Received: 1 August 2017 | Revised: 7 June 2018 | Accepted: 24 June 2018

DOI: 10.1111/vcp.12679

ORIGINAL RESEARCH

Aggregating resistant Staphylococcus aureus induces

hypocoagulability, hyperfibrinolysis, phagocytosis, and
neutrophil, monocyte, and lymphocyte binding in canine
whole blood

Anne K. H. Krogh1 | Jakob Haaber2 | Louise Bochsen1 | Hanne Ingmer2 | Annemarie

T. Kristensen1

1
Department of Veterinary Clinical
Sciences, University of Copenhagen,
Background: Staphylococcus aureus is an opportunistic pathogen with the ability to
Frederiksberg, Denmark form mobile planktonic aggregates during growth, in vitro. The in vivo pathophysio-
2
Department of Veterinary and Animal logic effects of S aureus aggregates on host responses are unknown. Knowledge of
Sciences, University of Copenhagen,
Frederiksberg, Denmark these could aid in combating infections.
Objective: This study aimed to investigate the effect of increasing concentrations
Correspondence
A.K.H. Krogh, Department of Veterinary of two different aggregating S aureus strains on the hemostatic and inflammatory
Clinical Sciences, University of Copenhagen, host responses in canine whole blood. The hypothesis was that aggregating bacteria
Frederiksberg, Denmark.
Email: akrk@sund.ku.dk would induce pronounced hemostatic and inflammatory responses.
Methods: Citrate‐stabilized whole blood from 10 healthy dogs was incubated with
two strains of aggregating S aureus at three different concentrations. Each sample
was analyzed using tissue factor‐thromboelastography (TF‐TEG) and the formed clot
was investigated with electron microscopy. The plasma activated partial thrombo-
plastin time (aPTT), prothrombin time (PT), fibrinogen, and D‐dimer tests were mea-
sured. Bacteria‐leukocyte binding was evaluated with flow cytometry, and
neutrophil phagocytosis was assessed using light and transmission electron micro-
scopy.
Results: The highest concentration of bacteria resulted in a significantly shortened
TF‐TEG initiation time, decreased alpha, maximum amplitude, global strength, and
increased lysis. In addition, significantly shortened PT, decreased fibrinogen, and
increased D‐dimers were demonstrated at the highest concentration of bacteria.
Lower concentrations of bacteria showed no differences in TF‐TEG when compared
with controls. The findings were similar for both S aureus strains. Increased concen-
tration‐dependent binding of bacteria and leukocytes and neutrophil bacterial
phagocytosis was observed.
Conclusions: Two strains of S aureus induced alterations of clot formation in con-
centrations where bacterial aggregates were formed. A concentration‐dependent
cellular inflammatory response was observed.

KEYWORDS
Canine, flow cytometry, hemostasis, immune system, MRSA, thromboelastography

560 | © 2018 American Society for wileyonlinelibrary.com/journal/vcp Vet Clin Pathol. 2018;47:560–574.
Veterinary Clinical Pathology
KROGH ET AL. | 561

1 | INTRODUCTION unknown. Both inflammation and hemostasis are part of the innate
immune response and will respond to bacterial pathogens with both
Gram‐positive bacteria are important pathogens in both human and
cellular and molecular mechanisms.19 The pathogen is recognized by
veterinary medicine causing both localized and systemic infections.1–
pattern recognition receptors on neutrophils, monocytes, and plate-
3
Staphylococcus aureus is a normal commensal bacterium of nasal
lets.19 Furthermore, activation of one cellular mechanism will initiate
and skin microflora in people.4 It is an opportunistic pathogen, which
responses from other mechanisms, and in concert, the pathogen can
in people is associated with colonization of surfaces such as lung
be eliminated.19 If a pathogen can evade the innate immune
and heart valves, and artificial surfaces such as medical implants.5 In
response via the expression of virulence factors such as planktonic
people, S aureus can cause infections ranging from benign soft tissue
growth, an infection can be established.17 Host responses to bacte-
infections to necrotizing pneumonia and sepsis.4 In veterinary medi-
ria, which can form planktonic aggregates might, therefore, be able
cine, the first S aureus infection was described in 1972 in cows6 and
to aid in combating resistant S aureus infections. Previously, inocula-
was later reported in horses, dogs, cats, and pigs.7–13 The increasing
tions with 1 mL/kg bodyweight saline containing 108 colony‐forming
incidence of human infections is thought to be a major factor in the
units (CFU)/mL of an S54F9 S aureus isolate resulted in hypercoagu-
rise of S aureus infection in companion animals.7,10,11,14 In animals,
lability, characterized by an increased strength of the formed clot,
as in humans, not all who encounter this bacteria will develop clinical
and an increased inflammatory response, characterized by increased
signs or illness. Most will eliminate the organism or simply have a
IL‐6 and C‐reactive protein (CRP) production and a neutrophilia.19,20
commensal association with the bacteria without developing clinical
It is unknown whether this S aureus strain can produce planktonic
signs. The most commonly reported clinical signs in animals are post-
aggregates. In dogs, sepsis caused by spontaneous gram‐negative or
operative wound infections. Less commonly, intravenous catheter
gram‐positive infections has been reported to cause hypercoagulabil-
site infections, urinary tract infections, pneumonia, and skin and ear
ity during the initial phases of disease, and hypocoagulability during
infections are observed.10 These infections can progress to a sys-
the latter progressive phases of disease.21,22
temic disease, such as sepsis.10,15 An increasing prevalence of methi-
Because the increasing incidence of infections in people is
cillin‐resistant S aureus (MRSA) has been reported in people
thought to be a major factor in the rise of S aureus infections in
worldwide.16
companion animals,7,10,11,14 an increased understanding of the
Our group recently showed that some S aureus strains do not
pathophysiologic reaction of the host toward this type of pathogen
grow as singlets, but as small clusters known as planktonic aggre-
is warranted, which will enable targeting therapies toward these
gates.17,18 A structural component of S aureus aggregates is the
infections in the future. The overall aim of the current study was to
polysaccharide intercellular adhesin (PIA) substance, as seen in bio-
investigate whether S aureus strains, with the ability to form plank-
films.17 Interestingly, planktonic aggregates were reported to have
tonic aggregates, would affect hemostatic and cellular inflammatory
increased antibiotic resistance, similar to bacteria forming stationary
host responses in canine whole blood, in vitro.
biofilms on artificial surfaces,17 and subinhibitory concentrations of
antibiotic treatment can influence the ability of these organisms to
form aggregates.17 Some antibiotics stimulate increases in aggregate 2 | MATERIALS AND METHODS
formation, and others inhibit aggregate formation, indicating that
antibiotics can differentially influence the formation of bacterial 2.1 | Study design
17
aggregates. Biofilm‐producing bacteria normally have a low meta-
Citrated whole blood from healthy dogs was incubated with either
bolic rate, while aggregating bacteria have a high metabolic rate,17
buffered saline (PBS) buffer or increasing concentrations (107, 108,
illustrating a difference between traditional biofilm‐forming bacteria
109 CFU/mL) of aggregating S aureus strains, WT1 and WT2. Blood
and aggregate‐forming bacteria. Planktonic aggregate formation
incubated with the different concentrations of aggregating bacteria
begins during the exponential growth phase of the bacteria where
were compared with blood incubated with PBS (Figure 1).
only a few small clusters of bacteria are observed at an OD600 of
0.1.17 Then, as bacterial concentrations increase, aggregates grow by
merging with additional clusters.17 2.2 | Study population
Based on the cell‐culture characteristics of aggregating bacteria,
Citrated whole blood was collected from 10 healthy privately owned
it is speculated that aggregates are a biofilm subtype that can
dogs, which were recruited specifically for this study. The inclusion
migrate through the body because of their mobility, resisting antibi-
criteria were as follows: dogs were older than 12 months, weighed
otic therapy and evading the host defense systems. Thus, aggregat-
more than 5 kg, had unremarkable physical examinations, hematol-
ing bacteria may cause seeding at distant sites, potentially leading to
ogy results, and serum biochemistry profiles, and no current medical
severe local and systemic infections, including sepsis.17
treatments. Microscopic evaluation of the blood smears confirmed
The pathophysiologic effect of planktonic bacterial aggregates,
the hematology results.
in vivo, is unknown since most studies have been performed under
The study was performed with owner consent and was approved
laboratory conditions. The innate immune response against aggregat-
by the Ethical and Administrative Committee, Department of Veteri-
ing S aureus, the first line of defense toward an infection, is
nary Clinical Sciences, University of Copenhagen, Denmark.
562 | KROGH
F I G U R E 1 A flow diagram illustrating the analysis of citrate‐stabilized whole blood from the dogs. All samples were based on the same pool
of citrate‐stabilized whole blood. The pool was separated in seven aliquots: an untreated control and increasing concentrations of bacteria: 107
CFU/mL, 108 CFU/mL, and 109 CFU/mL of the two different MRSA bacterial strains (WT1 and WT2)
2.3 | Bacterial strains
Two different strains of aggregating S aureus were selected, WT1
and WT2. The community‐acquired (CA)‐MRSA strain USA300 (LAC
300), designated WT1, is well‐characterized and a leading cause of
MRSA‐related diseases in human patients in the United States. WT1
has two plasmids that encode resistance to tetracycline and ery-
thromycin, and a third cryptic plasmid with no annotated phenotypic
traits and no known impact on virulence and growth.23,24 The
enhanced virulence of WT1 is linked, in part, to its ability to circum-
vent neutrophil killing and induce neutrophil lysis.26 A variant of this
strain has been isolated from a dog with a postoperative infection
after limb surgery and was reported to be transmitted from the vet-
erinarian to the dog.27 The WT2 strain also used in this study has
similar aggregating abilities as the W1 strain but does not contain
the three plasmids. To facilitate enumeration and colocalization of
bacteria with components of the blood by flow cytometry, both
strains were transformed with pCM29 constitutively expressing syn-
thetic green fluorescent protein (sGFP). The strains were inoculated
overnight in 10 mL of tryptic soy broth (TSB) and chloramphenicol
at 10 μg/mL. The following day the bacteria were resuspended in
phosphate buffered saline (PBS) after being centrifuged at 5000g for
5 minutes. Ten cycles of sonication followed, and the suspension
was adjusted to an OD600 of 10, and subsequently diluted twice to
yield suspensions with an OD600 of 1 and 0.1, which corresponded
8 9 10
to 10 , 10 , and 10 CFU/mL. Bacterial aggregates begin to form at
an OD600 of 0.1 and become gradually more frequent as bacterial
concentrations increase.17
2.4 | Blood sample handling
All blood samples were collected in the morning following a 12‐hour
fasting period. Blood was collected from the jugular vein by an expe-
rienced technician with minimal stasis. A 21‐gauge butterfly catheter
was used to collect blood directly into vacutainer tubes in the fol-
lowing order: five trisodium citrate tubes (2.7 mL, BD Vacutainer;
ET AL.
Becton, Dickinson and Company, Franklin Lakes, NJ, USA), where
the first tube was used to establish good continuous blood flow and
the following four citrate tubes were used for the study, one EDTA
tube was used for the complete blood count (2 mL, BD Vacutainer;
Becton, Dickinson and Company) and one serum tube was used for
the biochemistry profile (4 mL, Vacuette; Greiner Bio‐One, Interna-
tional AG, Kremsmunster, Austria). During the collection procedure,
the tubes (citrate and EDTA) were inverted gently five times to allow
the anticoagulant to mix. All samples were kept at room temperature
(RT; 20‐24°C). The citrate‐stabilized blood was pooled in a 1 mL
polypropylene plastic tube.
2.5 | Effects of bacterial concentration and
planktonic aggregate formation on canine whole
blood hemostasis and inflammation, in vitro
The pooled blood from each dog was aliquoted and a bacterial sus-
pension of varying CFU/mL concentrations (108, 109, and 1010) was
added to the citrated whole blood at a 1:10 dilution (200 µL suspen-
sion +1800 µL blood), yielding final bacterial concentrations of 107,
108, and 109 CFU/mL respectively. The bacterial concentrations were
chosen based on the appearance of bacterial planktonic aggregates
where aggregates will begin to form at an OD600 of 0.1 (107 CFU/
mL) and then increase in number and size in a concentration‐depen-
dent manner with the hypothesis that planktonic aggregation would
lead to more pronounced hemostatic and inflammatory responses.17
Seven tubes were evaluated from each dog: one tube containing
PBS buffer (control), three tubes containing strain 1 (WT1) bacteria
with final bacterial concentrations of 107,108, and 109 CFU/mL,
respectively, and three tubes containing strain 2 (WT2) bacteria with
final bacterial concentrations of 107,108, and 109 CFU/mL, respec-
tively (Figure 1). All seven sample tubes were incubated for 30 min-
utes at RT, and then 400 µL of whole blood was transferred to a
cryovial tube for tissue factor (TF) dilutions. Three hundred and forty
microliters of TF mixed with whole blood were transferred to a TEG
cup for TF‐initiated thromboelastography (TF‐TEG), and samples
KROGH ET AL.
were analyzed immediately. Fifty microliters of whole blood were
transferred to test tubes for flow cytometric (FC) analysis and analy-
sis was performed immediately after sample processing. Twenty µL
of whole blood were applied to glass slides to make the smears for
microscopic evaluation. The smears were air‐dried for fixation,
stained later the same day, and evaluated within 7 days.
2.6 | Laboratory analyses
2.6.1 | Thromboelastography
Whole blood global hemostatic abilities were evaluated using TF‐
TEG, and the analyses were performed as described previously.28
Briefly, a computerized thromboelastograph (TEG 5000 Hemostasis
Analyzer System; Haemonetics Corporation, Braintree, MA, USA)
with continuous data acquisition was used. Each sample was acti-
vated using a solution of recombinant human TF (Innovin, Dade
Behring, Marburg, Germany) at a final TF dilution of 1:50 000.
Four hundred microliters of canine citrated whole blood were
mixed with prediluted TF. Three hundred and forty microliters of
the whole blood‐TF mixture were added to a prewarmed (37°C)
TEG cup (Haemonetics Corporation). Twenty microliters of CaCl2
were pipetted into the cup giving a total volume of 360 µL in
each cup. The pin was gently lowered into the cup and measure-
ments were initiated. The parameters chosen for further evalua-
tion were reaction time (R), which represents the time until the
first evidence of clot is formed, angle (α) representing the rate of
clot formation from the beginning of clot formation until the clot
reaches 20 mm, maximum amplitude (MA), which is a reflection of
clot strength, and global clot strength (G), which is a calculated
value also evaluating clot strength, and clot lysis after 30 and
60 minutes (LY30, LY60). TF‐TEG analyses were recorded for a
total of 120 minutes.
In companion animals, hypocoagulability, hypercoagulability, and
hyperfibrinolysis are not clearly defined in terms of TEG parame-
ters.25 In this study, hypocoagulability was defined as a significant
(P < 0.05) decrease in the rate of clot formation, a decrease in clot
strength, and a decrease in global clot strength compared with con-
trols. Hyperfibrinolysis was defined as significantly (P < 0.05)
increased clot lysis evaluated at 30 or 60 minutes after the initiation
of clot formation compared with controls.
2.6.2 | Plasma coagulation tests
Immediately after whole blood was aliquoted for TEG, flow cytome-
try and blood smear analysis, the seven tubes were centrifuged in
one batch at 4000g for 120 seconds at RT. Five hundred microliters
of plasma from each tube were transferred to appropriate vials for
coagulation analyses.
Activated partial thromboplastin time (aPTT), prothrombin time
(PT), fibrinogen concentration, and D‐dimer tests were evaluated in
plasma from each of the seven tubes. Plasma aPTT, PT, and fibrino-
gen were measured using an automated coagulometric analyzer (ACL
| 563
Top500; Instrumentation Laboratory, Bedford, MA, USA). For aPTT
analysis, a high‐quality synthetic phospholipid reagent (HemosIL,
SynthAFax; Instrumentation Laboratory) was used, and for PT and
fibrinogen analyses, a sensitive thromboplastin agent (HemosIL,
RecombiPlasTin 2G; Instrumentation Laboratory) was used. Plasma
D‐dimers were measured using an immunometric flow‐through prin-
ciple (D‐dimer Single Test, NycoCard READER II; Medinor A/S,
Broendby, Denmark) previously validated in dogs and according to
the manufacturer's instructions.28,29
Pooled plasma samples from five healthy dogs with known val-
ues, confirmed through serial measurements, were used as internal
quality controls for the plasma‐based coagulation tests.
2.7 | Flow cytometric analysis
Bacterial‐leukocyte binding, the presence of platelet‐neutrophil
heteroaggregates, and binding of bacteria to platelet‐neutrophil
heteroaggregates were analyzed by flow cytometry using a BD
FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA).
From each of the seven tubes containing whole blood incubated
with either PBS buffer or bacterial strains, WT1 or WT2, in increas-
ing concentrations (Figure 1), 50 µL aliquots were transferred imme-
diately after the 30‐minute bacterial incubation into BD falcon tubes,
and the red blood cells were lysed and discarded. The remaining cells
in each tube were washed once with PBS. Leukocytes were identi-
fied using a rat anti‐canine monoclonal antibody to the leukocyte
common antigen marker, CD45‐phycoerythrin (PE; clone
YKIX716.13; Bio‐Rad AbD Serotec GmbH, Puchheim, Germany). Pla-
telets were identified with a mouse anti‐human monoclonal antibody
to the surface protein integrin beta‐3, CD61‐allophycocyanin (APC;
clone Y2/51; Dako Cytomation, Glostrup, Denmark)30 and activated
platelets were identified with a mouse anti‐human monoclonal anti-
body to P‐selectin, CD62‐PE (clone 1E3; Santa Cruz Biotechnology,
Dallas, TX, USA).30 Isotype controls were used for each of the anti-
bodies. Samples were incubated in the dark for 20 minutes, after
which time they were centrifuged, supernatants were discarded, and
FacsFlow buffer (BD Biosciences) was added. Samples were analyzed
immediately, which led to a 60‐minute total time period from the
start of bacterial incubation. All tubes were analyzed with a thresh-
old at 45 000 to avoid background noise and a stopping gate of
10 000 cells. The flow cytometer instrument settings were con-
trolled by internal quality control beads.
The leukocytes were identified by differences in CD45 expres-
sion (dim and bright) and forward and side‐scatter profiles. The
bacteria were fluorescein (FITC) positive since they expressed syn-
thetic green fluorescent protein, and based on hierarchical gating
of the leukocyte types, neutrophil, lymphocyte, and monocyte per-
centages positive for bacterial binding (FITC) were identified.
Leukocyte‐platelet heteroaggregates were identified by CD45‐PE
and CD61‐APC dual staining and the FITC fluorescence repre-
sented the binding of bacteria to either the respective leukocyte,
platelets, leukocyte‐platelet heteroaggregates, or leukocytes positive
for P‐selectin.
564 | KROGH ET AL.

Formvar supporting membranes. Sections were stained with uranyl

2.8 | Microscopic evaluation of the bacterial‐
acetate and lead citrate. Several sections from each preparation were
leukocyte and the bacterial‐platelet interactions
evaluated by SEM and TEM, and images were taken.

2.8.1 | Microscopic evaluation of blood smears

The presence and quantification of intracellular bacteria in neu-
trophils were investigated by evaluation of blood smears using light
microscopy. The blood smears were prepared from each tube imme-
diately after the 30‐minute incubation period with the two bacterial
strains in increasing concentrations or buffer (Figure 1). The smears
were dried and stained with a modified Wright's stain in an auto-
matic Hematek 2000 slide‐staining machine (Siemens Healthineers,
Tarrytown, NY, USA). The quality of the smears was microscopically
analyzed with a Nikon E200 light microscope, and 200 intact leuko-
cytes in each smear were counted and morphologically evaluated in
the monolayer by the same clinical pathologist (AHK). The number
of neutrophils containing intracellular bacteria was recorded.
2.9 | Statistical analysis
The results were analyzed using GraphPad Prism version 7.0 for
Windows (GraphPad Software La Jolla, CA 92037 USA). The differ-
ence between the bacterial strains and concentrations was evaluated
by a two‐way ANOVA with Tukey's correction for multiple compar-
isons with regard to the different bacterial strains and concentra-
tions, or by a one‐way ANOVA test with Dunnett's correction for
multiple comparisons when comparing to control samples. The signif-
icance was set at a P < 0.05 in all tests.
3 | RESULTS

3.1 | Study population

2.8.2 | Scanning and transmission electron
microscopy of TEG clots
Scanning electron microscopy (SEM) was performed to visualize and
characterize the two aggregating bacterial strains in increasing concen-
trations that affect the structural surface of the clot formed in the
thromboelastograph. An XL 30 FEG scanning electron microscope
(Phillips, Eindhoven, Netherlands) was used, and the images were
described. Transmission electron microscopy (TEM) was performed
with a CM100 transmission electron microscope (Phillips, Eindhoven,
Netherlands), and TEM was used to confirm the presence of intracyto-
plasmic bacteria in neutrophils seen by light microscopy and to evalu-
ate if bacterial aggregates were present in neutrophils based on more
than one bacterium in an intracytoplasmic vacuole. In addition, the
presence of bacterial aggregates adhered to platelets was evaluated.
Seven randomly selected TF‐TEG clots including controls and the
samples incubated with 107, 108, and 109 CFU/mL of WT1 and WT2
were transferred to a 2% glutaraldehyde fixative immediately after
the TF‐TEG analysis was finished and processed for SEM and TEM,
as previously described.31 In brief, after the initial fixation, the sam-
ples were postfixed in 1% OsO4 and dehydrated in ethanol. Then
they were critical point‐dried using CO2, and sputter coated with
gold for SEM. For TEM, the samples were prepared similarly until
the ethanol dehydration step, after which the samples were trans-
ferred to propylene oxide and embedded in Epon according to stan-
dard procedures. Sections, 80‐nm thick, were cut with a Reichert‐
Jung Ultracut E microtome and collected on copper grids with
Whole blood was collected from 10 healthy dogs (unremarkable
CBCs and biochemistry panels including the major acute phase pro-
tein, CRP) including three mixed breeds, two Labrador Retrievers,
two Border Terriers, one Doberman Pinscher, one Australian Shep-
herd, and one Bichon Frisé. Of these, five were neutered females,
two were intact females, and two were intact males. Median age
was 7 years and 4 months (range: 1 year and 11 months to 10 years
and 11 months). Hematocrits were at the upper level of the refer-
ence interval (RI; median 0.485 L/L; range 0.46‐0.60 L/L; RI 0.39‐
0.59 L/L) possibly due to at least a 12‐hour fasting period. Platelet
numbers fell within the RI (median 252 × 109/L; range 171‐
465 × 109/L; RI 200‐500 × 109/L).
3.2 | Analyses
3.2.1 | Thromboelastography
Incubating the blood samples with the highest bacterial concentra-
tion of 109 CFU/mL of WT1 or WT2, where planktonic aggregates
were markedly present, resulted in a significantly faster clot forma-
tion compared with buffer controls as evidenced by a shortened R
(WT1 P < 0.001; WT2 P < 0.05; Figure 2). Furthermore, the highest
bacterial concentration (109 CFU/mL) from both WT1 or WT2 also
induced a significant decrease in the rate of clot formation charac-
terized by a decreased alpha (WT1 P < 0.01; WT2 P < 0.001), and a
decreased clot strength characterized by a decreased MA (WT1

F I G U R E 2 Thromboelastographic parameters for 10 canine blood samples. R equals (seconds) time from initiation of analysis to the
beginning of clot formation. Alpha equals (degrees) the rate of clot formation. MA (mm) and G (kilodynes/cm2) represent clot strength. LY 30
and 60 (percentages) represent fibrinolysis measured after 30 and 60 minutes. Each parameter was analyzed for each MRSA bacterial strain and
a control (phosphate buffered saline) sample. For all parameters, statistically significant differences were observed when the concentration of
bacteria reached 109 CFU/mL. No difference between the two bacterial strains was detected. The box and whiskers plots represent all data
including median and 25/75 percentiles, and the maximum and minimum values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared
with untreated controls, as calculated by the one‐way ANOVA with the Dunnett's multiple comparison test performed separately for each strain
KROGH ET AL. | 565
566 | KROGH
P < 0.001; WT2 P < 0.001) and G (WT1 P < 0.0001; WT2
P < 0.0001) compared with buffer controls. In addition, hyperfibri-
nolysis was present in samples incubated with the highest bacterial
concentrations, and a significant increase in fibrinolysis was present
after a 60‐minute incubation with WT2 (P < 0.05; Figure 2).
In contrast to the results of samples incubated with the highest
WT1 and WT2 bacterial concentrations, which had markedly differ-
ent clot formations compared with samples in buffer, the clot forma-
tion in samples incubated with 107 CFU/mL and 108 CFU/mL were
not significantly different from the buffer controls, and clot forma-
tion times, R values, rates of clot formation, alphas, strengths of the
formed clot, and MA and G values were all similar (Figure 2). No sta-
tistical differences were found between the two bacterial strains
regarding clot formation times, R values, rates of clot formation,
alphas, strengths of clot formation, and MA and G values. No differ-
ence between the two strains was also observed for clot lysis, the
LY30 and LY60 values (Figure 2).
3.2.2 | Plasma coagulation and D‐dimer tests
The highest WT1 or WT2 bacterial concentrations (109 CFU/mL)
resulted in a marked number of planktonic aggregates and induced
significantly shortened PTs (WT1 P < 0.01; WT2 P < 0.001),
decreased fibrinogen (WT1 P < 0.001; WT2 P < 0.0001), and
increased D‐dimers (WT1 P < 0.0001; WT2 P < 0.0001) compared
with buffer controls (Figure 3). Similar, but slightly lower effects,
were observed for the intermediate WT1 or WT2 bacterial concen-
trations (108 CFU/mL) compared with buffer controls, where the PT
was significantly shortened for WT2 (P < 0.001), fibrinogen was
decreased for WT1 and WT2 (WT1 P < 0.05; WT2 P < 0.05), and
D‐dimers were increased (WT1 P < 0.05; WT2 P < 0.05) (Figure 3).
At the lowest WT1 or WT2 bacterial concentration (107 CFU/mL),
where planktonic aggregates began to form, PT was significantly pro-
longed for both bacterial strains (WT1 P < 0.01; WT2 P < 0.001)
compared with buffer controls (Figure 3). No significant differences
were observed compared with buffer controls in the evaluation of
fibrinogen or D‐dimer concentrations (Figure 3). Interestingly, no sig-
nificant differences in aPTT measurements in samples incubated with
any of the WT1 or WT2 concentrations compared with buffer con-
trols were observed (Figure 3). As for TF‐TEG, no significant differ-
ences in aPTT, PT, fibrinogen, or D‐dimer concentrations between
the two strains were observed (Figure 3).
It was not possible to analyze aPTT, PT, or fibrinogen in the
plasma of one dog combined with 109 CFU/mL of WT1 or WT2. In
addition, it was not possible to analyze PT in the plasma combined
with 108 CFU/mL of WT2 from the same dog. These results showed
irregular absorbance curves, which could have been due to marked
sample clotting, and were discarded. The D‐dimer concentrations of
plasma samples from the same dog that contained 109 CFU/mL WT1
or WT2 bacteria and samples from one additional dog that contained
109 CFU/mL WT1 or WT2 bacteria could not be analyzed due to
high sample viscosities, which caused suction failure of the samples
and reagent materials.
ET AL.
3.3 | Flow cytometric analysis
Analysis of whole blood samples incubated with the highest WT1
and WT2 concentrations (109 CFU/mL) using flow cytometry demon-
strated that neutrophils, lymphocytes, and monocytes had statisti-
cally increased cell percentages that were positive for FITC
fluorescence compared with that of the buffer controls (P < 0.0001)
(Figure 4), which indicated that the cells were associated with the
green fluorescence bacteria. In addition, neutrophils had greater FITC
expression compared with that of the monocytes and lymphocytes.
The median percentage of neutrophils positive for FITC in samples
with 109 CFU/mL bacteria was 95% for both WT1 and WT2, the
median percentage of monocytes positive for FITC was 62% and
65% for WT1 and WT2, respectively, and the median percentages of
lymphocytes positive for FITC was 32% and 30% for WT1 and WT2,
respectively (Figure 4). An increase in the percentage of platelet‐neu-
trophil heteroaggregates was also seen at the highest bacterial con-
centration and was characterized by an increase in the percentage of
neutrophils positive for the platelet marker CD61‐APC (integrin beta
3) (Figure 4). Also, in samples containing the highest WT1 bacterial
concentrations (109 CFU/mL), a significantly (P < 0.0001) increased
percentage of neutrophils was positive for CD62 P‐selectin (PE) (Fig-
ure 4), which could represent neutrophils binding to activated plate-
lets. The population of P‐selectin positive neutrophils was further
analyzed, and the results revealed two subpopulations of neutrophils
positive for P‐selectin. One subpopulation was surprisingly negative
for the common platelet marker integrin beta 3 (PE) and the other
subpopulation was dual positive for the common platelet marker as
expected (Figure 5). An increasing number of bacterial aggregates at
the highest bacterial concentrations was seen as an increase in the
number of FITC‐positive events in the area below the size (forward
scatter) and complexity (side‐scatter) of the leukocytes (Figure 5).
Samples incubated with the WT1 and WT2 bacterial strains at
low and intermediate concentrations revealed significantly increased
positive neutrophils, monocytes, and lymphocytes (P < 0.0001) com-
pared with those incubated with PBS, but lower median positive
leukocyte percentages were found in those samples compared with
samples incubated with high bacterial concentrations (Figure 4).
No statistically significant differences between the two bacterial
strains were demonstrated for the different leukocytes and the FITC,
APC, or PE positivity. One exception was the population of neu-
trophils positive for P‐selectin (APC) in the samples with the WT1
strain presented in Figure 4.
3.4 | Microscopic evaluation of the bacterial‐
leukocyte and bacterial‐platelet interactions
3.4.1 | Light microscopy
In blood smears from samples incubated with the highest WT1 and
WT2 concentrations (109 CFU/mL), the median number of neutrophils
with intracellular bacteria was 33.5 and 32.5 for WT1 and WT2, respec-
tively (range: WT1, 18‐55 cells; WT2, 19‐59 cells) and varying numbers
KROGH ET AL. | 567

F I G U R E 3 Activated partial thromboplastin time, aPTT (seconds), partial thromboplastin time, PT (seconds), and fibrinogen (g/L) were
measured in whole blood samples of dogs that were incubated with three different aggregating bacterial concentrations (107, 108, and 109).
The sample from dog 2 incubated with the highest bacterial concentrations did not have any tests performed, and PT was performed only on
the sample from that dog incubated with the intermediate WT1 bacterial concentration (108 CFU/ml). D‐dimer (mg/L) concentrations were
analyzed in all dogs except dogs 1 and 2. A statistically significant increase in aPTT and D‐dimer concentrations and decreases in PT and
fibrinogen were seen. The box and whiskers represent the median and 25/75 percentiles and the maximum and minimum values, respectively.
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with untreated control, as calculated using the one‐way ANOVA with the
Dunnett's multiple comparison test for each strain

of intracellular bacteria were observed in the neutrophils with up to 35
intracellular cocci seen. In blood smears from samples incubated with
intermediate WT1 and WT2 concentrations (108 CFU/mL), the median
number of neutrophils with intracellular bacteria was 1.5 in the smears
from both strains (range: WT1, 0‐4 cells; WT2, 0‐5 cells). In blood
smears from samples incubated with the lowest WT1 and WT2 concen-
trations (107 CFU/mL), no intracellular bacteria were observed in the
neutrophils. Bacteria were also not detected in samples incubated with
PBS (control). These results demonstrated that the number of neu-
trophils with intracellular bacteria in samples incubated with the highest
and intermediate bacterial concentrations increased compared with
samples incubated with PBS (109 CFU/mL, P < 0.0001; 108 CFU/mL,
P < 0.05). No statistical differences were present between samples
incubated with the lowest bacterial concentration (107 CFU/mL) and
those incubated with PBS, for either of the bacterial strains.
3.4.2 | Electron microscopy
Assessing the scanning electron microscopy illustrations of the TF‐
TEG clot surfaces in samples incubated with the highest WT1 and
WT2 concentrations, erythrocytes and fibrin strands were visualized
and the surfaces appeared to contain a high number of single, small
568 | KROGH ET AL.

F I G U R E 4 Flow cytometric analysis of all 10 canine blood samples incubated with varying concentrations (107, 108, and 109) of aggregating
bacterial strains (WT1 and WT2). Analyses were performed on neutrophils, lymphocytes, and monocytes. The Y‐axis represents the percent of
cells positive for FITC, APC, and PE for each given leukocyte. FITC‐bacteria, APC‐CD61‐platelet marker, PE‐P‐selectin marker. Data are
depicted with all measurements and the box and whiskers represent the median and 25/75 percentiles and maximum and minimum values.
*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with untreated control, as calculated using the one‐way ANOVA with the
Dunnett's multiple comparison test for each strain

particles with diameters of 0.1‐0.3 µm (Figure 6). Additionally, ery-
throcytes appeared to be more closely packed, and fibrin strands
showing increased decomposition (Figure 6). Samples incubated with
108 CFU/mL WT1 or WT2 bacteria had a decreased number of
these particles compared with samples incubated with the highest
bacteria concentrations. In samples incubated with the lowest bacte-
rial concentrations; the outer membranes of the erythrocytes
appeared smooth with only a few single particles present and the
fibrin strands were clear (Figure 6).
Transmission electron microscopy revealed intracellular bacteria
in neutrophils both as singlets and as small bacterial aggregates
within intracytoplasmic vacuoles in the samples incubated with the
highest bacterial concentrations (Figure 7). Furthermore, aggregating
bacteria and platelets were observed in clusters close to erythro-
cytes and leukocytes in samples incubated with the highest bacterial
concentrations. This was not observed in samples incubated with
intermediate and low concentrations of bacteria (Figure 7). No sam-
ple differences were observed at any of the concentrations between
the two strains of bacteria.
4 | DISCUSSION
In the present study, incubating whole blood with S aureus at a con-
centration of 109 CFU/mL, where large planktonic aggregates are
KROGH ET AL. | 569

F I G U R E 5 Flow cytometric scatterplots from dog 7. A, Scatterplot showing APC‐positive cells vs PE‐P‐positive cells after hierarchical
gating of neutrophils with attached bacteria. Q4‐1 represents neutrophils positive for FITC‐bacteria and P‐selectin. Q2‐1 represents neutrophils
positive for FITC‐bacteria, PE‐ P‐selectin, and APC‐CD‐61. B, Scatterplot representing forward (size) and side‐scatter (granularity). P1—
neutrophils, P2—lymphocytes, P3—monocytes, P4—aggregates of bacteria/platelets [Color figure can be viewed at wileyonlinelibrary.com]

A B

F I G U R E 6 Scanning electron microscopy (5000×) of blood clot surfaces from a blood clot derived from the thromboelastography machine
after a sample had been analyzed. A, The surface of a blood clot from a blood sample incubated with 107 CFU/mL of aggregating
Staphylococcus aureus. Long arrow—red blood cell, bold arrow—a platelet, arrowhead—fibrin strand. B, The surface of a blood clot from a
blood sample incubated with 108 CFU/mL of S aureus. Long arrow—red blood cell, arrowhead—fibrin strand. C, The surface of a blood clot
from a blood sample incubated with 109 CFU/mL of S aureus. Long arrow—red blood cell
570 | KROGH ET AL.

A B

F I G U R E 7 A, A photomicrograph of transmission electron microscopy showing a neutrophil containing several intracellular coccoid bacteria
marked with arrows. The neutrophils are from a blood clot derived from a thromboelastograph after testing a blood sample that was incubated
with 109 CFU/mL of Staphylococcus aureus. Black arrows indicate bacteria. B, A photomicrograph of transmission electron microscopy showing
a platelet‐bacteria aggregate from a blood sample incubated in the same way as in (A). Black arrows indicate bacteria; white arrows indicate
platelets

present, resulted in pronounced hemostatic changes in canine whole
blood, in vitro, which was characterized by hypocoagulability, hyper-
fibrinolysis, and poor clot quality. Clear inflammatory cell changes
were also seen, as characterized by the binding of bacteria to leuko-
cytes (neutrophils > monocytes > lymphocytes) and the presence of
intracellular bacteria in neutrophils.
The thromboelastographic response was hypocoagulable as iden-
tified by significantly decreased alpha, MA, and G values, and hyper-
fibrinolysis was indicated by a significantly increased LY60. In
addition, plasma coagulation analysis demonstrated shortened PTs,
decreased fibrinogen concentrations, and increased D‐dimer concen-
trations.
An increased concentration‐dependent inflammatory cell
response was also present and characterized by increased binding of
bacteria to neutrophils, monocytes, and lymphocytes at the highest
bacterial concentrations. Also, increased leukocyte‐platelet heteroag-
gregates were observed at the highest bacterial concentration. These
findings were confirmed by light microscopy and TEM, which
showed that neutrophils internalized bacteria in a concentration‐de-
pendent manner.
These results confirmed the hypothesis of a pronounced hemo-
static and inflammatory response to aggregating S aureus. The results
are important because it underscores that strains of S aureus capable
of producing aggregates might elicit more pronounced hemostatic
and inflammatory responses than singlets of bacteria, suggesting that
aggregating bacteria have an additional important virulence factor
affecting host hemostatic and inflammatory systems.
9
The aggregating bacteria at the highest concentration (10 CFU/
mL) exhibited a drastic effect on the thromboelastographic hemo-
static response. This could reflect that a certain number of bacteria
is required to elicit a response and that once this threshold has been
reached, the hemostatic and inflammatory systems are strongly acti-
vated. Such a marked threshold effect caused by aggregating bacte-
ria has not been described before but can be supported by earlier
studies using a different type of bacteria (gram‐positive Bacillus cer-
eus) artificially grouped and placed in flow chambers. In this system,
bacteria could only initiate coagulation in human plasma when pre-
sent in aggregates, but could not as single cells.32 On the other
hand, opposed to the hypocoagulable TEG profiles reported in our
study, in vivo experiments in pigs infected with a porcine‐derived S
aureus strain resulted in hypercoagulable TEG profiles.19,20 However,
given the unknown propensity of this porcine S aureus strain to form
aggregates, and the potential difference in virulence factors com-
pared to the strain used here, it is possible that different bacterial
traits cause different host responses. Interestingly, our results are
more in line with a severe hemostatic response seen in sponta-
neously septic dogs in the late hypocoagulable state of disseminated
intravascular coagulopathy (DIC).21 At this stage of DIC, consump-
tion of coagulation factors and increased fibrinolytic activity are pre-
sent.33
In addition to activation of the hemostatic system observed by
TEG, a shortened PT was also seen and induced by aggregating bac-
teria at the highest concentration. Although preanalytical activation
of platelets and coagulation factors during sample collection has
been reported to cause a shortened PT,34 which was not the case in
this study, since all samples originated from the same stock of
pooled citrate‐stabilized whole blood. Instead, our results indicate
that aggregating S aureus can cause activation of the extrinsic path-
way, possibly through increased TF expression as reported previ-
ously in monocytes activated by S aureus.35 In contrast to PT, aPTT
levels were similar to buffer controls even in samples incubated with
the high bacterial concentrations. One explanation for this finding
KROGH ET AL.
could be that aggregating bacteria can induce coagulation primarily
through factor VII rather than factors XII, XI, and IX.35 It has also
been reported that in both septic dogs and dogs with DIC, aPTT can
21,36
vary greatly. This could possibly be due to the varying sources
of bacteria in the different studies. In our study, a reserve capacity
of clotting factors could also be present since the samples were from
healthy dogs, and these reserve clotting factors could mask a mild
36
consumption of coagulation factors seen in septic dogs, in vivo.
When looking at fibrinogen, a marked decrease was observed in
samples with the highest bacteria concentration. This is likely caused
by fibrinogen consumption as part of fibrin production in the hemo-
21,37,38
static process as reported in other canine studies. In addition,
S aureus bacteria are also known to react with fibrinogen directly
and thereby cause a decrease in fibrinogen concentrations.39 The
reaction is through membrane‐bound or secreted proteins, bacterial
coagulase, or von Willebrand binding protein, which converts plasma
prothrombin to staphylothrombin, resulting in the conversion of fib-
39
rinogen to fibrin.
The fibrinolytic system was also activated at the highest bacterial
concentrations, as seen by significantly increased D‐dimer concentra-
tions and increased TEG LY30 and LY60. A similar D‐dimer response
has been reported previously in induced and spontaneously septic
dogs,36,37,40 and increased LY30 was seen in dogs with endotox-
emia.37 This could indicate that the local concentration of bacteria
during sepsis in vivo, could reach levels comparable to the ones
reported here, and thus provoke a similar response. Thus, it is tempt-
ing to speculate that the hemostatic and inflammatory responses
observed in some septic patients, could indeed be caused by very
high concentrations of (aggregating) bacteria localized in foci within
the body of affected animals.
In one dog, we could not analyze aPTT, PT, and fibrinogen con-
centrations at the highest bacterial concentrations of both strains,
and PT could not be measured at the intermediate WT2 bacterial
concentrations based on irregular absorbance curves. These results
were likely caused by marked sample clotting and were supported
by the TEG traces of the same samples showing markedly shortened
R times compared with the controls.
In this study, significant neutrophil‐bacteria binding and phagocy-
tosis were demonstrated with both flow cytometry and microscopy,
indicating recognition of the bacteria by the innate immune system,
and also in the presence of aggregate formation. Leukocytes bind
bacteria through the recognition of pathogen‐associated molecular
patterns (PAMPs) by pattern recognition receptors (PRRs)41 facilitat-
42–44
ing an inflammatory response. Specifically, S aureus is recog-
nized by TLR2, which is highly expressed on activated neutrophils,
45,46
monocytes, and T‐cell lymphocytes. This differential expression
of TLR2 is completely in line with our results, where flow cytometry
revealed that neutrophils contained the most bacteria, but also that
monocytes and lymphocytes were capable of bacterial binding.
These results indicate that leukocytes were activated by the bacteria
and that a cellular inflammatory response is ongoing in the cells.
Additional investigations that evaluated the products of a cellular
| 571
inflammatory response, such as specific cytokines or oxidative burst
activity in neutrophils, could be interesting.
Another interesting finding was that a subpopulation of neu-
trophils was P‐selectin positive, but negative for the platelet marker
CD61. A potential source of this population could be neutrophil‐pla-
telet microparticle‐positive cells as described in septic humans.47
These microparticles can be markedly procoagulant.47,48 Platelet
microparticles would be expected to be CD61 positive, but the lack
of CD61 reported here could be explained by internalization of the
CD61 receptor on platelets, as previously described.49 Also, it is pos-
sible that the CD61 epitope could simply be covered by neutrophil
binding and thus be unable to react with the antibody.
Flow cytometric analysis also confirmed the presence of large
bacterial aggregates at the highest bacterial concentrations. Bacterial
aggregates can directly react with platelets and form bacteria‐platelet
clumps. Indeed, S aureus mediates the binding of protein A to plate-
let Fc receptors,50 and could also use fibrinogen,51 clumping factors
A and B (ClfA and ClfB), and proteins A and B (FnbpA and FnbpB)
to bind platelets.52,53 This binding was confirmed visually by electron
microscopy, and it could indicate that aggregated bacteria contribute
to DIC not only through the hemostatic reactions discussed above
but also through the direct activation of platelets.
Kuipers et al demonstrated that bacterial fibrin formation
reduced the innate immune response by inhibiting phagocytosis.54,55
Thus, bacterial/fibrin aggregates might be too large to be phagocy-
tized, allowing the bacteria to escape the cells of the innate immune
system.56 From a host perspective, this would have a devastating
effect, since these aggregates could be a continuous source of sys-
temic infections. In the current study, light microscopy and TEM
demonstrated neutrophil phagocytosis of smaller bacterial aggregates
and that multiple bacteria were present in the same neutrophilic
intracytoplasmic vacuole. This result suggests a potential size limit of
phagocytosis, where a relatively mild infection with smaller aggre-
gates may be overcome by the innate immune system through
phagocytosis, whereas a stronger infection with large aggregates
might be able to surpass the innate immune defense system.
SEM of the TEG clot revealed a large number of small particles
attached to erythrocytes and fibrin strands in samples incubated
with the highest concentration of bacteria. The increased P‐selectin
expression indicates that these particles are likely platelet microparti-
cles. Alternatively, the particles observed by SEM could consist of a
mixture of fibrin degradation products, including D‐dimers from
degradation of fibrin fragments.57 Another possibility regarding the
origin of these particles could be in the components from neutrophil
extracellular traps (NETs), which are nuclear and mitochondrial DNA
released into the extracellular space.58 When neutrophils and mono-
cytes are activated, they can generate NETs or macrophage extracel-
lular traps (METs), respectively.59 Generation of NETs has been
shown both in purified neutrophils stimulated with S aureus60 and in
mouse studies where activated platelets stimulate neutrophils. The
formed NETs subsequently entrap bacteria and promote intravascu-
lar coagulation.61,62
572 | KROGH
In this study, the combination of electron microscopy with
thromboelastography provides a unique way of investigating both
visual and physical changes simultaneously. Thus, the results showed
that visible changes in the blood clot were apparent in conditions
where a strong hemostatic response could be detected.
Generally, no difference between the two bacterial strains was
observed, indicating that none of the three plasmids had any effect
on the hemostatic or cellular inflammatory system. This suggests that
the genetic factors required for aggregate‐mediated induction of the
hemostatic and inflammatory responses are situated elsewhere in
the bacterial genome.
A limitation of this in vitro study is the high bacterial concentra-
tion used does not reflect systemic bacterial concentrations reported
in septic patients, in vivo,63 but would rather reflect the local con-
centrations at specific foci, and aggregate “thrombi” embolizing from
these sites could result in significant and potentially life‐threatening
hemostatic and inflammatory reactions and complications.17,64
To our knowledge, this is the first study describing hemostatic and
cellular inflammatory responses to planktonic aggregating MRSA in
canine whole blood. With the information presented in this study,
future in vitro studies that can identify and investigate genetic factors
of S aureus responsible for planktonic bacterial aggregation should be
performed regarding its ability to influence or evade the innate immune
system. Also, it would be interesting to perform phenotypic evaluations
of bacteria isolated from septic dogs and correlate the disease outcome
with the ability of the bacteria to form aggregates, in vitro.
5 | CONCLUSIONS
This study demonstrated a clear effect of aggregating S aureus on the
hemostatic and cellular inflammatory response in dogs, in vitro. The
hemostatic response was hypocoagulable, hyperfibrinolytic, and of
poor clot quality. The inflammatory response demonstrated the bind-
ing of bacteria to neutrophils, monocytes, and lymphocytes, and bac-
terial phagocytosis was present. These effects were observed when
large bacterial aggregates were present, which suggests that the ability
of bacteria to form planktonic aggregates could be an important viru-
lence factor in infections, in vivo, resulting in marked hemostatic and
inflammatory host responses and more severe clinical phenotypes.
ACKNOWLEDGMENTS
We thank Klaus Qvortrup and Zhila Nikrozi, Core Facility for Inte-
grated Microscopy, Faculty of Health and Medical Sciences, Univer-
sity of Copenhagen, and Lene Theil Skovgaard, Department of Public
Health, University of Copenhagen.
DISCLOSURE
The authors have indicated that they have no affiliations or financial
involvement with any organization or entity with a financial interest
in, or in financial competition with, the subject matter or materials
discussed in this article.
ET AL.
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