A Mouse Model of Acinetobacter baumannii-Associated Pneumonia

Using a Clinically Isolated Hypervirulent Strain

Greg Harris,a Rhonda Kuo Lee,a Christopher K. Lam,b Gregory Kanzaki,b Girishchandra B. Patel,a H. Howard Xu,b Wangxue Chena,c
Vaccine Program, Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canadaa; Department of Biological Sciences, California State
University, Los Angeles, Los Angeles, California, USAb; Department of Biology, Brock University, St. Catharines, Ontario, Canadac

Acinetobacter baumannii is an important emerging pathogen in health care-acquired infections and is responsible for severe
nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor
colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h postintranasal inoculation. Infected mice showed severe
bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of
C57BL/6 and BALB/c mice succumbed to 108 CFU of LAC-4 inoculation within 48 h. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice
from a lethal (100 100% lethal dose) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of
therapeutic agents and vaccines against A. baumannii pneumonia in humans.

cinetobacter baumannii infection has recently emerged as a

major cause of health care-associated (hospital- and community-acquired) infections worldwide (1). The overall 30-day mortality of Acinetobacter infection can be as high as 49%, with the
respiratory tract being an important portal of entry (2). Indeed,
the United States National Nosocomial Infections Surveillance
data indicate that Acinetobacter infections were responsible for 7%
of intensive care unit (ICU) nosocomial cases of pneumonia in
2003 (3). Moreover, A. baumannii infections have become increasingly difficult to treat because of the rapid development of
resistance to multiple antibiotics by the pathogen (4). Therefore, there is an urgent need for the development of novel therapeutics and other intervention strategies to combat this important pathogen.
Animal models are crucial to the development of new therapeutics and vaccines and play critical roles in the assessment of the
efficacy and safety of the new products before they enter human
clinical trials. Over the years, a number of animal models of A.
baumannii pneumonia have been developed, with the mouse being the most widely used model (5). However, most laboratory
strains and clinical isolates of A. baumannii do not infect immunocompetent mice well and induce only a self-limiting pneumonia with no or very limited local bacterial replication and systemic
dissemination, even when a large inoculum is used (610). To
overcome these shortcomings, several laboratories use immunocompromised (such as neutropenic) mice or treat mice with mucin or agar to increase host susceptibility to A. baumannii and
bacterial virulence. Despite their limitations, these models have
been used for many years and have been instrumental in studies of
disease pathogenesis and product development (7, 9, 1121).
However, these models fail to mimic the natural course of human
infection and generally are not suitable for studying the host response to the infection or evaluating antimicrobials that involve
the host immune system.

August 2013 Volume 57 Number 8

The aim of this study was to evaluate the ability of several

clinical isolates and laboratory strains of A. baumannii to reproducibly infect commonly used laboratory mouse strains (BALB/c
and C57BL/6) and to cause disease that would more closely resemble the spectrum of the human infections. We identified one clinical isolate (LAC-4) that caused 100% mortality in conventional,
immunocompetent BALB/c and C57BL/6 mice when intranasally
(i.n.) infected at a dose of 108 CFU. To test the utility of our model,
we also assessed the efficacy of imipenem and tigecycline against a
lethal intranasal challenge of A. baumannii.
MATERIALS AND METHODS
Mice. Eight- to 12-week-old, specific-pathogen-free, male or female
C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). The animals were maintained and
used in accordance with the recommendations of the Canadian Council
on Animal Care Guide to the Care and Use of Experimental Animals, and
experimental procedures were approved by the institutional animal care
committee.
A. baumannii isolates and species confirmation. The clinical isolates
and ATCC strains used in the study are listed in Table 1. The species of all
Los Angeles County (LAC) isolates were further confirmed by DNA sequencing analysis of the intergenic spacer (ITS) between 16S and 23S
rRNA genes based on the methods of Chang et al. (22). Sequences were
analyzed through comparison of the ITS sequence to those of multiple
species of the Acinetobacter genus considered closest to the A. calcoaceticus-A. baumannii complex, including the A. baumannii strains AYE,

Received 3 May 2013 Accepted 12 May 2013

Published ahead of print 20 May 2013
Address correspondence to Wangxue Chen, wangxue.chen@nrc.gc.ca.
Copyright 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00944-13

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TABLE 1 A. baumannii isolates and strains used in the present study and initial bacterial burden screens following their i.n. inoculation in C57BL/6
mice
Bacterial burdenb
(log10 CFU/organ)

Strain/isolate

Strain/isolate reference

Source

Multidrug
resistancea

Lung

Spleen

LAC-1
LAC-4
LAC-5
LAC-6
LAC-7
LAC-11
LAC-15
LAC-16
AYE
SDF
NYB (isolate B)
ATCC 19606
ATCC 17978
ATCC 17961
15839 (ATCC item 202080)

23
23
23
23
23
23
23
23
36
36
37
23
23
38
23

Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Clinical outbreak
Human body lice
Clinical isolate
Clinical isolate; type strain
Clinical isolate; reference strain
Clinical isolate; reference strain
Clinical isolate

Not determined

6.5 0.9***
8.1 0.8***
5.6 0.4**
6.0 0.3***
4.7 0.1
5.0 0.1
6.1 1.0***
5.2 0.2
3.9 0.4
3.7 0.4
4.9 0.1
2.9 0.3
4.0 0.3
5.2 0.2
5.3 0.3

3.1 1.1
6.6 0.6***
1.5 0.2
3.0 1.0
1.9 0.5
1.3 0.0
3.1 1.8
1.8 0.4
2.0 0.3
1.5 0.3
1.4 0.2
1.4 0.2
1.8 0.2
1.3 0.0
1.3 0.0

a
Multidrug resistance of the strains against a panel of 17 known antibiotics was determined using the broth microdilution protocols of the Clinical and Laboratory Standards
Institute as described previously (23). , multidrug resistant (resistant to three or more different classes of antibiotics); , not multidrug resistant.
b
Female C57BL/6 mice (n 3) were intranasally inoculated with 2 107 CFU of various strains of A. baumannii. The mice were sacrificed 24 h later, and the bacterial burdens
in the lungs and spleen were determined by quantitative bacteriology. Data are presented as means SD. The detection limit for bacterial burdens was 1.3 log10 CFU/organ. P
0.01 (**) and P 0.001 (***) versus results for ATCC 17961.

ATCC 17978, and ATCC 19606 (strain BCRC 10591), the A. calcoaceticus
strain LMG 1046, the genomic species 3 strain LMG 1035, and the
genomic species 13TU strain BCRC 15417.
In vitro antimicrobial susceptibility testing. An antimicrobial susceptibility profile of select clinical isolates and laboratory strains against a
panel of 17 antimicrobials was determined using the broth microdilution
protocols of the Clinical and Laboratory Standards Institute as described
previously (23).
Intranasal A. baumannii inoculation and sample collections. For
intranasal (i.n.) inoculation of mice, fresh inocula were prepared for each
experiment from frozen stocks of A. baumannii isolates as previously described (7). Mice were anesthetized by intraperitoneal (i.p.) injection of
xylazine and ketamine and then inoculated intranasally with appropriate
numbers of various A. baumannii isolates in 50 l of saline. Actual inocula
in each experiment were determined by plating 10-fold serial dilutions on
brain heart infusion agar plates. The clinical appearance of the mice was
monitored and scored as described previously (7). Groups of three to six
infected mice were sacrificed 0, 4, and 24 h postinoculation (hpi). The
relevant organs (such as lungs, spleens, and lymph nodes) were aseptically
removed and used for quantitative bacteriology or histopathology. In
some experiments, blood samples were collected for serum separation and
lungs were lavaged, as described below, to collect bronchoalveolar lavage
(BAL) fluid.
Quantitative bacteriology and histopathology. Lungs and spleens
were homogenized in sterile saline using aerosol-proof homogenizers.
Lymph nodes were pressed through a 70-m cell strainer (BD Falcon,
Mississauga, Ontario) in sterile saline. Aliquots (100 l) of 10-fold serial
dilutions of the homogenates were cultured on brain heart infusion agar
plates to quantify the number of viable A. baumannii organisms in the
respective organs (7). In some experiments, blood samples were similarly
cultured. For histopathology, lungs, spleens, livers, hearts, and kidneys
were fixed immediately in 10% neutral buffered formalin and processed
by standard paraffin embedding methods (7). Sections were cut 4 m
thick, stained with hematoxylin-eosin (HE) (Department of Laboratory
Medicine, University of Ottawa, Ottawa, Canada), and examined by light
microscope.
BAL fluid. Lungs were lavaged five times with 1.0 ml saline supplemented with 3 mM EDTA and 1% fetal bovine serum as previously de-

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scribed (24). The total number of cells in BAL fluid was determined with
a hemacytometer, and differential cell counts were determined by examining 200 cells on cytospin slides (Cytospin 3; Shandon, Pittsburgh, PA)
stained with Hema-3 (Fisher Scientific, Kalamazoo, MI). The lavage fluid
was centrifuged at 3,200 g for 7 min, and the supernatant were collected,
supplemented with protease inhibitors (Complete protease inhibitor
cocktail tablets; Roche Applied Sciences, Laval, Quebec, Canada), and
stored at 80C.
Clinical blood chemistry. In some experiments, blood samples were
collected by incision of the posterior vena cava from mice sacrificed 24 h
after infection. The sera were separated and assayed for the levels of total
protein, albumin, and globulin, the ratio of albumin to globulin, and
levels of -glutamyl transpeptidase, aspartate aminotransferase (AST),
alanine aminotransferase (ALT), bilirubin, creatinine, urea, and alkaline
phosphatase (ALP) using the Roche Hitachi 917 analyzer (Vita-Tech,
Markham, Canada) (25).
Determination of cytokine and chemokine levels. The levels of cytokines and chemokines in the sera and lung homogenate supernatant were
measured using the 21-plex Milliplex MAP mouse cytokine/chemokine
kits (Millipore, Ltd., Billerica, MA) on a Luminex 100IS system (Luminex,
Austin, TX) as specified by the manufacturer. Samples were assayed in
duplicate, and cytokine/chemokine concentrations were calculated
against the standards using Beadview software (version 1.03; Upstate) (7).
In vivo antibiotic treatment efficacy studies. Groups of 10 female
C57BL/6 mice were intranasally inoculated with LAC-4 and treated with
imipenem (100 mg/kg of body weight/day, twice a day [b.i.d.], i.p.), amikacin (15 mg/kg/d, b.i.d., i.p.), tigecycline (10 mg/kg/d, b.i.d., subcutaneously) or diluent (placebo; i.p.) starting 3 h after the LAC-4 inoculation.
The blood and tissue (lungs and spleen) bacterial burdens at 24 hpi were
determined, and clinical signs and body weight changes were observed for
7 days.
Vaccination and protection studies. Groups of 5 female BALB/c mice
were intranasally immunized with either 1 108 CFU of formalin-fixed
LAC-4 (ffLAC-4) in 50 l saline or saline only (sham-immunized mice).
ffLAC-4 was prepared by incubating freshly grown LAC-4 in buffered 4%
formaldehyde solution (Formalde-Fresh; Fisher Scientific, Ottawa, Ontario, Canada) for 24 h. The sterility of the preparation was confirmed by
bacterial culture. The immunizations were performed under isoflurane

Antimicrobial Agents and Chemotherapy

Mouse Model of A. baumannii-Associated Pneumonia

FIG 1 PCR amplification of intergenic spacer (ITS) regions of four isolates:

LAC-4 (lane 2), LAC-15 (lane 3), ATCC 17978 (lane 4), and AYE (lane 5). A
DNA ladder is shown in lane 1. The ITS regions were amplified via PCR using
the 1512F and 6R primers based on the method of Chang et al. (22). Due to the
primers used, the amplicons are approximately 800 bp in length. Removal of
23S and 16S rRNA gene sequences from the sequencing reads will generate an
ITS of 607 to 638 bp in range, depending on the species (22).

inhalation anesthesia at 0, 14, and 21 days. At 28 days, fecal pellets as well

as vaginal wash and blood (submandibular bleed) samples were collected
from individual mice for enzyme-linked immunosorbent assays (ELISAs)
to determine A. baumannii-specific antibody (IgA and IgG) responses. All
of the mice were intraperitoneally challenged with 108 CFU (approximately 100 the 100% lethal dose [LD100]) of freshly grown LAC-4 on
day 47, and the survival of the mice was monitored for 7 days.
A. baumannii whole-cell-specific IgA and IgG ELISAs. Levels of A.
baumannii whole-cell-specific antibodies in serum and mucosal samples
were measured by ELISA (26). Briefly, 96-well microplates were precoated
with 106 ffLAC-4 cells/well in 100 l of sodium bicarbonate buffer (pH
9.6). Samples were prediluted before assays (1:20 for vaginal wash samples, 1:100 for serum IgG and IgA). Pooled samples collected from mice
that had been intranasally immunized with ffLAC-4 or from naive mice
were used as positive or negative controls, respectively.
Serum bactericidal assay. The serum bactericidal assay was performed based on the method of Luke et al. (14). The assays were set up in
triplicate in 5-ml polystyrene tubes. Briefly, 100 l mid-log-phase A. baumannii culture (5 107 CFU) in tryptic soy broth was mixed with 900 l
of either pooled normal human serum (NHS) (Valley Biomedical, Winchester, VA) or heat-inactivated serum (HIS; 56C for 30 min). The mixtures were incubated at 37C with rotation, and aliquots of 100 l were
removed from the culture at the indicated time for the determination of
viable bacterial counts. Results were expressed as percent survival, with
100% being the number of viable bacteria grown in HIS.
Statistical analysis. Data are presented as means standard deviations (SD) for each group, unless otherwise specified. Differences in quantitative measurements were assessed by Students t test or one- or two-way
analysis of variance (ANOVA), followed by Bonferronis post hoc multiple-comparison tests when appropriate. Differences were considered significant when P 0.05.

RESULTS

In vivo selection of the LAC-4 isolate in C57BL/6 mice. To develop a reproducible mouse model of A. baumannii pneumonia
that is more representative of natural human infection, we first
evaluated the relative virulence of a panel of 11 clinical isolates and
4 ATCC type strains of A. baumannii in our collection, using the
mouse model of i.n. infection (7). We found that LAC-4 and, to a
lesser degree, LAC-1, LAC-5, LAC-6, and LAC-15, demonstrated
significantly higher bacterial burdens than did ATCC 17961 in
both the lungs and the spleen at 24 hpi (P value of 0.01 to
0.001) (Table 1).
Although LAC-4 was initially determined to belong to A. baumannii species using an API 20NE kit and the temperature test

August 2013 Volume 57 Number 8

(23), we reconfirmed its species designation in this study using a

more quantitative ITS sequencing assay (22). The ITS regions of
LAC-4 are 607 bp long (Fig. 1), identical to known A. baumannii
strains (22). The LAC-4 ITS shares 99% (606/607) and 100% (607/
607) sequence identity with the sequence of AYE (another clinical
strain) and ATCC 19606 strains, respectively. LAC-4 was multidrug resistant (i.e., resistant to three or more different classes of
antibiotics) according to antibiotic MICs and breakpoint designations. Its multidrug resistance lies between AYE, which shows resistance to 12 of the 17 antibiotics tested, and ATCC 17978, which
is susceptible to all 17 antibiotics tested (Table 2).
Susceptibility of C57BL/6 and BALB/c mice to intranasal inoculation with LAC-4. To determine the relative susceptibility
of mice to i.n. infection with LAC-4, groups of male or female
BALB/c and C57BL/6 mice were intranasally inoculated with
105 to 108 CFU of freshly grown LAC-4. Clinical signs, body
weight, and survival of the mice were monitored. Female
BALB/c and C57BL/6 mice showed little or no clinical signs
following i.n. inoculation with 105 or 106 CFU of LAC-4,
whereas their male counterparts showed transient but mild
clinical signs at day 1 postinoculation (Fig. 2A). Mice inoculated with 107 CFU displayed various degrees of clinical signs,
with the clinical scores of female mice being generally more
severe than those of male mice. All mice developed severe clinical signs when inoculated with 108 CFU of LAC-4 (Fig. 2A).
Similarly, LAC-4-inoculated mice lost about 5 to 15% of their
initial body weight at 24 h, depending on the size of the inocula
received (Fig. 2B). The body weights of those mice that received
the lower inocula (105 or 106 CFU) generally recovered to their
preinoculation level by day 3, and the mice continued to gain
weight throughout the 6 to 7 days of the observation period
(Fig. 2B). The body weights of the surviving mice from the
group at 107 CFU were more variable, presumably reflecting
the various severities of the acute infection. Most significantly,
i.n. inoculation with 108 CFU of LAC-4 resulted in 100% mortality by day 2 after inoculation, regardless of the mouse strain
or sex (Fig. 2C). Up to 80% of the mice that received 107 CFU of
LAC-4 succumbed to the infection, whereas all mice that re-

TABLE 2 Antibiotic resistance profile of LAC-4, ATCC 17978, and AYE

strains
LAC-4

ATCC 17978

AYE

Antibiotic

MIC
MIC
MIC
(g/ml) Breakpointa (g/ml) Breakpoint (g/ml) Breakpoint

Amikacin
Gentamicin
Tobramycin
Imipenem
Meropenem
Piperacillin
Cefepime
Cefotaxime
Ceftazidime
Ceftriaxone
Ciprofloxacin
Gatifloxacin
Levofloxacin
Doxycycline
Minocycline
Tetracycline
Tigecycline

32
16
32
2
4
256
16
256
128
256
16
2
2
0.5
0.25
8
0.25

I
R
R
S
S
R
I
R
R
R
R
S
S
S
S
I
S

2
2
2
2
2
16
4
8
8
8
0.5
2
2
2
0.25
4
0.25

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

64
256
64
2
2
256
256
256
256
256
128
8
4
4
0.5
128
1

R
R
R
S
S
R
R
R
R
R
R
R
I
S
S
R
S

R, resistant; I, intermediate; S, susceptible.

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FIG 2 Clinical sign scores (A), body weight (B), and survival rates (C) of male and female BALB/c and C57BL/6 mice following i.n. inoculation with LAC-4.

Groups of BALB/c or C57BL/6 mice (n 5) were intranasally inoculated with 105 to 108 CFU of LAC-4 as indicated, and their clinical outcome was monitored
daily for up to 7 days. The overall clinical assessment for each mouse was scored as 0 (normal, active, healthy), 1.0 (slightly sick, slightly ruffled fur, otherwise
normal), 2.0 (sick, ruffled fur, slow movement, hunching), 3.0 (very sick, ruffled fur, very slow movement, hunched, eyes shut), 4.0 (moribund), or 5.0
(dead). Clinical scores and body weights are expressed as means SD.

ceived 105 or 106 CFU survived the infection (Fig. 2C). This is
in contrast to previous studies with other A. baumannii strains,
such as ATCC 17961 (7, 8), and other clinical isolates (9, 12),
where no mice died after i.n. inoculation with 108 CFU. These
results suggest that the i.n. 50% lethal dose (LD50) for LAC-4
infection in mice is about 107 CFU, and that, in both C57BL/6
and BALB/c mice, LAC-4 is at least 100 times more lethal than
most ATCC type strains and clinical isolates, including AYE
and SFD (data not shown). Moreover, there were no significant
differences in susceptibility due to mouse strain or sex. Consequently, we focused our subsequent characterization of this
model primarily in female C57BL/6 mice, with the consideration that the majority of transgenic mice currently available to
the research community are on the C57BL/6 background.
Kinetics and extrapulmonary dissemination of LAC-4 in
mice following i.n. inoculation. As the first step toward the characterization of our mouse model of A. baumannii-associated
pneumonia, we determined the kinetics and extrapulmonary dissemination of LAC-4 in different organs of female C57BL/6 mice
over the course of the infection following i.n. inoculation with 108

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CFU. At 4 hpi, the A. baumannii burden in the lungs was about 8.0
log10 CFU, while the burdens in the spleen and blood were barely
above the detectable limit (Fig. 3A). Bacteria continued to replicate rapidly in the lungs, reaching 10.0 log10 CFU in some of the
infected mice by 24 hpi. More significantly, at 24 hpi infected mice
showed significant extrapulmonary LAC-4 dissemination, with
5.0 to 6.0 log10 CFU cultured from the blood and spleen at this
time point (Fig. 3A).
To further characterize the local replication and systemic dissemination of LAC-4, we also determined the bacterial burdens in the
tracheobronchial lymph nodes (TBLN; the major lung draining
lymph nodes) and inguinal lymph nodes (IGN; non-lung draining
lymph nodes). As shown in Fig. 3B, significantly more LAC-4 organisms were cultured from the TBLNs at 4 hpi than from the blood (P
0.001) and spleen (P 0.01) (Fig. 3A), while no LAC-4 organisms
were detected in the IGN at this early stage of infection. At 24 hpi, high
numbers of LAC-4 were recovered from TBLNs, while the IGNs harbored far fewer bacteria. These data suggest that LAC-4 exerts a
unique capability for pulmonary multiplication and extrapulmonary
dissemination via local draining lymph nodes.

Antimicrobial Agents and Chemotherapy

Mouse Model of A. baumannii-Associated Pneumonia

FIG 3 A. baumannii bacterial burdens in the lungs, spleen, and blood (A) and tracheobronchial (TBLN) and inguinal (IGN) lymph nodes (B) of female C57BL/6

mice (n 3 to 6) intranasally inoculated with 108 CFU of LAC-4. Bacterial burdens in the blood and respective organs were determined by quantitative
bacteriology at 4 and 24 h postinoculation. The data are presented as means SD and represent one of at least two experiments with similar results. The detection
limits (dotted lines) for bacterial burdens were 1.3 log10 CFU/organ for lung and spleen and 1.0 log10 CFU/ml blood or lymph nodes, respectively. Compared to
results for the 4-hpi group, P 0.05 (*) and P 0.001 (***).

Pulmonary inflammatory cell responses to i.n. LAC-4 inoculation. To characterize the host response to i.n. LAC-4 infection in
this model, we next determined the pulmonary recruitment of
inflammatory cells in response to LAC-4 infection. As anticipated,
the major BAL fluid cell populations following i.n. A. baumannii
infection were alveolar macrophages and neutrophils (Fig. 4),
with negligible numbers of lymphocytes and eosinophils (data not
shown). The number of total and differential (macrophages and
neutrophils) BAL fluid cells in LAC-4-infected mice at both 4 and
24 hpi were comparable to those reported previously by us and
others for mice infected with ATCC 17961 or other clinical strains
(79), with 90% being neutrophils at 24 hpi (Fig. 4). This suggests that the enhanced virulence of LAC-4 was not due to the
inhibition of the pulmonary recruitment of neutrophils, the key
effector cell in host defense against A. baumannii (7, 27).
Histopathology and blood clinical chemistry. Histopathologically, the lungs from all mice at 24 hpi showed mild to moderate
bronchopneumonia consisting of small to moderate numbers of
mixed neutrophils and mononuclear cells in the airway and alveolar lumen (Fig. 5A). In addition, there was severe dilation and
mixed inflammatory cell infiltration in the perivascular and peribronchial space in the lung (Fig. 5B). The spleen from infected
mice showed the presence of multiple small clusters of degenerated or necrotic leukocytes in the white pulp areas and moderate
congestion in the medullar region (Fig. 5C). The liver showed
mild increases in the number and size of Kupffer cells, whereas the
hepatocytes showed no remarkable changes in their appearance
(Fig. 5D). However, there were no abnormal changes in the kidneys or heart of any infected mice.

August 2013 Volume 57 Number 8

Blood chemistry analysis showed that LAC-4 infection induced

significant elevation of serum urea (P 0.001) and moderate
elevation of serum bilirubin, ALT, and AST at 24 hpi compared to
sera from uninfected mice (Fig. 6). On the other hand, the serum
ALP level was significantly reduced in the infected mice (P
0.001). There were no differences in serum levels of total protein,
albumin, globulin, the ratio of albumin to globulin, or -glutamyl
transpeptidase between LAC-4-infected and uninfected mice
(data not shown).
Systemic and local cytokine and chemokine responses to i.n.
LAC-4 inoculation. Systemic (serum) and local (lung) levels of a
panel of 21 cytokines and chemokines, including those that have
previously been implicated in the pathogenesis of respiratory A.
baumannii infection in mice, rats, and human skin or peripheral
blood mononuclear cell culture (79, 20, 28), were determined at
0, 4, and 24 h after i.n. LAC-4 inoculation. Compared to uninfected mice (0 h), the levels of granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-1 (IL-1), IL-1,
IL-6, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein
1 (MIP-1), tumor necrosis factor alpha (TNF-), and vascular
endothelial growth factor (VEGF) in the lung of LAC-4-infected
mice increased substantially at 4 hpi, with many being significantly higher (P 0.05) (Fig. 7A). The levels of most of these
cytokines and chemokines maintained similar magnitudes at 24
hpi, while the levels of IL-17 and RANTES were substantially or
significantly increased at this time point. On the other hand, the
IL-13 levels were significantly reduced after the infection (P
0.001). There were no significant changes in the levels of the re-

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FIG 4 Pulmonary inflammatory cell responses in female C57BL/6 mice (n 6) intranasally inoculated with 108 CFU of LAC-4. (Left) The cellular composition
in the bronchoalveolar lavage fluid. Groups of six mice were killed at 0, 4, and 24 h postinoculation, their lungs were lavaged, and the total and differential cell
counts were determined. Data are presented as means SD and are representative of at least two independent experiments with similar results. ***, P 0.001
versus results for the 0-h group. (Right) Photomicrographs of cells from BAL fluid from mice killed at the indicated times following i.n. inoculation with LAC-4.
(a) Cells from BAL fluid from a mouse killed at 0 h, showing the presence of predominantly resting alveolar macrophages without neutrophils; (b) cells from BAL
fluid from a mouse killed at 4 hpi, showing the presence of alveolar macrophages admixed with moderate numbers of neutrophils; and (c) cells from BAL fluid
from a mouse killed at 24 hpi, showing predominantly neutrophils with horseshoe-shaped nuclei admixed with very few alveolar macrophages. Hema-3 staining
was used. For photomicrographs in panels a and b, the BAL fluid was used for cytospin preparation without dilution. For the photomicrograph shown in panel
c, the BAL fluid was diluted 1:20 before being used for cytospin preparation.

maining cytokines or chemokines (Fig. 7A). Similarly, LAC-4infected mice showed significantly higher serum levels of IL-6,
KC, and MIP-1 at both 4 and 24 hpi (P 0.05) (Fig. 7B), while
significant increases in the levels of GM-CSF, IL-10, IL-12p40,
IL-17, IL-1, MCP-1, RANTES, and TNF- were observed only at
24 hpi (P 0.05). A slight but significant decrease in the serum
level of RANTES was observed at 4 hpi (P 0.01). On the other
hand, the serum levels of gamma interferon (IFN-), IL-2, IL-3,
IL-4, IL-5, and VEGF were not significantly changed and generally
were at the margin of or below the detection limits at either time
point (Fig. 7A and B).
Effect of in vitro passage or frozen storage on LAC-4 virulence. To determine the relative stability of LAC-4 virulence after
in vitro passage, we compared the bacterial burdens and survival
rates in mice that were intranasally inoculated with 5 107 CFU
of either LAC-4 that had been subcultured 10 times in vitro or the
parent stock. There were no significant differences between the
two groups, as assessed by the clinical scores and body weight
changes at 24 h after i.n. inoculation (data not shown). Moreover,
the bacterial burdens in the lungs, spleen, and blood were generally comparable between the two groups of mice (Fig. 8A). Similar
results were also observed when the mice were inoculated with 106

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CFU of subcultured or parental LAC-4 (data not shown). We also

compared the clinical signs and bacterial burdens in mice that
were intranasally inoculated with 2 107 CFU of either LAC-4
that had been stored in 20% glycerol at 80C for 5 years or
freshly grown bacterial cells. There were no significant differences
in either the clinical scores (data not shown) or the tissue and
blood bacterial burdens (Fig. 8B) between the two groups of mice.
These results indicate that repeated in vitro subculture or longterm frozen storage of LAC-4 did not significantly affect its virulence in mice. This implies that the phenotype required for the
mouse infection and dissemination is relatively stable.
In vivo efficacy of antimicrobials and vaccination against i.n.
A. baumannii infection. Since our mouse model of A. baumannii
pneumonia develops many clinical and histopathological features
of human A. baumannii pneumonia, we next evaluated whether
the model is useful for the evaluation of novel therapeutics and
vaccine candidates. Groups of C57BL/6 mice were intranasally
inoculated with 5 107 CFU of LAC-4. Beginning 3 hpi, the mice
were treated twice a day with imipenem, amikacin, tigecycline, or
diluent (saline) only. LAC-4 burdens in the blood and tissues
(lungs and spleen) were determined at 24 hpi, and clinical signs
and body weight changes were monitored for 7 days. In agreement

Antimicrobial Agents and Chemotherapy

Mouse Model of A. baumannii-Associated Pneumonia

FIG 5 Photomicrographs of lung (A and B), spleen (C), and liver (D) histopathology in LAC-4-infected mice. Groups of female C57BL/6 mice (n 5) were

intranasally inoculated with 108 CFU of LAC-4 and sacrificed at 24 h postinoculation. The lungs from LAC-4-infected mice showing severe inflammatory cell
infiltration in the perivascular and peribronchial areas (A, arrowheads) and the dilatation of perivascular lymphatics (B, arrows). An asterisk indicates bronchial
lumen. (C) The spleen from an LAC-4-infected mouse showing the presence of multiple clusters of degenerated leukocytes in the white pulps (arrowheads) and
moderate congestion of the medullar region. (D) The liver from an LAC-4-infected mouse showing a mild increase in the number and size of Kupffer cells. H&E
staining was used. Bar, 100 m in A and B and 50 m in C and D.

with in vitro antimicrobial susceptibility observations (Table 2),

treatment of LAC-4-infected mice with imipenem or tigecycline
significantly reduced blood and tissue bacterial burdens compared to those of saline-treated mice (P 0.001) (Fig. 9A). To a
lesser extent, amikacin treatment also significantly reduced LAC-4
burdens in the blood (P 0.05) and spleen (P 0.001) but not in
the lungs (P 0.05). This was expected based on the intermediate
in vitro susceptibility of LAC-4 to amikacin. Moreover, none of the
imipenem- or tigecycline-treated mice showed any clinical signs
of the disease (Fig. 9B, middle) and survived the infection, while
40% of amikacin-treated mice succumbed to the infection (Fig.
9B, lower). As expected, all placebo-treated mice died of this dose
of infection by 48 hpi.
We also assessed the potential of this model for the development and evaluation of candidate vaccines against A. baumannii
infection. Groups of BALB/c mice were intranasally immunized
with 108 CFU ffLAC-4, or sham immunized with phosphate-buffered saline (PBS), and i.p. challenged with 108 CFU (100 LD100)

August 2013 Volume 57 Number 8

of freshly grown LAC-4. As shown in Fig. 10, immunized mice

developed LAC-4-specific serum IgG and IgA responses as well as
vaginal IgA responses (an indication of mucosal immune responses). More importantly, 40% of the immunized mice survived a lethal (100 LD100) LAC-4 challenge. As expected, no
antibody response or protection was observed in sham-immunized mice. These data indicate the potential utility of this model
for the development and evaluation of vaccines and therapeutics
for A. baumannii-associated pneumonia.
High resistance of LAC-4 to serum killing. As a first step to
elucidate the hypervirulence of the LAC-4 isolate, we compared
the serum sensitivity of LAC-4 to that of another A. baumannii
strain (ATCC 17961) that has been used by us and others (710) in
mice, since serum sensitivity has been implicated in the virulence
mechanisms of A. baumannii (19, 20). As shown in Fig. 11, LAC-4
was highly resistant to the killing action of serum with 95% or
greater survival after 3 h of incubation. In contrast, the ATCC
17961 strain was much more serum sensitive, showing a survival

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FIG 6 Selected blood chemistry profiles of LAC-4-infected mice. Groups of

female C57BL/6 mice (n 5) were intranasally inoculated with 108 CFU of
LAC-4 and sacrificed at 24 h postinoculation. Serum samples were prepared
and analyzed for the levels of bilirubin, alkaline phosphatase (ALP), alanine
aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine. Data are presented as means SD. ***, P 0.005 versus uninfected
mice.

of 50%. Similarly, ATCC 17978 and AYE strains were also more
serum sensitive than the LAC-4 strain, showing a survival of 25%
or lower after a 6-h incubation (data not shown).
DISCUSSION

The respiratory tract is an important site of A. baumannii colonization and the most common site of infection. Moreover, the incidence of A. baumannii colonization in the respiratory tract increases during stays in the intensive care unit (5). The crude
mortality rate of ventilation-associated and community-acquired
pneumonia caused by A. baumannii has been estimated to be between 40 and 70% (5). As a result of its increasing resistance to
many currently available antibiotics (29), A. baumannii is now
one of the most difficult-to-treat pathogens. Over the years, a
number of animal models of A. baumannii pneumonia, including
laboratory rodents and model organisms, have been developed
and used for studying the disease pathogenesis and for evaluating
the efficacies of new antimicrobials and novel intervention strategies. In this study, we described a mouse model of acute A. baumannii pneumonia using a clinical isolate of A. baumannii that is
highly virulent in immunocompetent mice when inoculated intranasally. This model offers a reproducible acute course of pneumonia in immunocompetent, common mouse strains, such as
C57BL/6 and BALB/c; therefore, it is suitable for the study of antibiotic pharmacokinetics/pharmacodynamics within the first 48
h of infection. Similar to the clinical course of respiratory infection
with A. baumannii, the bacteria replicated in the lung and disseminated rapidly to extrapulmonary tissues (Fig. 3). Bacteremia and
pneumonia were fulminant in all cases, with up to 100% mortality, depending on the inoculum size. The mean duration of symptoms was 3 days for survivors and 2 days or less for those who died.
The results of the present study clearly show that LAC-4 is
significantly more virulent than other clinical isolates and type
strains in the mouse model of i.n. infection. To the best of our
knowledge, this is the first report of a clinical isolate of A.
baumannii which is hypervirulent to conventional mice and

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causes 100% mortality in both C57BL/6 and BALB/c mice

within 48 h of infection when administered intranasally. Previous studies generally have utilized special mouse strains
(such as C3H/HeN, A/J, and phoxgp91/) and/or induction of
neutropenic status by various means to induce severe pneumonia and lethal infection (6, 7, 10, 16, 17). Thus, our results
support those of Eveillard et al. (17), the only study to date that
has systematically compared in vivo virulence among different
clinical isolates of A. baumannii (17), showing that different
clinical isolates of A. baumannii display significantly different degrees of virulence following respiratory inoculation.
Our quantitative bacteriology results (Fig. 3) also suggest that
LAC-4 possesses unique capabilities in rapid localized replication
and extrapulmonary dissemination. The presence of high bacterial burdens in the spleen is noteworthy, because various A. baumannii strains/isolates reported to date do not normally disseminate in large numbers to the spleens of immunocompetent mice
(7) and only rarely do so in immunosuppressed mice (6, 17). The
reasons for this high level of extrapulmonary dissemination by
LAC-4 are currently unknown. It is possible that the high LAC-4
burden in the lungs of infected mice exacerbated systemic dissemination. However, the fact that ATCC 17961-infected mice can
harbor similar levels of bacteria in the lungs (8.5 log10) at 4 hpi
with very few in the spleen (3 log) (unpublished data) refutes
this possibility. The bacterial burdens in the spleens of the LAC4-infected mice at 4 hpi in this study (Fig. 3A) appear comparable
to those observed in mice similarly infected with other A. baumannii isolates of less virulence (7). This suggests that the total numbers of bacteria that initially disseminated to spleens were comparable between LAC-4 and other strains, but the host innate
immunity was less effective in controlling LAC-4 replication. Indeed, it has recently been suggested that the ability to suppress
innate immune responses may be associated with the virulence of
A. baumannii (20). Our detailed studies on the kinetics of bacterial
burdens in the lung draining (TBLNs) and nondraining (IGNs)
lymph nodes (Fig. 3B) also suggests that lymphogenic dissemination is the predominant route of extrapulmonary spread at the
early stage of the LAC-4 infection. The numbers of bacteria in the
TBLNs at 4 hpi were about 100 higher than those in the blood or
IGN, where LAC-4 was only occasionally detected at this time.
Despite the fact that the respiratory route of A. baumannii infection is the most common form of hospital-acquired infection
(1), the manifestation of human A. baumannii-associated pneumonia is not well defined in the medical literature. It is generally
referred to as being similar to other ventilation-associated pneumonia induced by other nosocomial pathogens (1). Leung et al.
(30) described fever, cough, and sputum production as some of
the common features of A. baumannii pneumonia. In addition,
limited clinical data on chest radiography suggest that lobar consolidation and small pleural effusions are consistently found in
outpatients with A. baumannii-associated pneumonia. However,
pulmonary cavitation is absent, probably reflecting the relatively
acute nature of the infection. The patients with community-acquired A. baumannii pneumonia have a high incidence of bacteremia, acute respiratory distress syndrome, and death (30). Others
have proposed that the pertinent features of pneumonia caused by
Gram-negative bacteria are bacterial growth and clearance, host
inflammatory response, acute lung injury, and death associated
with respiratory failure (31). In this regard, the mouse model of A.
baumannii pneumonia described here mimics many aspects of

Antimicrobial Agents and Chemotherapy

Mouse Model of A. baumannii-Associated Pneumonia

FIG 7 Cytokine and chemokine levels in the lung homogenate supernatants (A) and sera (B) of LAC-4-infected mice. Groups of female C57BL/6 mice (n 5

to 6) were intranasally inoculated with 108 CFU of LAC-4 and sacrificed at 0, 4, and 24 h postinoculation. Cytokine and chemokine levels were determined in the
sera and lung tissue homogenate supernatants using the 21-plex Milliplex mouse cytokine/chemokine kits on a Luminex 100IS system. The detection limit for all
cytokines and chemokines is 10 pg/ml. All values are means SD. Compared to results at 0 h, P 0.05 (*), P 0.01 (**), and P 0.001 (***).

human infection. Naturally, this newly described mouse model

suffers from certain inherent shortcomings due to the anatomy
and physiology of the mouse respiratory tract. For example, cough
and sputum production are very common in human patients with
A. baumannii-associated pneumonia but are absent from mice. In
this regard, it is probably unrealistic to expect that a single animal
model will reproduce the entire spectrum of the human disease
features and process.
An additional, but equally critical, parameter for an ideal animal model of A. baumannii infection is the bacterial strain used.
Ideally, a strain with a proven record to cause human infections

August 2013 Volume 57 Number 8

and a well-characterized virulence mechanism should be used.

However, all currently sequenced strains and well-defined clinical
isolates of A. baumannii display relatively low virulence in rodents
(reviewed in reference 5). The LAC-4 isolate used in this study was
initially described in a 2008 study of 20 multidrug-resistant isolates obtained from an outbreak at hospitals in Los Angeles
County, California (23). It was one of the outbreak isolates obtained from a patient in January 1997. Among the 20 outbreak
isolates, LAC-4 was the least resistant to antimicrobials, and its
genomic profile is distinct from that of the rest of the outbreak
isolates studied, as well as from ATCC 17978, ATCC 19606, and

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Harris et al.

FIG 8 Effect of repeated in vitro passage on the in vivo virulence of LAC-4 in

mice. (A) Groups of female C57BL/6 mice (n 5) were intranasally inoculated

with 5 107 CFU of either the parent stock (LAC-4) or a culture of LAC-4 that
had been subcultured 10 times in vitro (PASS 10). The bacterial burdens in
tissue (lungs and spleen) and blood at 24 h after inoculation were determined
and compared. Data are presented as mean log10 CFU SD and are representative of two independent experiments with similar results. The confirmed
inocula for the parent and the subcultured LAC-4 were 5.4 107 and 4.3 107
CFU, respectively. (B) Effect of frozen storage on the in vivo virulence of LAC-4
in mice. Groups of female C57BL/6 mice (n 5) were intranasally inoculated
with 2 107 CFU of either the freshly grown (Fresh) LAC-4 or cells directly
taken from stock frozen for 5 years (Frozen). The bacterial burdens in tissue
(lungs and spleen) and blood at 24 h after inoculation were determined and
compared. Data are presented as mean log10 CFU SD. The confirmed inocula for the fresh and frozen LAC-4 were 2.6 107 and 3.9 107 CFU, respectively.

AYE strains of A. baumannii (23). Using multilocus sequencing

typing (32), LAC-4 was found recently to belong to sequence type
10 of A. baumannii (W. A. Warner and H. H. Xu, unpublished
data). One of the shortcomings of our mouse model is that the
virulence genes and mechanisms of the LAC-4 strain remain unknown. Although preliminary studies have shown that LAC-4 is
more resistant to serum than several other A. baumannii strains
(Fig. 11), a complete understanding of the virulence mechanism
of LAC-4 would provide additional novelty to our mouse model.
In this regard, the recent development of genetic tools for targeted
mutagenesis in certain strains of A. baumannii is encouraging.
All mouse models of A. baumannii pneumonia reported to
date employ various tactics to render the animals temporarily immunosuppressed (reviewed in reference 5). The rat model of A.
baumannii pneumonia developed by Russo et al. is probably the
only other laboratory rodent model that does not require the immunosuppression (31). Therefore, our mouse model can be a
complementary tool to the rat model for the future identification
of novel therapeutic targets and their preclinical evaluation. However, compared to the rat model, the mouse model has advantages,
such as a lower cost and ease of handling, the use of i.n. inocula-

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tion rather than surgical, intratracheal instillation, and the availability of abundant immunological reagents and transgenic
strains for mechanistic studies. One of the initial concerns among
some in the research community was the superior consistency of
the infection induced by intratracheal versus the previous i.n. inoculations. However, the relevant tissue and blood bacterial burdens in our mouse model were very consistent, as evident in this
study (Fig. 3A, 8, and 9A) and in our previous publications regarding other A. baumannii strains (7, 10, 16). In addition, by appropriate manipulation of the inoculum size, various degrees of mortality (up to 100%) can be induced readily. Thus, the current
mouse model offers a high-throughput, economical animal model
for A. baumannii research and product development.
LAC-4 also induced a number of pathological features that
mimic the human diseases, and these may partially contribute to
the high mortality of the infected animals. The lungs of LAC-4infected mice showed severe perivascular inflammatory infiltration and severe lymphatic dilatation (Fig. 5). Although vascular
congestion was observed in the lungs of mice infected with other
virulent clinical strains (such as SAN) of A. baumannii, such
perivascular changes were not reported (17). The more severe
tissue pathology in LAC-4-infected mice was also corroborated by
blood biochemical analysis which showed significantly increased
urea concentration in these mice. The observed moderate elevation of the serum liver enzymes (ALT and AST) was in agreement
with the relatively mild histopathology in the livers of the infected
mice (Fig. 5D). However, although the blood biochemistry suggests some degree of renal impairment in LAC-4-infected mice, no
abnormal histopathology was observed in their kidneys, despite
careful examination. Collectively, these results suggest that the
infected mice died primarily of severe pneumonia and subsequent
bacteremia.
The cytokine/chemokine levels in the lungs and sera were significantly elevated in LAC-4-infected mice compared to those of
uninfected mice. This is probably due to the high bacterial burdens which stimulate cytokine/chemokine responses, as has also
been seen in other A. baumannii infections (7, 10, 16, 17, 31).
Variations in cytokine responses to different A. baumannii strains
(17) or challenge doses (31) have been reported, but their levels
(such as those of TNF-) generally are not directly related to mortality. Although MIP-2 level was observed to positively correlate
with the mortality of the mice, it is not clear if this was directly
responsible for the death (17). In fact, we have previously detected
10,000 pg/ml of MIP-2 in the BAL fluid of mice that survived i.n.
A. baumannii challenge (10). However, the use of neutropenic
mice in that study may have further complicated the cytokine/
chemokine responses and the clinical outcome of the infection.
Unfortunately, the MIP-2 level was not assayed in the current
study because it is not part of the 21-plex Luminex kit that was
used.
The utility of our mouse model of A. baumannii pneumonia
for the evaluation of new antimicrobials and therapeutics was
tested by comparing the efficacies of imipenem, tigecycline, and
amikacin. Imipenem is indicated for treatment of pneumonia
caused by susceptible Acinetobacter spp. As for tigecycline, while it
is not yet indicated for the treatment of A. baumannii or hospitalacquired pneumonia, several clinical trials have been initiated to
expand its use for these indications. In this regard, the effectiveness of tigecycline in treating pneumonia caused by A. baumannii
has been reported recently (3335). As expected, we observed re-

Antimicrobial Agents and Chemotherapy

Mouse Model of A. baumannii-Associated Pneumonia

FIG 9 Effect of antibiotic treatment on pulmonary LAC-4 infection in mice. Groups of female C57BL/6 mice (n 10) were intranasally inoculated with 5 107
CFU of LAC-4. Beginning at 3 h postinoculation, the mice were treated twice a day with imipenem (100 mg/kg/day, i.p.), amikacin (15 mg/kg/day, i.p.),
tigecycline (10 mg/kg/day, subcutaneously), or saline diluent only (i.p.). (A) Half of the infected mice were killed at 24 h postinoculation for the determination
of the bacterial burdens in the blood and tissues (lungs and spleen) by quantitative bacteriology. The data are presented as mean log10 CFU per organ or per ml
of blood SD. The detection limits (dotted lines) for bacterial burdens were 1.3 log10 CFU/organ (lung and spleen) and 1.0 log10 CFU (ml blood), respectively.
P 0.05 (*) and P 0.001 (***) versus results for the saline-treated group. The effect of antibiotic treatment on clinical scores, body weight, and survival rates
was determined in the remaining half of the LAC-4-infected mice (B). The clinical scores were assigned as described in the legend to Fig. 2. Body weights and
clinical scores are expressed as means SD.

markably improved clinical scores and survival rates and significantly reduced bacterial burdens in the antimicrobial-treated
mice compared to placebo-treated mice (Fig. 9). We also demonstrated that imipenem and tigecycline offered significantly better

FIG 10 Protection of vaccinated mice against LAC-4 challenge. Groups of

female BALB/c mice were intranasally vaccinated with 108 CFU ffLAC-4 cells
(immunized) or sham immunized with PBS (naive) on days 0, 14, and 21.
Blood and vaginal wash samples were collected at day 28, and LAC-4-specific
IgA and IgG responses (mean optical density SD) were determined by
ELISA. The mice were challenged intraperitoneally with 108 CFU (100
LD100) of freshly grown LAC-4 at day 47. The survival rates of the challenged
mice were recorded.

August 2013 Volume 57 Number 8

FIG 11 Sensitivity of LAC-4 and ATCC 17961 to normal human serum

(NHS). LAC-4 and ATCC 17961 were grown in the presence of 90% NHS or
90% heat-inactivated NHS as controls. Viable bacterial counts were determined after 0, 1, and 3 h of incubation at 37C with rotation. Data are presented as percent survival, with 100% being the number of viable bacteria
grown in heat-inactivated serum. All values are means SD from triplicate
samples and are representative of three independent experiments.

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Harris et al.

protection than amikacin, an observation that is consistent with in

vitro MIC values (Table 2). Our data also show that imipenem and
tigecycline were significantly more efficient in controlling pulmonary bacterial multiplication than amikacin. Similarly, our prophylactic vaccination and challenge experiment showed that vaccination of mice with inactivated LAC-4 cells protects against
lethal challenge with this pathogen.
In conclusion, we have described a mouse model of A. baumannii pneumonia which offers a reproducible, acute course of
pneumonia that, in many aspects (high tissue bacterial burdens,
severe pulmonary pathology, and high mortality), mimics human
disease. One of the major limitations of our study is that the relationship of LAC-4 to the severity of clinical infection in patients
could not be established due to the unavailability of clinical information from the hospital where LAC-4 was initially isolated. Because of the difficulties in treating health care-acquired infections
caused by A. baumannii, this model should be a useful tool for
future identification and characterization of important virulence
factors, pathogenesis studies, and the evaluation of novel therapeutics and vaccines for this emerging infectious agent.
ACKNOWLEDGMENTS
This work was partially funded by the intramural program (A-base) of the
National Research Council Canada (to W.C. and G.B.P.) and by the
United States Department of Homeland Security (2009-ST-062-000018),
the U.S. Army Research Laboratory, and the U.S. Army Research Office
(W911NF-12-1-0059) (to H.H.X.).
We are grateful to Pfizer for providing tigecycline. We thank Didier
Raoult (The University of the Mediterranean Aix-Marseille II, France),
Carl Urban (New York Hospital Queens), and Sheena Chu (Los Angeles
County Public Health Laboratory) for providing clinical isolates of A.
baumannii used in this study. We thank Tom Devecseri for assistance in
the preparation of photomicrography, as well as Sonya Valentine, Melissa
Chong, Maria Gonzalez, and Wayne Warner for technical assistance.

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