SUSCEPTIBILITY

crossm

Survival of Carbapenem-Resistant
Klebsiella pneumoniae Sequence Type

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
258 in Human Blood
Frank R. DeLeo,a Scott D. Kobayashi,a Adeline R. Porter,a Brett Freedman,a
David W. Dorward,b Liang Chen,c Barry N. Kreiswirthc
Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Hamilton, Montana, USAa; Research Technologies Branch, Rocky Mountain
Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton,
Montana, USAb; Public Health Research Institute Tuberculosis Center, New Jersey Medical School-Rutgers
University, Newark, New Jersey, USAc

ABSTRACT Klebsiella pneumoniae is a prominent cause of nosocomial infections

worldwide. Bloodstream infections caused by carbapenem-resistant K. pneumoniae, Received 29 November 2016 Returned for
modification 19 December 2016 Accepted
including the epidemic lineage known as multilocus sequence type 258 (ST258), are
18 January 2017
difficult to treat, and the rate of mortality from such infections is high. Thus, it is im- Accepted manuscript posted online 23
perative that we gain a better understanding of host defense against this pathogen January 2017
as a step toward developing novel therapies. Here we tested the hypothesis that the Citation DeLeo FR, Kobayashi SD, Porter AR,
resistance of ST258 to bactericidal components of human blood, such as serum Freedman B, Dorward DW, Chen L, Kreiswirth
BN. 2017. Survival of carbapenem-resistant
complement, is linked to virulence capacity in the context of bacteremia. There was Klebsiella pneumoniae sequence type 258 in
significant variance in the survival of ST258 clinical isolates in heparinized human human blood. Antimicrob Agents Chemother
blood or normal human serum. The rate of survival of ST258 isolates in human 61:e02533-16. https://doi.org/10.1128/
AAC.02533-16.
blood was, in general, similar to that in normal human serum, suggesting a promi-
Copyright © 2017 American Society for
nent role for complement (rather than leukocytes) in the healthy host defense Microbiology. All Rights Reserved.
against ST258 isolates and related organisms. Indeed, deposition of serum comple- Address correspondence to Frank R. DeLeo,
ment—the C5b to C9 (C5b-C9) membrane attack complex— onto the surface of fdeleo@niaid.nih.gov.

ST258 isolates accompanied serum bactericidal activity. Human serum treated with
pharmacological inhibitors of complement, depleted of antibody, or heated at 56°C
for 30 min had significantly reduced or absent bactericidal activity. In contrast to
heparinized blood from humans, that from BALB/c mice lacked bactericidal activity
toward the ST258 isolates tested, but the virulence of these ST258 isolates in a
mouse bacteremia model was inexplicably limited. Our data highlight the impor-
tance of the complement system in host defense against ST258 bacteremia, and we
propose that there is the potential to enhance complement-mediated bactericidal
activity using an antibody-based approach.

KEYWORDS Klebsiella pneumoniae, antibiotic resistance, bactericidal activity,

bloodstream infections, carbapenem resistance, complement

K lebsiella spp. are significant nosocomial pathogens in the United States and ac-
count for ⬃10% of hospital-acquired infections (1). Klebsiella pneumoniae is second
only to Escherichia coli as a cause of Gram-negative bacteremia (2) and is also an
etiologic agent of pneumonia, urinary tract infections, wound infections, peritonitis,
and meningitis (1). K. pneumoniae strains are notoriously resistant to antibiotics, and
virtually all strains are resistant to ampicillin (3). Moreover, the incidence of disease
caused by multidrug-resistant (MDR) strains, including those whose genomes en-
code extended-spectrum ␤-lactamases, is on the rise. More recently, a class A
␤-lactamase termed Klebsiella pneumoniae carbapenemase (KPC) has been described
(4), and these elements confer resistance to virtually all ␤-lactam antibiotics (5). K.

April 2017 Volume 61 Issue 4 e02533-16 Antimicrobial Agents and Chemotherapy aac.asm.org 1
DeLeo et al. Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 1 Differential survival of K. pneumoniae ST258 isolates in human blood. The bactericidal activity of
human blood is presented as the number of CFU of K. pneumoniae (K. p.) recovered (A) or the percent
K. pneumoniae survival (B). Results are presented as the mean ⫾ standard error of the mean (SEM) from
6 separate experiments (with blood from 6 random blood donors), as indicated. *, P ⬍ 0.05 versus the
starting inoculum (0 min) for each strain using a repeated-measures one-way ANOVA and Dunnett’s
posttest. The asterisks are color coded to match the associated strain.

pneumoniae strains harboring KPC are currently endemic in New York City and have
been reported in over 30 U.S. states (6–9). In addition, carbapenem-resistant K. pneu-
moniae strains have spread globally and are highly prevalent in Israel, Greece, Italy,
China, and South America (10–13). Infections with MDR K. pneumoniae strains are
associated with high rates of morbidity and mortality, particularly among persons with
prolonged hospitalization, critically ill patients, and individuals with invasive devices (7,
14–22). The expanded drug resistance profile of K. pneumoniae KPC strains has severely
limited the treatment options available following infection (23, 24). Although recent
studies indicate that ceftazidime-avibactam is relatively effective against diverse KPC-
containing K. pneumoniae strains (25, 26), the development of ceftazidime-avibactam
resistance is a major concern (26, 27).
Molecular epidemiology studies of K. pneumoniae suggest that multilocus sequence
type 258 (ST258) is the predominant KPC lineage in the United States and other parts
of the world (6, 10, 12, 28). The basis for the success of this organism, outside of
resistance, is not known, and the virulence capacity of ST258 isolates is incompletely
characterized. As a step toward addressing these deficiencies in knowledge, we inves-
tigated the ability of selected ST258 clinical isolates to survive in normal human blood
and normal human serum (NHS) and evaluated the virulence of these isolates in a
mouse model of bacteremia.

RESULTS AND DISCUSSION

Differential survival of ST258 clinical isolates in human blood. To better under-
stand the success of ST258 as a human pathogen, we tested the ability of selected
clinical isolates to survive in heparinized human blood (Fig. 1A and B). Although all
isolates contain genes encoding capsule polysaccharide (cps-1 or cps-2), there was a
relatively wide range in the rate of survival among the 6 isolates tested (e.g., at 60 min

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 2

Survival of K. pneumoniae in Blood Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 2 Differential survival of K. pneumoniae isolates in NHS. The bactericidal activity of human serum is
presented as the number of CFU of K. pneumoniae (K. p.) recovered (A) or the percent K. pneumoniae
survival compared to the starting inoculum (0 min) (B). The results are presented as the mean ⫾ SEM
from 3 separate experiments (with blood from 3 random donors), as indicated. *, P ⬍ 0.05 versus the
starting inoculum (0 min) for each strain using a repeated-measures one-way ANOVA and Dunnett’s
posttest. Asterisks are color coded to match the associated strain. (C) Bactericidal activity of NHS toward
20 selected ST258 clinical isolates or single-locus variants (ST379, ST418, and ST512) (from reference 30).
The isolates contain cps-1 or cps-2 gene clusters, as indicated. (D) Bactericidal activity of 100% NHS after
depletion of IgG using protein G-Sepharose (Ab-depl.) or control Sepharose beads (Ctl). *, P ⬍ 0.05 versus
the control using a ratio paired t test.

the rate of survival was 105.9% ⫾ 11.8% for isolate 34446 but 0.04% ⫾ 0.01% for isolate
NJST258_1) (Fig. 1B). The limited survival of 3 ST258 isolates (NJST258_1, NJST258_2,
and 28577) in these assays is not likely explained by killing by phagocytic cells, since
previous studies have demonstrated that neutrophil phagocytosis of these isolates is
limited (29). Therefore, components in human blood other than neutrophils, such as
serum complement, contribute to the observed bactericidal activity.
Killing of K. pneumoniae ST258 in human blood in vitro is attributed largely to
serum complement. To elucidate the basis of the observed bactericidal activity in
human blood, we next evaluated the ability of ST258 clinical isolates to survive in NHS.
All isolates but one (NJST258_1) grew in the presence of serum at concentrations of up
to 25% (Fig. 2A and B). In contrast, there was significant killing of 4 of the 6 isolates
tested in 100% NHS (Fig. 2A and B), and NJST258_1 was destroyed in the presence of
NHS at concentrations greater than 5% (Fig. 2 and 3). Notably, the survival of K.
pneumoniae isolates (except 35602) in 100% NHS was, in general, similar to that in
human blood at 30 or 60 min (compare Fig. 1B and 2B). Results with 20 selected clinical
isolates—including the 6 isolates tested in blood—revealed that survival in NHS was
similar for the two major ST258 clades (30), which are defined by gene clusters
encoding the capsule polysaccharide biosynthesis machinery (cps-1 or cps-2) (31) (Fig.
2C). Depletion of IgG from NHS significantly increased the survival of NJST258_1 in
serum, a finding consistent with the presence of naturally occurring K. pneumoniae-
specific antibody (Ab) in NHS (32) and the known ability of antibody to activate the
complement cascade (Fig. 2D). The finding that these K. pneumoniae clinical isolates
were susceptible to components in NHS is intriguing, because ST258 is well-known to

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 3

DeLeo et al. Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 3 Destruction of isolate NJST258_1 by components of NHS. K. pneumoniae isolate NJST258_1 or
35602 was incubated in 50% NHS for 0 min and 30 min, and samples were processed for transmission
electron microscopy. Representative images are shown. Bars ⫽ 1 ␮m. The brightness and contrast of the
images were adjusted in Adobe Photoshop CC software.

cause fatal bacteremia, albeit in individuals with significant comorbidities, such as

immunosuppression (14–20).
To determine whether serum complement components contribute to the ob-
served killing of K. pneumoniae in serum, we first measured the surface association
of C5b to C9 (C5b-C9), complement molecules that form the membrane attack
complex (MAC) (33), by flow cytometry (Fig. 4). Although there was a varied
association of the MAC with these K. pneumoniae isolates after incubation in 5%
NHS (Fig. 4A and B), all isolates had surface-bound C5b-C9 after incubation in 50%
NHS (Fig. 4A to C). Thus, the decreased survival of 4 K. pneumoniae isolates in NHS
correlates with surface-bound C5b-C9 (MAC).
We next evaluated the survival of K. pneumoniae isolates in heat-inactivated serum
or serum pretreated with cobra venom factor (CVF) (34) or FUT-175 (35, 36), widely used
inhibitors of the complement system (Fig. 5A to C). The rate of survival of all ST258
isolates in heat-inactivated serum was increased significantly (⬎100% survival) com-
pared to that in untreated NHS (Fig. 5A). Consistent with these findings, pretreatment
of NHS or human heparinized blood with CVF or FUT-175 increased the rate of survival
of some of the isolates compared to that in untreated serum or blood (Fig. 5B to E). The
basis for the observed differences in survival for isolates in NHS pretreated with CVF or
FUT-175 is not clear, but they inhibit complement via distinct mechanisms. Taken
together, these data provide support to the idea that the killing of ST258 in human
blood is mediated in part by the complement MAC, results compatible with those
presented in previous reports (37, 38).
Survival of ST258 in mouse blood in vitro and near mortality in a mouse
bacteremia model. Although carbapenem-resistant K. pneumoniae ST258 causes bac-

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 4

Survival of K. pneumoniae in Blood Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 4 Deposition of complement on the surface of ST258 isolates. K. pneumoniae strains were opsonized
in 5% or 50% NHS, as indicated, and the surface association of complement C5b-C9 was determined by
flow cytometry. (A) Representative histograms of bacteria stained with isotype control MAb (IgG2a) or
anti-C5b-C9 (␣C5b-9) are shown. (B, C) Quantitation of the mean fluorescence (geometric mean) for
bacteria opsonized in 5% (B) or 50% (C) NHS. The results are the mean ⫾ SEM from 3 separate
experiments (with blood from 3 random donors).

teremia that is associated with high rates of morbidity and mortality in humans, it
remains unclear whether resistance to serum complement contributes to disease
severity in these patients. As a first step toward addressing this issue, we tested the
hypothesis that the survival of ST258 isolates in mouse blood correlates with and is
linked to virulence in a mouse bacteremia model. Unexpectedly, there was greater than
100% survival (growth) for all ST258 isolates tested in heparinized mouse blood (Fig.
6A). These findings are at variance with data obtained using human blood (compare
Fig. 1A and 6A) but could be explained in part by the paucity of antibody that
recognizes K. pneumoniae (the mouse is naive) and thus fails to contribute to comple-
ment activation. Consistent with this idea, addition of rabbit IgG specific for K. pneu-
moniae to heparinized mouse blood reduced the rate of survival of NJST258_2 by
20.8% ⫾ 2.9% after 120 min compared to that in assays containing IgG from preim-
mune sera (n ⫽ 3, P ⬍ 0.05, using a one-way analysis of variance [ANOVA] and Tukey’s
posttest).
Inasmuch as there was 100% survival of each of the K. pneumoniae strains in mouse
blood, including NJST258_1 (which was readily killed in human blood), we predicted
that these isolates would cause similar levels of mortality in a mouse bacteremia model.
However, at 7 days postinfection there was 100% survival for mice infected with 3 of
the 6 isolates (NJST258_1, 34446, and 28577) and ⬃50% survival for mice infected with
the other 3 isolates (NJST258_2, 35602, and 15692) (Fig. 6B). Thus, bactericidal activity
data from human or mouse blood in vitro had little or no correlation with lethality in
the mouse bacteremia model.
Conclusions. Carbapenem-resistant K. pneumoniae infections are often associated
with bacteremia. The rate of mortality among these patients is high (⬃50%), primarily
because of patient comorbidities and limited treatment options (7, 14–22). In the

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 5

DeLeo et al. Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 5 Complement-mediated killing of K. pneumoniae isolates. NHS (A to C) or heparinized human blood
(D, E) was heat inactivated (HI; NHS only) (A), pretreated with CVF (B, D), or pretreated with FUT-175 (C,
E) as described in Materials and Methods, and the survival of K. pneumoniae in each assay was
determined after 30 min. The results are the mean ⫾ SEM from the indicated number of experiments
(with blood from the same number of different donors). *, P ⬍ 0.05 versus untreated (no heat inactivation
or no inhibitor) serum or blood controls (open boxes) using a ratio paired t test.

United States and elsewhere, many of these infections are caused by ST258 and closely
related strains (6, 10, 12). The relatively high burden of ST258 strains compared with
that of other multidrug-resistant lineages of K. pneumoniae is not readily explained by
comorbidities or antibiotic resistance. Therefore, other characteristics, including resis-
tance to serum complement or a lack of complement-mediated killing in infected
patients, likely contribute to the success of ST258. To that end, we evaluated the ability
of ST258 clinical isolates to survive in normal human blood and serum.
We found that there is differential survival of ST258 clinical isolates in human blood
and serum, results partially at variance with those from two recent studies (37, 38). The
finding that some of the isolates tested were readily killed in human blood or serum
was surprising to us, since bacteremia is relatively common (among individuals with
infections caused by carbapenem-resistant K. pneumoniae isolates) and ST258 isolates
whose genomes encode the same capsule polysaccharide subtype (e.g., cps-2) are
genetically very similar (30, 39). Previous studies have shown that the Klebsiella lipo-
polysaccharide (LPS) O antigen and/or the capsule polysaccharide contributes to
resistance to the bactericidal activity of human serum (40–45). Merino et al. proposed
that the deposition of complement C3b occurs too far from the membrane of serum-
resistant strains of K. pneumoniae, and thus, the C5b-C9 MAC fails to form (43).
However, we found that the C5b-C9 complex is present on the surface of serum-
resistant and -susceptible K. pneumoniae isolates, and therefore, the segregation of the
C5b-C9 MAC from the bacterial membrane cannot fully explain the serum resistance
phenotype of some of the ST258 isolates.
Isolates NJST258_1 and NJST258_2 were killed to varied degrees in human blood,
and NJST258_2 was significantly less sensitive than NJST258_1 to the bactericidal

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 6

Survival of K. pneumoniae in Blood Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
FIG 6 Survival of ST258 isolates in mouse blood and mouse bacteremia model. (A) K. pneumoniae was
combined with heparinized mouse blood for the indicated times, and survival was determined as
described in Materials and Methods. (B) BALB/c mice (15 mice per strain group, 5 mice for the uninfected
saline control group) were infected i.v. with the indicated K. pneumoniae isolates (7 ⫻ 107 CFU), and
mouse survival was determined as described in Materials and Methods. Data were analyzed using a
log-rank test (GraphPad Prism software, version 6.05), and none of the curves were significantly different
from the others at a P value of ⬍0.05 after correction for multiple comparisons.

activity of normal human serum (Fig. 2A and B). There was no detectable C5b-C9 on the
surface of NJST258_2 after opsonization in 5% NHS, whereas it was detected on the
surface NJST258_1 under the same conditions (Fig. 4A and B). This observation is
notable, because the genomes of NJST258_1 and NJST258_2 encode the same capsule
polysaccharide subtype (cps-2) and the genes encoding proteins reported to be in-
volved in LPS O-antigen biosynthesis (wzm, wzt, wbbM, glf, wbbN, and wbbO) (46) are
100% identical between these two strains (30). The molecular basis for the difference
in serum susceptibility between these isolates merits further investigation.
On the basis of the observed killing of ST258 isolates in human blood (Fig. 1), the
inability of mouse blood to kill these bacterial isolates during the same time frame (up
to 120 min) was unexpected. Shankar-Sinha et al. reported similar results with mouse
blood during their studies of the role of K. pneumoniae O antigen in bacteremia (47). It
is possible that the presence of naturally occurring K. pneumoniae-specific antibodies in
normal human serum (32), which are presumably absent in the naive mouse, contrib-
utes in part to the noted difference in bactericidal activity. The results of our IgG
depletion assays (Fig. 2D) and preliminary data indicating that K. pneumoniae-specific
IgG promotes the bactericidal activity of mouse blood support this idea. Differences
between mouse and human serum complement systems or other components of the
innate immune system may also contribute to the differences in bactericidal activity
observed between human and mouse blood. Nonetheless, there was no correlation

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 7

DeLeo et al. Antimicrobial Agents and Chemotherapy

between survival in mouse blood in vitro and the ability of these clinical isolates to
cause mouse mortality (measured as near-death euthanasia) in a bacteremia model.
Although the rate of mortality for mice infected with isolate 35602 or NJST258_2 was
significantly different from that for control mice given saline (P ⱕ 0.035 for each isolate
using a log-rank test) (Fig. 6B), there was no significant difference among the strains
after correction for multiple comparisons (Bonferroni’s posttest). Thus, the virulence of
these clinical isolates in the mouse bacteremia model is, in general, limited. It is

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
noteworthy that K. pneumoniae is normally a colonizer of humans that causes no
symptoms and causes disease only in individuals with comorbidities. Indeed, ST258 has
not been reported to be a significant cause of infection outside of the health care
setting.
Our finding that ST258 clinical isolates are killed in normal human blood and serum,
albeit to varied degrees, is compatible with the notion presented above that the
virulence capacity of this lineage is limited (37). Importantly, the complement system of
healthy individuals is important for host defense against organisms such as ST258
strains. The ability of naturally occurring antibody to contribute to the killing of K.
pneumoniae in serum is intriguing in the context of potential antibody-based thera-
peutic approaches. An enhanced understanding of the healthy host response to this
pathogen is needed as the field moves forward to develop immune-based therapies to
prevent or treat infections caused by carbapenem-resistant K. pneumoniae strains.

MATERIALS AND METHODS

Human subjects and ethics statement. Blood and serum were obtained from 17 different healthy
subjects (age range, 24 to 64 years). All subjects (blood donors) were healthy and immunocompetent and
had no known recent illness. Studies with human blood and serum were approved by the Institutional
Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases (protocol
01-I-N055). The studies conformed to the principles set forth in the Declaration of Helsinki. Subjects gave
written informed consent prior to participation in the study.
K. pneumoniae strains/clinical isolates. K. pneumoniae clinical isolates NJST258_1, NJST258_2,
35602, 34446, 15692, and 28577 were selected for these studies on the basis of their multilocus sequence
type (they were ST258 or single-locus variant ST379 [isolate 28577]), carbapenemase resistance profile
(KPC-2 or KPC-3), and resistance to neutrophil phagocytosis (29, 30). The general characteristics of
NJST258_1, NJST258_2, 34446, 28577, and 16 selected ST258 isolates or single-locus variants (cps-1 and
cps-2) were reported previously (29, 30). Isolates 35602 and 15692 are ST258 isolates (each carries KPC-3
and cps-2) from patients in Florida (isolated in 2012) and New Jersey (isolated in 2005), respectively.
Bacteria from frozen stocks were cultured in Luria-Bertani (LB) broth overnight at 37°C with shaking (220
rpm). Overnight cultures were diluted 1/200 and recultured to mid-exponential growth phase (optical
density at 600 nm, 0.75) for all experiments unless stated otherwise.
Bactericidal activity assays with blood and serum. The survival of bacteria in heparinized blood
was determined using a standard assay (48), but with a few modifications. For assays with human blood,
K. pneumoniae was added to heparinized human blood (5 U heparin/ml blood) in 1.7-ml tubes (1.7-ml
Ultra Clear microtubes [polypropylene] certified to be RNase, DNase, and pyrogen free; Phenix Research
Products) in a final volume of 550 ␮l (⬃1 ⫻ 106 CFU/ml blood). For some experiments, the complement
inhibitor FUT-175 (50 ␮g/ml, incubated for 30 min on ice) or cobra venom factor (CVF; 10 ␮g/ml,
incubated for 60 min at 37°C) was added to heparinized blood for the times indicated above and in the
figures before adding bacteria. For assays with mouse blood, K. pneumoniae was added to heparinized
mouse blood (10 U heparin/ml blood) in 1.7-ml tubes (Ultra Clear microtubes; Phenix Research Products)
in a final volume of 210 to 220 ␮l (⬃1 ⫻ 106 CFU/ml blood). In some experiments, purified rabbit IgG
specific for K. pneumoniae (29) or control IgG from rabbit nonimmune serum was added to the assay
mixtures at a 5-␮g/ml final concentration. Assay mixtures were rotated slowly at 37°C until the desired
time point. At the times indicated above and in the figures, K. pneumoniae was plated on LB agar and
the colonies were enumerated the next day. Percent survival was calculated with the equation (CFUtn/
CFUt0) ⫻ 100, where CFUt0 is the number of CFU in the starting inoculum at time zero (t0) and CFUtn is
the number of CFU of bacteria recovered at the specified time point (tn).
Normal human serum (NHS) was prepared fresh or frozen and thawed once for bactericidal activity
assays. Bacteria were suspended at ⬃1 ⫻ 108 CFU/ml in NHS, and 320 to 400 ␮l of the bacterium-serum
suspension was aliquoted and placed into a 1.7-ml tube (Ultra Clear microtubes; Phenix Research
Products), which was then incubated for the times specified above and in the figures at 37°C with
shaking. In some experiments, FUT-175 (50 ␮g/ml, incubated for 30 to 45 min on ice) or CVF (10 ␮g/ml,
incubated for 60 min at 37°C) was added to serum prior to adding bacteria. Alternatively, NHS was
inactivated by heating at 56°C for 30 min prior to use in assays or antibody was depleted from serum
prior to use in assays. Protein G-Sepharose was used to deplete antibody from NHS as follows. A 1-ml
protein G-Sepharose (Abcam) slurry was centrifuged to pellet the Sepharose, the supernatant was
aspirated, and the Sepharose was resuspended in 1 ml of NHS. The mixture was rotated for 40 min at
room temperature. The Ab-depleted serum was recovered following centrifugation and transferred to a

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 8

Survival of K. pneumoniae in Blood Antimicrobial Agents and Chemotherapy

tube containing fresh protein G-Sepharose. The process was repeated for 3 sequential rounds in total to
deplete the Ab from serum. The Ab depletion process was performed using fresh NHS for each of the 3
separate experiments (with blood from a separate donor for each experiment). An aliquot of protein
G-Sepharose from each round of Ab depletion was washed twice in Dulbecco’s phosphate-buffered
saline (DPBS; Sigma) and analyzed by SDS-PAGE for the depletion of Ab. Percent survival for all serum
assays was calculated as described above for the assay with blood.
Transmission electron microscopy. K. pneumoniae was cultured/opsonized in 50% NHS for 30 min
at 37°C as described above, and samples were processed for transmission electron microscopy as
described previously (29). The brightness and contrast of images were adjusted in Adobe Photoshop CC

Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
software.
Analysis of serum complement deposition by flow cytometry. K. pneumoniae (2 ⫻ 107 CFU) at
mid-exponential phase of growth was pelleted by centrifugation (2,400 relative centrifugal force [RCF] for
4 min at room temperature), washed once in sterile DPBS, and resuspended in DBPS. Bacteria were
opsonized in 5% or 50% fresh NHS for 30 min at 37°C and then pelleted by centrifugation. The bacteria
were washed once in DPBS, resuspended in blocking buffer (DPBS containing 5% normal goat serum
[NGS]), and placed on ice for 1 h. Bacteria were pelleted as described above and resuspended in DPBS,
and the cells were incubated with anti-C5b-C9 or IgG2a isotype control monoclonal antibodies (MAbs;
each at 0.1 ␮g/ml) for 30 min on ice. DPBS (0.8 ml) containing 2% NGS was added to each tube, and the
bacterium-MAb mixtures were centrifuged at 4,300 RCF for 8 min at room temperature to pellet the cells.
Bacteria were resuspended in DPBS, a fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse
secondary Ab was added to the tubes, and the bacteria were incubated on ice for 30 min. The bacteria
were centrifuged, resuspended in 200 ␮l DBPS containing 2% NGS, and analyzed by flow cytometry
(FACSCalibur flow cytometer; BD Biosciences). Ten thousand events were collected for each sample, and
the geometric mean was used for quantitation.
Mouse infection studies. All animal studies were reviewed and approved by the Institutional Animal
Care and Use Committee, Rocky Mountain Laboratories, NIAID/NIH (protocol RML 2016-022-E). BALB/c
mice (age, 6 to 8 weeks) were infected intravenously (i.v.) with 7 ⫻ 107 CFU K. pneumoniae (in 100 ␮l
injection-grade saline) by tail vein inoculation. Fifteen mice were infected with each strain, and 5 mice
were used as uninfected controls treated with DPBS. Following inoculation, animal health was monitored
every 3 h for the first 24 h, every 6 h for the second 24 h, and then every 24 h thereafter for up to 7 days
(the end of the experiment). Weight was monitored daily. The criteria for determining morbidity/sickness
in mice included hunched posture, decreased activity, ruffled fur, and labored breathing. Animals were
euthanized if they were unable to eat or drink, exhibited respiratory distress, had a ⱖ20% decrease in
body weight, or became immobile. Mice that required euthanasia (near mortality) were scored as a death.
All remaining mice were euthanized at 7 days postinfection.
Statistics. All statistics were determined using GraphPad Prism (version 6.05) software (GraphPad
Software, Inc.). Analysis of two data groups was performed using a ratio paired t test. Comparisons of
three or more data groups were performed using a repeated-measures one-way ANOVA and Dunnett’s
or Tukey’s posttest. Survival data were analyzed using a log-rank test and were corrected for multiple
comparisons using Bonferroni’s posttest.

ACKNOWLEDGMENTS
This work was supported by the Intramural Research Program of the National
Institute of Allergy and Infectious Diseases, National Institutes of Health, and grants
from the National Institutes of Health (R01AI090155 to B.N.K. and R21AI117338 to L.C.).

REFERENCES
1. Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA,
Lynfield R, Maloney M, McAllister-Hollod L, Nadle J, Ray SM, Thompson
DL, Wilson LE, Fridkin SK, Emerging Infections Program Healthcare-
Associated Infections and Antimicrobial Use Prevalence Survey Team.
2014. Multistate point-prevalence survey of health care-associated
infections. N Engl J Med 370:1198 –1208. https://doi.org/10.1056/
NEJMoa1306801.
2. Bone RC. 1993. Gram-negative sepsis: a dilemma of modern medicine.
Clin Microbiol Rev 6:57– 68. https://doi.org/10.1128/CMR.6.1.57.
3. Haeggman S, Lofdahl S, Burman LG. 1997. An allelic variant of the
chromosomal gene for class A beta-lactamase K2, specific for Klebsiella
pneumoniae, is the ancestor of SHV-1. Antimicrob Agents Chemother
41:2705–2709.
4. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW,
Steward CD, Alberti S, Bush K, Tenover FC. 2001. Novel carbapenem-
hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain
of Klebsiella pneumoniae. Antimicrob Agents Chemother 45:1151–1161.
https://doi.org/10.1128/AAC.45.4.1151-1161.2001.
5. Livermore DM. 2009. Has the era of untreatable infections arrived? J
Antimicrob Chemother 64(Suppl 1):i29 –i36. https://doi.org/10.1093/jac/
dkp255.
6. Kitchel B, Rasheed JK, Patel JB, Srinivasan A, Navon-Venezia S, Carmeli Y,
Brolund A, Giske CG. 2009. Molecular epidemiology of KPC-producing
Klebsiella pneumoniae isolates in the United States: clonal expansion of
multilocus sequence type 258. Antimicrob Agents Chemother 53:
3365–3370. https://doi.org/10.1128/AAC.00126-09.
7. Guh AY, Bulens SN, Mu Y, Jacob JT, Reno J, Scott J, Wilson LE, Vaeth E,
Lynfield R, Shaw KM, Vagnone PM, Bamberg WM, Janelle SJ, Dumyati G,
Concannon C, Beldavs Z, Cunningham M, Cassidy PM, Phipps EC, Ken-
slow N, Travis T, Lonsway D, Rasheed JK, Limbago BM, Kallen AJ. 2015.
Epidemiology of carbapenem-resistant Enterobacteriaceae in 7 US com-
munities, 2012-2013. JAMA 314:1479 –1487. https://doi.org/10.1001/jama
.2015.12480.
8. Endimiani A, Hujer AM, Perez F, Bethel CR, Hujer KM, Kroeger J,
Oethinger M, Paterson DL, Adams MD, Jacobs MR, Diekema DJ, Hall GS,
Jenkins SG, Rice LB, Tenover FC, Bonomo RA. 2009. Characterization of
blaKPC-containing Klebsiella pneumoniae isolates detected in different
institutions in the eastern USA. J Antimicrob Chemother 63:427– 437.
https://doi.org/10.1093/jac/dkn547.
9. Endimiani A, Depasquale JM, Forero S, Perez F, Hujer AM, Roberts-
Pollack D, Fiorella PD, Pickens N, Kitchel B, Casiano-Colon AE, Tenover
FC, Bonomo RA. 2009. Emergence of blaKPC-containing Klebsiella pneu-
moniae in a long-term acute care hospital: a new challenge to our

April 2017 Volume 61 Issue 4 e02533-16 aac.asm.org 9

DeLeo et al.
healthcare system. J Antimicrob Chemother 64:1102–1110. https://
doi.org/10.1093/jac/dkp327.
10. Grundmann H, Livermore DM, Giske CG, Canton R, Rossolini GM, Campos
J, Vatopoulos A, Gniadkowski M, Toth A, Pfeifer Y, Jarlier V, Carmeli Y,
CNSE Working Group. 2010. Carbapenem-non-susceptible Enterobacte-
riaceae in Europe: conclusions from a meeting of national experts. Euro
Surveill 15(46):pii⫽19711. http://www.eurosurveillance.org/View
Article.aspx?ArticleId⫽19711.
11. Nordmann P, Naas T, Poirel L. 2011. Global spread of carbapenemase-
producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. https://
doi.org/10.3201/eid1710.110655.
12. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M,
Cornaglia G, Garau J, Gniadkowski M, Hayden MK, Kumarasamy K, Liver-
more DM, Maya JJ, Nordmann P, Patel JB, Paterson DL, Pitout J, Villegas MV,
Wang H, Woodford N, Quinn JP. 2013. Clinical epidemiology of the global
expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 13:
785–796. https://doi.org/10.1016/S1473-3099(13)70190-7.
13. Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN.
2014. Carbapenemase-producing Klebsiella pneumoniae: molecular and
genetic decoding. Trends Microbiol 22:686 – 696. https://doi.org/
10.1016/j.tim.2014.09.003.
14. Ben-David D, Kordevani R, Keller N, Tal I, Marzel A, Gal-Mor O, Maor Y,
Rahav G. 2012. Outcome of carbapenem resistant Klebsiella pneumoniae
bloodstream infections. Clin Microbiol Infect 18:54 – 60. https://doi.org/
10.1111/j.1469-0691.2011.03478.x.
15. Clancy CJ, Chen L, Shields RK, Zhao Y, Cheng S, Chavda KD, Hao B, Hong
JH, Doi Y, Kwak EJ, Silveira FP, Abdel-Massih R, Bogdanovich T, Humar A,
Perlin DS, Kreiswirth BN, Hong Nguyen M. 2013. Epidemiology and
molecular characterization of bacteremia due to carbapenem-resistant
Klebsiella pneumoniae in transplant recipients. Am J Transplant 13:
2619 –2633. https://doi.org/10.1111/ajt.12424.
16. Kontopidou F, Giamarellou H, Katerelos P, Maragos A, Kioumis I, Trikka-
Graphakos E, Valakis C, Maltezou HC, Group for the Study of KPC-
Producing Klebsiella pneumoniae Infections in Intensive Care Units.
2014. Infections caused by carbapenem-resistant Klebsiella pneumoniae
among patients in intensive care units in Greece: a multi-centre study on
clinical outcome and therapeutic options. Clin Microbiol Infect 20:
O117–O123. https://doi.org/10.1111/1469-0691.12341.
17. Alicino C, Giacobbe DR, Orsi A, Tassinari F, Trucchi C, Sarteschi G, Copello
F, Del Bono V, Viscoli C, Icardi G. 2015. Trends in the annual incidence of
carbapenem-resistant Klebsiella pneumoniae bloodstream infections: a
8-year retrospective study in a large teaching hospital in northern Italy.
BMC Infect Dis 15:415. https://doi.org/10.1186/s12879-015-1152-0.
18. Diaz A, Ortiz DC, Trujillo M, Garces C, Jaimes F, Restrepo AV. 2016.
Clinical characteristics of carbapenem-resistant Klebsiella pneumoniae
infections in ill and colonized children in Colombia. Pediatr Infect Dis J
35:237–241. https://doi.org/10.1097/INF.0000000000000987.
19. Falcone M, Russo A, Iacovelli A, Restuccia G, Ceccarelli G, Giordano A,
Farcomeni A, Morelli A, Venditti M. 2016. Predictors of outcome in ICU
patients with septic shock caused by Klebsiella pneumoniae
carbapenemase-producing K. pneumoniae. Clin Microbiol Infect 22:
444 – 450. https://doi.org/10.1016/j.cmi.2016.01.016.
20. Pouch SM, Satlin MJ. 28 July 2016. Carbapenem-resistant Enterobacte-
riaceae in special populations: solid organ transplant recipients, stem
cell transplant recipients, and patients with hematologic malignancies.
Virulence. https://doi.org/10.1080/21505594.2016.1213472:1-12.
21. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, Schlaeffer
F, Sherf M. 2009. Attributable mortality rate for carbapenem-resistant
Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 30:
972–976. https://doi.org/10.1086/605922.
22. Viale P, Giannella M, Lewis R, Trecarichi EM, Petrosillo N, Tumbarello M.
2013. Predictors of mortality in multidrug-resistant Klebsiella pneu-
moniae bloodstream infections. Expert Rev Anti Infect Ther 11:
1053–1063. https://doi.org/10.1586/14787210.2013.836057.
23. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong
B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu
JH, Shen J. 2016. Emergence of plasmid-mediated colistin resistance
mechanism MCR-1 in animals and human beings in China: a microbio-
logical and molecular biological study. Lancet Infect Dis 16:161–168.
https://doi.org/10.1016/S1473-3099(15)00424-7.
24. Du H, Chen L, Tang YW, Kreiswirth BN. 2016. Emergence of the mcr-1
colistin resistance gene in carbapenem-resistant Enterobacteriaceae.
Lancet Infect Dis 16:287–288. https://doi.org/10.1016/S1473-3099(16)
00056-6.
April 2017 Volume 61 Issue 4 e02533-16
Antimicrobial Agents and Chemotherapy
25. Shields RK, Clancy CJ, Hao B, Chen L, Press EG, Iovine NM, Kreiswirth BN,
Nguyen MH. 2015. Effects of Klebsiella pneumoniae carbapenemase
subtypes, extended-spectrum beta-lactamases, and porin mutations on
the in vitro activity of ceftazidime-avibactam against carbapenem-
resistant K. pneumoniae. Antimicrob Agents Chemother 59:5793–5797.
https://doi.org/10.1128/AAC.00548-15.
26. Shields RK, Potoski BA, Haidar G, Hao B, Doi Y, Chen L, Press EG,
Kreiswirth BN, Clancy CJ, Nguyen MH. 2016. Clinical outcomes, drug
toxicity, and emergence of ceftazidime-avibactam resistance among
patients treated for carbapenem-resistant Enterobacteriaceae infections.
Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
Clin Infect Dis 63:1615–1618. https://doi.org/10.1093/cid/ciw636.
27. Spellberg B, Bonomo RA. 2016. Editorial commentary: ceftazidime-
avibactam and carbapenem-resistant Enterobacteriaceae: “we’re gonna
need a bigger boat.” Clin Infect Dis 63:1619 –1621. https://doi.org/
10.1093/cid/ciw639.
28. Cuzon G, Naas T, Truong H, Villegas MV, Wisell KT, Carmeli Y, Gales AC,
Venezia SN, Quinn JP, Nordmann P. 2010. Worldwide diversity of Kleb-
siella pneumoniae that produce beta-lactamase blaKPC-2 gene. Emerg
Infect Dis 16:1349 –1356. https://doi.org/10.3201/eid1609.091389.
29. Kobayashi SD, Porter AR, Dorward DW, Brinkworth AJ, Chen L, Kreiswirth
BN, DeLeo FR. 2016. Phagocytosis and killing of carbapenem-resistant
ST258 Klebsiella pneumoniae by human neutrophils. J Infect Dis 213:
1615–1622. https://doi.org/10.1093/infdis/jiw001.
30. DeLeo FR, Chen L, Porcella SF, Martens CA, Kobayashi SD, Porter AR,
Chavda KD, Jacobs MR, Mathema B, Olsen RJ, Bonomo RA, Musser JM,
Kreiswirth BN. 2014. Molecular dissection of the evolution of
carbapenem-resistant multilocus sequence type 258 Klebsiella pneu-
moniae. Proc Natl Acad Sci U S A 111:4988 – 4993. https://doi.org/
10.1073/pnas.1321364111.
31. Chen L, Chavda KD, Findlay J, Peirano G, Hopkins K, Pitout JD, Bonomo
RA, Woodford N, DeLeo FR, Kreiswirth BN. 2014. Multiplex PCR for
identification of two capsular types in epidemic KPC-producing Klebsiella
pneumoniae sequence type 258 strains. Antimicrob Agents Chemother
58:4196 – 4199. https://doi.org/10.1128/AAC.02673-14.
32. Lepper PM, Moricke A, Held TK, Schneider EM, Trautmann M. 2003.
K-antigen-specific, but not O-antigen-specific natural human serum anti-
bodies promote phagocytosis of Klebsiella pneumoniae. FEMS Immunol
Med Microbiol 35:93–98. https://doi.org/10.1016/S0928-8244(02)00459-5.
33. Serna M, Giles JL, Morgan BP, Bubeck D. 2016. Structural basis of
complement membrane attack complex formation. Nat Commun
7:10587. https://doi.org/10.1038/ncomms10587.
34. Vogel CW, Fritzinger DC. 2010. Cobra venom factor: structure, function,
and humanization for therapeutic complement depletion. Toxicon 56:
1198 –1222. https://doi.org/10.1016/j.toxicon.2010.04.007.
35. Inagi R, Miyata T, Maeda K, Sugiyama S, Miyama A, Nakashima I. 1991.
FUT-175 as a potent inhibitor of C5/C3 convertase activity for production
of C5a and C3a. Immunol Lett 27:49 –52. https://doi.org/10.1016/0165
-2478(91)90243-4.
36. Ikari N, Sakai Y, Hitomi Y, Fujii S. 1983. New synthetic inhibitor to the
alternative complement pathway. Immunology 49:685– 691.
37. Tzouvelekis LS, Miriagou V, Kotsakis SD, Spyridopoulou K, Athanasiou E,
Karagouni E, Tzelepi E, Daikos GL. 2013. KPC-producing, multidrug-
resistant Klebsiella pneumoniae sequence type 258 as a typical oppor-
tunistic pathogen. Antimicrob Agents Chemother 57:5144 –5146.
https://doi.org/10.1128/AAC.01052-13.
38. Chiang TT, Yang YS, Yeh KM, Chiu SK, Wang NC, Lin TY, Huang LY, Chang
FY, Siu LK, Lin JC, Chen JH. 2016. Quantification and comparison of
virulence and characteristics of different variants of carbapenemase-
producing Klebsiella pneumoniae clinical isolates from Taiwan and the
United States. J Microbiol Immunol Infect 49:83–90. https://doi.org/
10.1016/j.jmii.2015.08.011.
39. Chen L, Mathema B, Pitout JD, DeLeo FR, Kreiswirth BN. 2014. Epidemic
Klebsiella pneumoniae ST258 is a hybrid strain. mBio 5:e01355-14.
https://doi.org/10.1128/mBio.01355-14.
40. Tomas JM, Benedi VJ, Ciurana B, Jofre J. 1986. Role of capsule and O
antigen in resistance of Klebsiella pneumoniae to serum bactericidal
activity. Infect Immun 54:85– 89.
41. Williams P, Lambert PA, Brown MR, Jones RJ. 1983. The role of the O and
K antigens in determining the resistance of Klebsiella aerogenes to serum
killing and phagocytosis. J Gen Microbiol 129:2181–2191.
42. Yeh KM, Chiu SK, Lin CL, Huang LY, Tsai YK, Chang JC, Lin JC, Chang FY,
Siu LK. 2016. Surface antigens contribute differently to the pathophys-
iological features in serotype K1 and K2 Klebsiella pneumoniae strains
aac.asm.org 10
Survival of K. pneumoniae in Blood
isolated from liver abscesses. Gut Pathog 8:4. https://doi.org/10.1186/
s13099-016-0085-5.
43. Merino S, Camprubi S, Alberti S, Benedi VJ, Tomas JM. 1992. Mechanisms
of Klebsiella pneumoniae resistance to complement-mediated killing.
Infect Immun 60:2529 –2535.
44. Alvarez D, Merino S, Tomas JM, Benedi VJ, Alberti S. 2000. Capsular
polysaccharide is a major complement resistance factor in lipopolysac-
charide O side chain-deficient Klebsiella pneumoniae clinical isolates.
Infect Immun 68:953–955. https://doi.org/10.1128/IAI.68.2.953-955.2000.
45. Szijarto V, Guachalla LM, Hartl K, Varga C, Banerjee P, Stojkovic K,
Kaszowska M, Nagy E, Lukasiewicz J, Nagy G. 2016. Both clades of the
epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a
modified galactan O-antigen type. Int J Med Microbiol 306:89 –98.
https://doi.org/10.1016/j.ijmm.2015.12.002.
April 2017 Volume 61 Issue 4 e02533-16
Antimicrobial Agents and Chemotherapy
46. Guan S, Clarke AJ, Whitfield C. 2001. Functional analysis of the galacto-
syltransferases required for biosynthesis of D-galactan I, a component of
the lipopolysaccharide O1 antigen of Klebsiella pneumoniae. J Bacteriol
183:3318 –3327. https://doi.org/10.1128/JB.183.11.3318-3327.2001.
47. Shankar-Sinha S, Valencia GA, Janes BK, Rosenberg JK, Whitfield C,
Bender RA, Standiford TJ, Younger JG. 2004. The Klebsiella pneumoniae
O antigen contributes to bacteremia and lethality during murine pneu-
monia. Infect Immun 72:1423–1430. https://doi.org/10.1128/IAI.72
.3.1423-1430.2004.
48. Eraso J, Olsen RJ, Beres SB, Kachroo P, Porter AR, Nasser W, Bernard PE,
Downloaded from http://aac.asm.org/ on March 15, 2018 by INST OF MOLECULAR & CELL BIO
DeLeo F, Musser JM. 2016. Genomic landscape of intrahost variation in
group A Streptococcus: repeated and abundant mutational inactivation
of the fabT gene encoding a regulator of fatty acid synthesis. Infect
Immun 84:3268 –3281. https://doi.org/10.1128/IAI.00608-16.
aac.asm.org 11