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85
Interaction between Human Polymorphonuclear Leukocytes
and Streptococcus milleri Group Bacteria
Anna Wanahita,1 Elizabeth A. Goldsmith,1
Daniel M. Musher,1,3,4 Jill E. Clarridge III,2,3,5
Jose Rubio,6 Bhuvaneswari Krishnan,2,5
and JoAnn Trial1,6
1
Medical and 2Laboratory Services, Veterans Affairs Medical Center,
and Departments of 3Medicine, 4Molecular Virology
and Microbiology, 5Pathology, and 6Immunology,
Baylor College of Medicine, Houston, Texas
Because Streptococcus milleri group (SMG) bacteria—Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus—exhibit a striking propensity to cause
abscesses, the interaction of these organisms with human polymorphonuclear leukocytes
(PMNL) was examined. After incubation in pooled normal human serum, SMG stimulated
less chemotaxis than did Staphylococcus aureus, in contrast to viridans streptococci, which
caused greater chemotaxis than did S. aureus. PMNL ingested greater numbers of SMG and
viridans streptococci than S. aureus but killed these organisms more slowly and less completely. Relative resistance to killing by PMNL is expected in organisms that cause abscesses,
and inhibition of chemotaxis may contribute to pathogenicity, because delayed arrival of
PMNL gives a head start to proliferating bacteria. This study helps explain the capacity
of SMG to cause abscesses. It is unclear, however, why viridans streptococci, bacteria that
rarely produce abscesses, share some of these same properties.
The Streptococcus milleri group (SMG) consists of 3
species of bacteria (Streptococcus constellatus, Streptococcus
intermedius, and Streptococcus anginosus) that share a
remarkable propensity to cause abscesses [1– 3]. We recently
showed [3] that the great majority of clinical isolates of
S. constellatus and S. intermedius are obtained from patients
in whom these organisms have caused abscesses. S. intermedius tended to cause pleuropulmonary infections, central
nervous system abscesses, and abscesses of deep soft tissues,
whereas S. constellatus tended to cause odontogenic, soft
tissue, pleuropulmonary, and intra-abdominal abscesses. A
smaller proportion of S. anginosus isolates are associated with
abscesses (generally involving soft tissue, the pleural space,
and the peritoneal cavity), which reflects the more frequent isolation of this species from blood, infected soft tissues, and urine.
We hypothesized that the remarkable capacity to cause
abscesses might reflect a unique interaction between SMG
organisms and human polymorphonuclear leukocytes (PMNL).
We reasoned that their capacity to stimulate PMNL migration
(chemotaxis) and/or to resist ingestion or killing by PMNL
might be involved. Accordingly, we undertook the present study
to investigate this interaction; we compared isolates of Staphylococcus aureus, a well-recognized abscess-forming bacteri-
Received 24 May 2001; revised 29 August 2001; electronically published
6 December 2001.
Reprints or correspondence: Dr. Daniel Musher, Infectious Disease Section,
Veterans Affairs Medical Center, 2002 Holcombe Blvd., Bldg. 10, Rm. 4B-370,
Houston, TX 77030 ([email protected]).
The Journal of Infectious Diseases
2002;185:85–90
q 2002 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2002/18501-0011$02.00
um, and viridans streptococci, a relatively avirulent bacterial
group.
Materials and Methods
Bacteria
Twenty-two SMG isolates (7 S. constellatus, 7 S. intermedius,
and 8 S. anginosus ) from different patients were selected to reflect
the clinical spectrum of disease caused by these 3 species (table 1)
[3]. These isolates had been stored at 2 70 C after their original
isolation in the Microbiology Laboratory of the Veterans Affairs
Medical Center of Houston. Assignment to SMG was based on
results of the API 20 Strep system (BioMérieux Vitek), with further speciation by polymerase chain reaction amplification and sequence analysis of a segment of the 16S rRNA gene [4]. A convenience sample of 8 viridans streptococci isolates was obtained
from sputum cultures of 8 individual patients; these strains were
speciated by the API 20 Strep system (BioMérieux Vitek) and
were found to include Streptococcus mitis (5 strains), Streptococcus
parasanguis (2 strains), and Streptococcus sanguis (1 strain). Two
strains of S. aureus (ATCC 29213 and 25923) and 4 clinical isolates
of S. aureus obtained from blood culture were also studied.
Bacteria were cultured overnight in Todd-Hewitt broth supplemented with 5% yeast extract (Difco). Then they were collected
by centrifugation, were washed in Hanks’ balanced salt solution
(HBSS; Life Technologies), were collected by centrifugation, and
were sonicated (Ultrasonic Cleaner; Fisher Scientific) for 5 min.
Microscopic examination showed that individual cocci predominated; pairs and short chains persisted to a greater or lesser extent,
depending on the isolate. Sonication was followed by an increase
in colony-forming units. Bacteria were collected again by centrifugation and were resuspended to the desired concentration, as
determined by serial dilution and plating of aliquots on 5% sheep
blood agar (BBL; Becton Dickinson).
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Wanahita et al.
Table 1. Sources of isolates in study of interaction between human polymorphonuclear leukocytes and Streptococcus milleri group
bacteria.
Bacteria
Abscessa
Blood
Urine
Colonizing
6
5
2
0
1
2
3
0
0
0
3
0
0
0
0
8
Streptococcus constellatus
Streptococcus intermedius
Streptococcus anginosus
Viridans streptococci
NOTE. Data are no. of isolates.
Including intracerebral, intrathoracic, intra-abdominal, and perirectal sites.
a
Human Serum and PMNL
Blood obtained from 6 healthy adults was allowed to clot at room
temperature, after which it was promptly centrifuged. Serum was
removed, was pooled for use in this study (pooled human serum
[PHS]), and was stored in aliquots at 2 70 C until immediately
before use. Bacteria were opsonized by incubating an estimated
108 cfu/mL in 40% PHS in HBSS for 30 min in a shaking water
bath at 37 C. A single donor provided PMNL for all studies reported
here.
Chemotaxis Assay
PMNL. PMNL were isolated from whole blood by sedimentation in Histopaque 1119 and 1077 (Sigma). Red blood cells were
lysed by brief exposure to 0.2% saline, after which PMNL were
resuspended in HBSS with 0.1% gelatin, to yield 2 £ 106 cells/mL.
Bacteria. Triplicate samples of opsonized bacteria were
pipetted into wells in a multiwell plate that served 1 chamber of a
modified Boyden apparatus (NeuroProbe). A filter was placed
over the plate, and 50 mL of a suspension that contained
2 £ 106 PMNL/mL was pipetted onto the filter above each well.
Preliminary studies showed that 50 min of incubation at 37 C
maximized the difference between random and directed migration.
Accordingly, after 50 min, the reaction was stopped. Excess cells
were removed, after which filters were stained with Hema-Diff
(StatLab Medical Products). Cells at the lower margin of the filter
were counted under £100 magnification. Twenty fields were examined at random by 2 experienced observers who were blinded to the
conditions under which each filter had been incubated. The result
for each condition was calculated by averaging triplicate determinations. For each isolate, a range of 3-fold dilutions was studied, and a graph was constructed to extrapolate the attraction of
PMNL at 107 cfu of bacteria/mL. Migration under each condition
was reported as the percentage of cells at the margin after exposure
to S. aureus. Migration of PMNL in response to HBSS (random
migration) was subtracted before calculating actual migration in
response to other stimuli.
JID 2002;185 (1 January)
which a final sonication was done for 5 min. Pilot studies showed
that, if bacteria were not used the same day, the kinetics of phagocytosis shifted; accordingly, for the experiments reported here,
bacteria were used on the same day that the staining process was
completed.
Phagocytosis. Aliquots that contained 5 £ 106 cfu of Texas
red– labeled bacteria were added to 100 mL of heparinized blood
(preservative-free heparin; ESI) to yield a final ratio of 10 bacteria
to 1 PMNL. Tubes containing bacteria in blood were incubated for
60 min at 37 C in a shaking water bath. Ten microliters of
fluorescein-labeled anti-CD16 (CD16-FITC; Beckman Coulter)
was added, after which tubes were placed in a refrigerator for
30 min. Three milliliters of Dulbecco’s PBS lacking Mg or Ca
(Life Technologies) was added, and cells were collected by centrifugation (200 g for 5 min at 4 C). Red blood cells were lysed by use
of FACS lysing solution (Becton Dickinson), and cells were again
collected by centrifugation, were washed, and were resuspended in
500 mL of PBS containing 1% paraformaldehyde.
Flow cytometry. Flow cytometry (Epics XL-MCL; Beckman
Coulter) was used to quantitate cell-associated bacteria, by modification of a method we reported elsewhere [5]. The instrument was
standardized before each experiment by use of Flow-check
Fluorospheres (Beckman Coulter) and Flow-set Fluorospheres
(Beckman Coulter). Granularity (side scatter) was recorded on the
X-axis, and particle size (forward scatter) was recorded on the
Y-axis. The gate was set to show PMNL, both unassociated and
associated with bacteria. Events from this gate were plotted on a histogram in which the X-axis represented CD16-FITC– labeled
PMNL and the Y-axis represented Texas red– labeled bacteria.
Determination of the mean number of organisms per PMNL was
based on the ratio of the fluorescence intensity of cell-associated
organisms to the mean fluorescence intensity of each bacterium, as
assessed in a separate sample. PMNL-associated bacteria were
determined by the ratio of CD16+ PMNL that ingested bacteria to
the total number of CD16+ PMNL and were reported as a percentage.
Phagocytosis Assay
Labeling of bacteria. Opsonized bacteria were collected by
centrifugation and were resuspended in carbonate buffer (pH 9)
that contained 0.7 mg/mL Texas red sulfonyl chloride (Molecular
Probes). This suspension was incubated on a shaking platform in
the dark at 4 C for 18 h. Texas Red–labeled bacteria were washed
6 times and were resuspended in HBSS to yield 108 cfu/mL, after
Figure 1. Chemotaxis induced by Streptococcus milleri group bacteria (Streptococcus constellatus, Streptococcus intermedius, and
Streptococcus anginosus) and viridans streptococci (Y-axis), shown as
percentage of chemotaxis stimulated by reference strain Staphylococcus aureus ATCC 29213 (heavy vertical line, 100%). Error bars indicate SE.
JID 2002;185 (1 January)
S. milleri Group Bacteria and PMNL
Figure 2. Mean numbers of Streptococcus milleri group bacteria
(Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus), Staphylococcus aureus, and viridans streptococci
associated with polymorphonuclear leukocytes (PMNL) after incubation in whole blood at a 10:1 ratio of bacteria to PMNL for 60 min
at 37 C. Error bars indicate SE.
Bacterial Killing by Human PMNL
Opsonized bacteria were added to freshly obtained blood from a
human donor, to yield a ratio of bacteria to PMNL of 1:1, and this
suspension was incubated at 37 C. Aliquots were removed at time
0 and after 30, 60, 90, and 120 min. Tenfold dilutions were made in
distilled water, with ample vortexing to allow for disintegration of
PMNL, and 10-mL aliquots were streaked onto blood agar for
bacterial enumeration.
Statistics
Mean data (^ SE) are reported. The Kruskal-Wallis test was used
to determine whether there were significant differences among the
3 species of SMG. The Mann-Whitney U test was used to make
pairwise comparisons between species in phagocytosis and
chemotaxis studies. Survival of bacteria in bactericidal assays was
analyzed by the Kaplan-Meier method. To test for equality between
curves, the log rank test, x2 probability was used. For all tests,
a ¼ :05.
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by opsonized SMG species ranged from 33:5% ^ 9:8% to
55:1% ^ 27:6% of that induced by S. aureus. Differences
among the 3 SMG species were not significant (P ¼ :9, KruskalWallis test). The differences compared with S. aureus were significant for S. constellatus and S. anginosus (P , :05 for each
comparison; P ¼ :07 for S. intermedius ). In contrast, chemotaxis
induced by viridans streptococci greatly exceeded that induced by
S. aureus (166:9% ^ 23:1%; P , :005).
Phagocytosis. Pilot studies showed that the number of cellassociated bacteria and the proportion of PMNL that had .1
associated bacterium were similar for 2 ATCC strains and 4
clinical isolates of S. aureus. Each flow cytometry experiment
thereafter included S. aureus ATCC 29213, .1 isolate of each
species from SMG, and a viridans streptococcus. The number
of organisms per PMNL and the percentage of PMNL with .1
cell-associated S. aureus were consistent from day to day and
were similar to previous reports from this laboratory [5]. The
mean (^ SE) number of organisms per PMNL for S. aureus was
4:7 ^ 2:4. Greater numbers of organisms per PMNL were
found for SMG bacteria and viridans streptococci, with means
ranging from 7:0 ^ 2:8 organisms/PMNL for S. intermedius to
14:3 ^ 2:8 organisms/PMNL for S. constellatus (figure 2). The
differences compared with S. aureus were significant (P , :05)
for S. constellatus, S. anginosus, and viridans streptococci (P ¼
:07 for S. intermedius ).
The mean (^ SE) number of PMNL with cell-associated
S. aureus was 77:6% ^ 7:7% (figure 3). After incubation with
SMG organisms, a greater proportion of PMNL had cell-associated bacteria, ranging from 91:9% ^ 4:2% to 97:9% ^ 2:0%,
with the differences being significant for S. constellatus and
S. anginosus (P , :05 for each comparison); in the case of S.
intermedius, the difference did not reach statistical significance
Results
Chemotaxis. Preliminary experiments examined migration
of PMNL in response to HBSS, PHS, unopsonized bacteria,
and PHS after bacteria had been incubated in it and removed
by centrifugation (spent serum). Identical results were obtained
with S. aureus ATCC 29213 and a clinical S. aureus isolate
after incubation in PHS; accordingly, ATCC 29213 served as the
internal laboratory reference to be run in each assay and against
which all other bacteria would be compared. Chemotaxis in
response to pooled human serum, unopsonized bacteria, and
spent serum was , 15% of that observed with opsonized S. aureus. As shown in figure 1, chemotaxis (mean ^ SE) induced
Figure 3. Percentage of polymorphonuclear leukocytes (PMNL)
with . 1 cell-associated bacterium of Streptococcus milleri group bacteria (Streptococcus constellatus, Streptococcus intermedius, and
Streptococcus anginosus), Staphylococcus aureus, or viridans streptococci after 1 h of incubation at 37 C. Error bars indicate SE.
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Wanahita et al.
JID 2002;185 (1 January)
nearly identical, with the mean percentage killed at 120 min
ranging from 65% to 71%. There was no killing of viridans
streptococci under these conditions.
Electron microscopic examination. To verify that the association between PMNL and bacteria, as shown in phagocytosis
assays, was accompanied by actual bacterial ingestion, we examined PMNL after they had been incubated for 30 min at 37 C
with opsonized bacteria at a ratio of 10 bacteria to 1 PMNL.
Figure 5 shows representative results—in this case, phagocytosis of S. intermedius by PMNL. One isolate of each bacterial
species described here was studied; ingestion was found to occur in each case (data not shown).
Figure 4. Killing of Streptococcus milleri group bacteria (Streptococcus constellatus, Streptococcus intermedius, and Streptococcus
anginosus), Staphylococcus aureus, and viridans streptococci during
incubation in whole blood for 120 min.
(P ¼ :1). For viridans streptococci, 91:8% ^ 6:1% of PMNL
had ingested bacteria (P ¼ :03, vs. S. aureus ).
Killing of bacteria by human PMNL. Time-dependent killing of S. aureus occurred during incubation with PMNL; at 120
min, 99% of organisms were killed (figure 4). Results were
consistent from day to day. In contrast, killing of each of the
SMG species proceeded at a substantially slower rate (P ,
:001, Kaplan-Meier method). Results for these 3 species were
Discussion
To cause an abscess, bacteria must release substances that
attract PMNL and, to a greater or lesser extent, resist ingestion
and killing by PMNL. The present study examined the interaction of 3 species of SMG, a taxonomic group of bacteria that
all share a remarkable propensity to cause abscesses, with human PMNL. We examined the chemotactic effect of these organisms, as well as their capacity to resist phagocytosis and killing by
PMNL after opsonization in complement-rich, pooled human
serum.
The observation that SMG bacteria attracted significantly
fewer PMNL than did S. aureus in an in vitro assay of chemotaxis might appear to be paradoxical. It should be noted,
Figure 5. Electron microscopic view (original magnification, £12,000) after incubating opsonized Staphylococcus intermedius with polymorphonuclear leukocytes (PMNL) for 30 min at a ratio of 10 bacteria per PMNL. Cytoplasmic projections are seen surrounding and about to ingest bacteria. Many bacteria are seen within cytoplasm, contained in phagocytic vacuoles. Some bacteria show signs of replicating; others have lost integrity,
consistent with loss of viability. Most bacteria appear to be intact.
JID 2002;185 (1 January)
S. milleri Group Bacteria and PMNL
however, that S. aureus, with its well-recognized capacity to
cause abscesses, has been shown to inhibit the ingress of PMNL
in vivo [6] and in vitro [7], which is consistent with the
even greater degree of chemotaxis after exposure to viridans
streptococci. Leukocidin(s), probably identical with PantonValentine leukocidins that directly damage PMNL and/or red
blood cells [8, 9], have been found to be responsible; these leukocidins are especially likely to be present in strains that cause furunculosis [10]. Inhibition of chemotaxis is thought to contribute
to pathogenicity of S. aureus [11] by delaying the ingress of
PMNL, thereby giving a head start to invading bacteria [6].
S. intermedius produces intermedilysin [12], a toxin that may
share certain characteristics with staphylococcal leukocidins.
Although the gene that encodes this toxin is not present in
S. constellatus or S. anginosus [13], other toxins may exert a
leukocidin-like effect in these species.
In the case of SMG, the number of bacteria per PMNL and the
number of PMNL that had cell-associated organisms after
incubation exceeded that of S. aureus, an extensively studied
bacterium that also causes abscesses. By contrast, a bacterial
killing study showed that, after ingestion by PMNL, SMG
bacteria were killed at a rate of only 3% of that of S. aureus. It is
unclear why SMG are so highly resistant to PMNL killing.
Seven or 8 distinct isolates were studied from each bacterial
species, and some were studied on as many as 4 occasions, with
remarkably uniform results.
Relative resistance to killing by PMNL is an expected characteristic of organisms that cause abscesses. Because S. aureus is
well known to survive relatively well within human leukocytes
[14], the substantially better survival of SMG bacteria, at least
during 2 h of study in vitro, is quite impressive. Organisms as
diverse as Mycobacterium tuberculosis [15], Brucella abortus
[16], and Bacteroides gingivalis [17] have been shown to produce substances that inhibit activation and/or discharge of granule enzymes into vacuoles within PMNL; a similar mechanism
might be responsible in the case of SMG. Electron microscopic
examination verified that streptococci were ingested by PMNL;
although some cocci appeared to be disintegrating, the morphologic appearance of the bacteria inside phagocytic vacuoles was
quite normal, which is consistent with a low degree of bacterial
killing.
Rakita et al. [18] suggested that the presence of large phagocytic vacuoles is associated with the failure of PMNL to kill Enterococcus faecalis. Although we detected large vacuoles after
ingestion of SMG bacteria, which were not killed, similar-sized
vacuoles were seen after ingestion of S. aureus, which were
killed.
An unexplained finding that emerged from these studies was
the resistance of viridans streptococci to killing by PMNL. Mandell [19] found a single strain of viridans streptococci to
be susceptible to PMNL-mediated killing, but our study used
8 separate clinical isolates, running each experiment .2 times,
with consistent results. Electron microscopy showed that viri-
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dans streptococci had been internalized, so the fact that they
were not killed remains unexplained.
Acknowledgment
We thank Edward A. Graviss for statistical analysis.
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