitis, C57BL/6J mice were gavaged 5 consecutive days with 108–1010 CFU of KpIA565S at day minus 2, minus 1, 0, 1, and 2. At day 0, some of those KpIA565S treated mice and additional wild-type mice were gavaged with 108 CFU of C. rodentium. A group of KpIA565S treated only mice and C. rodentium only infected mice were used as controls. At day 14, animals were sacrificed and their small and large intestine and cecum were harvested for CFU analysis.

For the DSS induced model of murine colitis, C57BL/6J mice were gavaged 5 consecutive days with 108–109 CFU of KpIA565S at day minus 2, minus 1, 0, 1, and 2. At day 0, some of those KpIA565S treated mice received 3 % w/v DSS molecular weight 36,000–50,000 (ICN Biomedicals, Inc., Irvine, CA) in their drinking water ad libitum for 7 days. A group of KpIA565S treated only mice and 3 % DSS only treated mice were used as controls. At day 7, animals were sacrificed and their small and large intestine and cecum were harvested for CFU analysis.

2.4 Whole organ homogenates for CFU analysis

At designated time points, mice were euthanized by inhalation of CO. Excised whole organs were homogenized using a tissue homogenizer (Biospec Products, Bartlesville, OK) in 1 mL PBS for nasal cavity contents and 1 mL distilled water for small and large intestine and cecum. For organ CFU determination, a small aliquot of organ homogenate was serially diluted and plated on blood agar plates for KpIA565 CFU assessment and LB-Streptomycin (200 μg/ml) agar for KpIA565S CFU assessment. Plates were incubated for 24 hours at 37 °C and colonies counted. Colonies at 24 hours were uniformly mucoid in appearance. Random isolated colonies were biotyped using Enterotube II tubes (BD Diagnostics). All colonies tested were K. pneumoniae.

2.5 Histology

The colon of mice were removed, cut longitudinally and washing in 1x PBS, organized in a Swiss roll fashion, and placed in 4 % formalin. Paraffin sections were stained with hematoxylin and eosin.

2.6 Statistical Analysis

Statistical significance was determined using the unpaired, two-tailed student t test, ANOVA for multiple group comparisons using the Student-Newman-Keuls post-test, and Fisher’s Exact Test. Where appropriate, the unpaired, non-parametric Mann-Whitney test was utilized. Calculations were performed using InStat 3 (GraphPad Software, San Diego, CA).

3. Results

3.1 KpIA565 upper respiratory tract colonization is independent of host factors and the microbiota

Previously, we have reported that K. pneumoniae strain IA565 is rapidly cleared from lower lung airspaces following intratracheal inoculation without any subsequent mortality [20]. We wished to determine the ability KpIA565 to colonize the mucosal surfaces of the nasal cavity. C57BL/6J mice were intranasally inoculated with 1×106 CFU and the kinetics of nasal cavity colonization determined at weeks 1, 2, and 3 post-inoculation (Fig. 1A). Although lower than seen at week 1, KpIA565 was consistently detected in the nasal cavity at 3 weeks post-inoculation. These data indicate that KpIA565 is able to stably colonize the murine nasal cavity.

Figure 1. KpIA565 nasal cavity colonization in wild-type and germ-free mice.

Figure 1

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(A) C57BL/6J mice were inoculated with 1×106 CFU of KpIA565 and the nasal cavity CFU was determined. Data were generated from two independent experiments for a total of 10 mice per group. (B) C57BL/6J, Swiss Webster, and Swiss Webster Germ-Free mice were intranasally inoculated with 1×106 CFU of KpIA565 and the nasal cavity was harvested at the indicated time points for CFU determination. Data were generated from two independent experiments with 4–10 mice per group. Nasal cavity bacterial numbers are for the entire organ and are presented as the bacterial CFU mean +/− SEM. LOD, limit of detection.

The murine nasal cavity is colonized with endogenous microbiota. To determine whether this microbiota can influence or regulate K. pneumoniae colonization, Swiss Webster GF mice were intranasally inoculated with KpIA565 and colonization levels determined on days 1, 7, and 21 post-inoculation (Fig. 1B). Low-level nasal cavity colonization in wild-type mice was independent of genetic background as C57BL/6J and Swiss Webster mice were equally colonized through day 21. Of interest, the absence of endogenous microbiota in GF animals did not impact KpIA565 nasal cavity colonization. Collectively, these data indicated that KpIA565 could persistently colonize the nasal cavity of both wild-type and GF mice, indicating that upper respiratory tract colonization with KpIA565 is independent of endogenous microbiota. It is worth noting that KpIA565 is rapidly cleared from the lungs of inoculated animals, with no detectable bacteria present by 24 hours post-inoculation [20].

3.2 KpIA565 gastrointestinal colonization is limited in the presence of endogenous microbiota

Endogenous microbiota have been shown to provide colonization resistance to exogenously administered microorganisms. The broad-spectrum antibiotic cefoperazone (third generation cephalosporin) has been shown to reduce colonization resistance in mice, thus increasing outgrowth of exogenous microorganisms [23]. In our first series of experiments, we wished to determine whether KpIA565S would be able to colonize the GI-tract at all. We therefore pretreated animals with cefoperazone in the drinking water for 4 days followed by 24 hours of antibiotic-free water in order to reduce the endogenous microbiota-mediated colonization resistance. To determine the ability of KpIA565 to colonize the murine GI-tract, a streptomycin resistant derivative of IA565 (IA565S) was generated. Mice were then orally gavaged with 1×107 CFU KpIA565S on 2 consecutive days. Bacterial colonization of the small and large intestine and the cecum was assessed at days 6, 13, and 20 post-gavage by serial dilution of GI-tract contents on streptomycin-containing LB agar plates (Fig. 2A). KpIA565S colonization was observed throughout the GI-tract, with higher levels in the cecum and large intestine. Steady-state levels were achieved by day 13 and persisted though day 20.

Figure 2. KpIA565S gastrointestinal tract colonization in wild-type and germ-free mice.

Figure 2

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(A) C57BL/6J mice were orally gavaged with 1×107 CFU of KpIA565S on two consecutive days following 4 days of cephoperazone treatment. On days 6, 13, and 20 post initial gavage, the small and large intestine and cecum were harvested for CFU analysis. Data were generated from one experiment with 4 mice per group. (B) Swiss Webster wild-type and germ-free mice were intranasally inoculated with 1×106 CFU of KpIA565S. Data were generated from 2 independent experiments with 10 mice per group. GI-tract organs were harvested on day 1, 7 and 21-post inoculation and assessed for CFU. Data are presented as bacterial CFU mean +/− SEM. Values were statistically significant at all time points and GI-tract organs (p < 0.001).

Upper respiratory tract colonization by KpIA565 was not enhanced in the absence of nasal microbiota, therefore we next determined whether GI tract colonization of GF mice was influenced by endogenous GI-tract microbiota. Since GF mice have no endogenous microbiota, these animals and their Swiss Webster wild-type controls were not pretreated with cefoperazone prior to K. pneumoniae inoculation. Mice were intranasally inoculated to allow monitoring of nasal cavity and GI-tract colonization. It has been well demonstrated that microorganisms administered intranasally largely end up being swallowed, with a majority of the inoculum being rapidly detected in the GI-tract [24, 25]. Swiss Webster wild-type and GF mice were intranasally inoculated and GI-tract colonization was analyzed at day 1, 7 and 21 post-challenge. Unlike in the nasal cavity (Fig. 1B), the absence of the GI-tract microbiota resulted in significantly increased colonization levels of KpIA565S in the GI-tract (Fig. 2B, conventional versus GF at each time point in each organ, p < 0.001).

Of interest, the level of long-term (21 days) GI-tract colonization by KpIA565 in non-antibiotic treated wild-type mice was similar to that seen in mice pre-treated with cefoperazone (compare Fig. 2A and Fig. 2B). This suggests that this strain of K. pneumoniae is able to successfully “compete” with the endogenous microbiota and is able to colonize the GI-tract, supporting the contention that KpIA565 represents a commensal strain of K. pneumoniae.

3.3 KpIA565S GI colonization does not alleviate acute murine colitis

Since KpIA565 was able to successfully colonize the GI-tract without the need for antibiotic pretreatment, mice in the following series of experiments examining the impact of KpIA565 colonization during colitis were not antibiotic treated prior to K. pneumoniae inoculation. We examined the impact of K. pneumoniae GI-tract colonization on non-infectious DSS-induced acute colitis. Mice were gavaged with KpIA565S and administered 3 % DSS in their drinking water for 7 days. A group of KpIA565S treated only mice and 3 % DSS only mice were used as controls. On day 7, animals were sacrificed and colons were removed for histological analyses to visualize the degree of inflammation. The colons of untreated (Fig. 3A) and KpIA565S treated alone mice (Fig. 3B) display characteristics of a normal colonic mucosa with the crypts being straight, well-defined, and sitting on the muscularis mucosae (the lighter pink staining regions). As seen in Fig. 3C, the colons from DSS-treated mice exhibited inflammatory cell infiltrate (black arrowheads), severe submucosal edema (line with arrow), and crypt destruction and elongation (double headed arrows). GI-tract colonization with KpIA565S did not affect the histological outcome of DSS treatment; colons from these animals displayed evidence of colitis that was indistinguishable from DSS treatment alone (Fig. 3D).

Figure 3. Representative colon histology of mice with induced colitis.

Figure 3

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Swiss rolls displaying the normal colonic mucosa were prepared from wild-type (A) and KpIA565S gavaged mice (B). Colon tissue for 3 % DSS (C) and 3 % DSS and KpIA565S (D) treated mice were prepared at 7 days post 3 % DSS treatment. Double-headed arrows show crypt destruction and elongation. Black arrows indicate edema. Black arrowheads indicate leukocyte infiltrate. Left-hand column images are 40X magnification while right-hand column images are at 1000X magnification.

As KpIA565S colonization had no impact on DSS-induced inflammation, we next examined the impact of K. pneumoniae GI-tract colonization on intestinal inflammation in C. rodentium infected mice. As expected, C. rodentium infected mice developed diarrhea by days 3–4 post treatment. Oral gavage of KpIA565S into C. rodentium infected mice did not reduce or eliminate diarrhea (data not shown). Similar to DSS-induced colitis, colonization with KpIA565S did not affect the histological outcome C. rodentium-infected mice (data not shown). Both animal groups displayed heavy inflammatory cell infiltrate and colonic hyperplasia. Thus, strain KpIA565S did not have any effect on the disease progression and outcome in either DSS or C. rodentium treated mice.

3.4 Non-infectious DSS-induced inflammation promotes GI-tract outgrowth of KpIA565

To determine the effect of GI-tract inflammation on KpIA565S colonization, we assessed K. pneumoniae GI-tract bacterial numbers in C. rodentium-infected or DSS-treated animals. C. rodentium infection-induced inflammation had no impact on GI-tract colonization with KpIA565; bacterial levels of KpIA565S were similar in the KpIA565S alone and KpIA565S plus C. rodentium groups (Fig. 4A). Similarly, KpIA565S colonization had little overall effect on C. rodentium bacterial numbers; similar small and large intestine-associated bacterial levels of C. rodentium were seen in the C. rodentium alone and IA565S plus C. rodentium group (Fig. 4B). In sharp contrast to animals undergoing C. rodentium-induced inflammation, the GI-tract K. pneumoniae colonization were significantly elevated in the DSS plus KpIA565S group when compared to the KpIA565S alone group, suggesting that the altered GI-tract microenvironment resulting from DSS-induced intestinal inflammation favors the outgrowth of K. pneumoniae (Fig. 4C).

Figure 4. KpIA565S gastrointestinal tract colonization in mice with colitis.

Figure 4

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(A) KpIA565 CFU’s from C57BL/6J mice infected with C. rodentium with or without KpIA565S treatment at day 14 post C. rodentium infection. (B) C. rodentium CFU’s from C57BL/6J mice infected with C. rodentium with or without KpIA565S treatment at day 14 post C. rodentium infection. (B) C57BL/6J mice were given 3 % DSS with or without KpIA565S treatment and sacrificed at day 7 post DSS treatment. GI-tract organs were harvested and assessed for bacterial CFU. Data were generated from 2 independent experiments with 8 mice per group and presented as bacterial CFU mean +/− SEM.. * p < 0.01.

4. Discussion

K. pneumoniae can be isolated from the upper respiratory and GI-tracts of both man and mouse and thus is considered to be a normal component of the microbiota at these mucosal sites [17–19, 26]. Normally, the lower airway (lungs) is kept sterile due to effective host defense mechanisms. Deposition of K. pneumoniae into the lower airspace can result in a severe pyogenic infection with high mortality rates without therapeutic intervention. Thus, K. pneumoniae is a considered to be a clinically significant opportunistic pathogen and is a leading cause of nosocomial and community-acquired gram-negative bacterial pneumonia. We have previously identified a strain of K. pneumoniae (KpIA565) that is rapidly cleared from the lungs of immunocompetent and immunosuppressed mice [20]. Here we report that KpIA565 can stably and persistently colonize the upper airway (nasal cavity) of wild-type mice for up to 3 weeks post inoculation (Fig. 1A). KpIA565 was also able to stably colonize the GI-tract of wild-type mice for up to 3 weeks post-inoculation (Fig. 2A). These results, coupled with the observation that K. pneumoniae is part of the normal microbiota of humans and mice, suggest that KpIA565 represents a commensal strain of K. pneumoniae.

It should be noted that KpIA565 colonization of the GI-tract occurred at relatively similar levels regardless of whether the mice were pretreated with oral cefoperazone prior to K. pneumoniae inoculation. A major protective role of the endogenous microbiota is to provide colonization resistance to exogenous pathogenic organisms. Cefoperazone has been shown to suppress this resistance, allowing growth of pathogenic bacteria. KpIA565 was able to successfully colonize the murine GI-tract without antibiotic pretreatment, in support of this strain representing a commensal rather than pathogenic strain of K. pneumoniae. Indeed, we have previously reported that KpIA565 was unable to induce pulmonary infection in wild-type or immunosuppressed mice and was rapidly cleared from lungs within 24 hours [20].

Stable GI-tract colonization by KpIA565 prompted us to investigate whether this commensal strain of K. pneumoniae could function as a probiotic organism eliciting health benefits during murine colitis. In murine DSS-induced colitis, oral gavage of a non-pathogenic commensal strain of E. coli (Strain Nissle 1917) in DSS-treated mice resulted in alleviation of colonic damage and body weight loss compared to DSS treatment alone [13, 14]. Administration of Bacillus subtilis spores, Lactobacillus rhamnosus, or L. acidophilus attenuated the effect of C. rodentium induced colitis as measured by decreased enteropathology in the colonic tissue and decreased levels of C. rodentium [8, 11, 12, 27]. However, oral administration of KpIA565S did not prevent or reduce diarrhea, weight loss (data not shown) or intestinal inflammation (Fig. 3) in either DSS-treated or C. rodentium-infected animals, indicating that KpIA565 does not behave like a probiotic organism in these models.

Interestingly, GI-tract levels of KpIA565 in DSS treated mice were significantly higher than those in DSS only treated mice (Fig. 4C). In contrast, C. rodentium infection did not alter KpIA565S CFU titers in the GI tract (Fig. 4A). This may be attributed to differences in initiation of inflammation during DSS and C. rodentium-induced colitis. The significant increase in KpIA565S colonization during DSS-induced inflammation may be due to epithelial cell damage not present during C. rodentium-induced inflammation, possibly creating an environment conducive for KpIA565S intestinal overgrowth.

Recently it has been reported that host-mediated inflammation resulted in overgrowth of Enterobacteriaceae family members in the GI-tract [28]. Using both DSS and C. rodentium models, host inflammation resulted in altered colonic microbiota with an outgrowth of the aerotolerant Enterobacteriaceae family. Our results using DSS are in agreement as increased K. pneumoniae growth was observed in mice receiving DSS in their drinking water for 7 days (Fig. 4C). In contrast, we did not observe increased K. pneumoniae growth during infection with C. rodentium. It must be noted however, that we were specifically measuring growth of K. pneumoniae. It is quite possible that the Enterobacteriaceae family as a whole was increased in our model of C. rodentium infection and would thus be in agreement with this report.

In the absence of the normal microbiota (GF mice), KpIA565 colonization was significantly increased in the GI-tract while nasal cavity colonization was similar to that seen in wild-type mice. There are several possibilities to explain this interesting observation. A bacterial genetic factor(s) could be limiting KpIA565 nasal cavity growth independent of the presence or absence of endogenous microbiota. Variation in oxygen tolerance of anaerobes has been reported to be related to levels of superoxide dismutase, an enzyme that neutralizes superoxide radicals generated from molecular oxygen [29]. K. pneumoniae is a facultative anaerobe but growth in the presence of high oxygen levels could be limiting growth in the nasal cavity of both wild-type and GF mice, imposing a colonization threshold for this bacterium. It has been reported that certain terminal carbohydrate structures in the mucin of the murine nasal cavity can dictate susceptibility to bacterial colonization [30]. Thus, nasal cavity colonization, even in the absence of normal microbiota, may be limited by adhesion site accessibility. In contrast, many microbes have been shown to monocolonize the GI-tract at levels approaching 109 CFU/g tissue [31–33]. While the CFU levels of normal microbiota are not quantifiable because of culturing limitations, these studies suggest that the GI-tract is capable of accommodating a maximum microbiota load.

Collectively, these data indicate that K. pneumoniae strain IA565 is a murine commensal organism capable of stably and persistently colonizing the upper respiratory and GI-tract. In two models of GI-tract inflammation, KpIA565 was shown to lack beneficial probiotic activity. Host-mediated inflammation induced by DSS-treatment, but not C. rodentium infection, resulted in excessive KpIA565 outgrowth throughout the GI-tract, suggesting that K. pneumoniae growth in the GI-tract can be modulated by intestinal epithelial damage.

Acknowledgments

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

We thank Dr. John Kao (Department of Internal Medicine) for his help with murine colitis models and Dr. Kate Eaton (Unit for Animal Laboratory Medicine) for the Swiss Webster Germ Free mice.

Footnotes

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