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Infection and Immunity, January 2001, p. 45-51, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.45-51.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Staphylococcus aureus agr Genotypes with Enterotoxin
Production Capabilities Can Resist Neutrophil Bactericidal
Activity
I. K.
Mullarky,1
C.
Su,1
N.
Frieze,1
Y. H.
Park,2 and
L. M.
Sordillo1,*
Department of Veterinary Science, Center for
Mastitis Research, The Pennsylvania State University, University
Park, Pennsylvania 16802-3500,1 and
Department of Microbiology, College of Veterinary Medicine,
Seoul National University, Seoul, Korea2
Received 30 June 2000/Returned for modification 20 August
2000/Accepted 3 October 2000
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ABSTRACT |
Staphylococcus aureus pathogenicity is mainly due to
the production of a number of secreted and cell surface-associated
proteins under the regulation of the agr gene. A region of
the agr gene was used to subgroup S. aureus
strains according to restriction fragment length polymorphisms.
Additionally, strains were subtyped according to the coagulase gene in
order to strengthen discriminatory power. Virulence capabilities of
agr genotype subgroups were evaluated using an in vitro
neutrophil bactericidal assay, which showed that prevalent genotypes
were significantly better at evading this primary host defense.
Multiplex PCR was then used to detect enterotoxin genes among the
genotype subgroups in order to determine possible virulence candidates
that enable strains to combat neutrophil killing. The prevalent
genotype strains were found to possess higher production capabilities
for enterotoxin A than did low-prevalence strains. The significance of
enterotoxin A production capabilities in affecting pathogenicity of
S. aureus strains was evaluated and found to have a
profound effect on neutrophil killing abilities. The use of a large
epidemiological database as a tool for subgrouping strains with varying
degrees of pathogenicity has allowed the identification of relevant and
previously undefined virulence factors that affect a pathogen's
capability to overcome host immune defenses.
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INTRODUCTION |
Staphylococcus aureus is
a gram-positive bacterium that has remained a persistent pathogen,
causing such infections as endocarditis, meningitis, and toxic shock
syndrome in humans. S. aureus also is the leading cause of
intramammary infections (mastitis), especially in dairy animals, from
whose milk it is frequently isolated (38). Neutrophils are
the principle line of defense during the initial stages of mastitis,
and the ability of these cells to phagocytize and kill invading
bacteria is critically related to the establishment of new intramammary
infections (26). Therefore, any bacterially derived
component that may compromise neutrophil function would constitute an
important virulence factor in the pathogenesis of S. aureus
mastitis. Although a number of different virulence factors involved in
the pathogenesis of S. aureus mastitis have been identified (38), the differential expression of these factors as it
relates to field strain prevalence of S. aureus genotypes
has not been investigated. A better understanding of the epidemiology
of S. aureus mastitis as it pertains to virulence will
provide insight concerning important host-pathogen interactions during
the pathogenesis of disease.
Subtyping is an important tool for epidemiologic investigation of
bacterial infections. In the past decade, numerous molecular techniques
such as multilocus enzyme electrophoresis, phage typing, plasmid DNA
restriction patterns, random amplified polymorphic DNA ribotyping, and
coagulase genotyping have proved useful in identification and
comparison of S. aureus isolates in epidemiological studies
(7, 21, 29, 35, 36). However, very few studies have
identified S. aureus isolates by the gene polymorphisms
among important virulence-related genes. Among the virulence-related genes in S. aureus, we were particularly interested in the
accessory gene regulator (agr), which has been shown to
regulate the synthesis of many virulence factors during bacterial
growth (5, 25). The agr system coordinately
down-regulates the production of cell wall-associated proteins and
up-regulates secreted proteins at late to stationary growth phase in
vitro (16, 24, 25, 27). The agr locus encodes a
two-component signal-transducing system consisting of two divergent
transcription units driven by promoters P2 and P3 (15).
The P3 operon encodes the transcript for RNAIII, the effector of the
agr response, while the P2 operon contains transcripts for
four open reading frames designated agrA, -B, -C, and -D (6). agrB and
-D generate an autoinducing peptide that acts as an
activating ligand for agrC. Interestingly, Ji et al.
(15, 16) have shown that variations in the gene sequences of agrB and -D result in variation of the
autoinducing peptide that, in turn, causes differences in the
activation of strains by one another (15, 16). In
contrast, mutations of wild-type S. aureus strains resulting
in agr deletions reduced persistence of infection, exotoxin
synthesis, and binding capabilities and decreased intracellular growth
of these strains (3, 11, 37), suggesting that
agr itself is an important virulence gene in S. aureus.
The aim of this study was to identify potential S. aureus
virulence factors based on their unique expression in predominant field
strains isolated from clinical cases of mastitis. In this study, we
developed a genotyping method for S. aureus strains based on
agr gene polymorphisms. We showed that enterotoxin
production capabilities were more pronounced in the prevalent
agr genotypes that were more resistant to neutrophil
bactericidal activities than were the low-prevalence genotypes. The
ability of enterotoxin A to directly modify neutrophil function
suggests an important role of these toxins in the pathogenesis of mastitis.
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MATERIALS AND METHODS |
Bacterial isolates.
The S. aureus strains used in
this study included RN6390B, a wild-type agr+
strain (25); RN6911, an agr mutant
(25); SA 502A (ATCC 27217); and 255 field isolates.
S. aureus field isolates were collected from clinical
mastitis bovine milk samples from the Czech Republic (n = 10), France (n = 34), Korea (n = 165), and several locations within the United States (n = 46), (including Indiana, Kentucky, Louisiana, Minnesota, New
York, Pennsylvania, Tennessee, Washington, and Wisconsin). The variety
of geographical locations from which isolates were collected provided
control for regional differences in herd management and herd
differences that result in variations in host resistance to disease.
All isolates were stored in Trypticase soy broth with 15% glycerol at
70°C until needed. Isolates were cultured on Trypticase soy agar
with 5% sheep blood (BiMed, St. Paul, Minn.) for identification based
on colony morphology, hemolysis, Gram stain, and acetoin and catalase
production. Coagulase production by isolates was determined in a tube
test using 0.5 ml of citrate-stabilized rabbit plasma. One colony from
an overnight culture on Trypticase soy agar-5% sheep blood was
inoculated into a plasma-containing tube and incubated at 37°C. A
positive test was determined by clot formation after 1, 4, or 24 h. To differentiate S. aureus from coagulase-positive
S. hyicus and S. intermedius, the acetoin test
(Voges-Proskauer test) was used as outlined previously
(1). All isolates with a questionable acetoin test result
were further identified by the API Staph system (bioMerieux Vitek,
Hazelwood, Mo.).
Bacterial DNA lysates.
Bacterial DNA lysates were prepared
from 1 ml of an overnight TSB culture. Bacteria were then pelleted and
resuspended in 500 µl of 50 mM Tris-HCl buffer (pH 8.3) that
contained 50 mM disodium EDTA. Lysis of cells was conducted using 15 U
of lysostaphin (Sigma, St. Louis, Mo.) and incubating at 37°C for 30 min. Lysis was completed by adding 1 ml of lysis buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 1% Triton
X-100, 0.45% Igepal CA-630, 0.45% Tween 20, and 0.6 µg of
proteinase K and incubating for 1 h at 56°C. Proteinase K was
inactivated by heating at 95°C for 10 min.
agr genotyping.
In order to amplify the variable
region of the agr gene in the S. aureus strains,
nested primers were designed using S. aureus sequences
available from GenBank and the DNASTAR (Madison, Wis.) software
program. The sequences of the outer primers were
5'-ACCAGTTTGCCACGTATCTCA-3' and
5'-AACCACGACCTTCACCTTTAGTAG-3'. Amplification was
conducted in a total volume of 40 µl consisting of 10 µl of
cell lysate, 4 µl of 10× buffer, 0.4 µl of deoxyribonucleoside
triphosphate (dNTP) (25 mM [each] dATP, dCTP, dGTP, and dTTP), a 2 µM concentration of each primer, 2.4 µl of MgCl2 (25 mM), 2.5 U of Taq DNA polymerase, and 8 µl of
nuclease-free water. Reactions were cycled as follows: 95°C for
30 s, 64°C for 60 s, and 72°C for 120 s for 34 cycles.
For the nested primer amplification, 1 µl of the first PCR mixture
was added to 39 µl of PCR mixture containing 2 µM concentrations of
the second set of primers: 5'-TGCCACGTATCTTCAAA-3' and
5'-ATAATCATGACGGAACTT-3'. The nested PCR amplification
conditions were the same as above with an annealing temperature of 54 instead of 64°C.
Ten microliters of the second PCR mixture was digested at 37°C for
1 h with 2 U of the restriction endonuclease
AluI
(Promega,
Madison, Wis.) according to the manufacturer's instruction.
The
digested DNA fragments were separated in a 3% agarose gel (Sigma)
and visualized in the presence of ethidium bromide under UV
light.
Coagulase genotyping.
The coagulase genotyping was performed
by a previously described method (1). Ten microliters of
DNA lysates was added to a mixture containing a 1 µM concentration of
each primer (COAG1, 5'-ATACTCAACCGACGACACCG-3', and COAG4,
5'-GATTTTGGATGAAGCGGATT-3'), 50 mM KCl, 1.5 mM
MgCl2, 10 mM Tris-HCl, 1% Triton X-100, a 200 µM
concentration of each dNTP, and 1 U of Taq polymerase to a final reaction volume of 40 µl. Each sample was subjected to 40 PCR
cycles, consisting of 30 s at 95°C, 2 min at 55°C, and 2 min at 72°C. For the nested-PCR amplification, 1 µl of the first PCR mixture was added to 39 µl of PCR mixture containing a 1 µM
concentration of the second set of primers (COAG2,
5'-ACCACAAGGTACTGAATCAACG-3', and COAG3,
5'-TGCTTTCGATTGTTCGATGC-3'). The nested-PCR amplification was performed using the same conditions as the first PCR. Ten microliters of the second PCR mixture was digested at 37°C for 1 h with 2 U of the restriction endonuclease AluI according to the manufacturer's instruction. The digested DNA fragments were separated in 4% NuSieve GTG agarose gel (FMC BioProducts, Rockland, Maine) and detected in the presence of ethidium bromide under UV illumination.
Detection of enterotoxin genes by multiplex PCR.
Enterotoxin
typing was conducted according to the following methodology. In brief,
staphylococcal genomic DNA was extracted from lysostaphin-treated cells
and processed as described previously (20). The DNA was
extracted with phenol-chloroform (1:1, vol/vol) and chloroform and then
precipitated with ethanol according to standard techniques
(28). Specific primers for staphylococcal enterotoxin A
(SEA), SEE, and toxic shock syndrome toxin 1 (TSST-1) were synthesized
with a DNA synthesizer (Expedite 8905; Perseptive Co.) as described
previously by Johnson et al. (17, 18, 28). The
oligonucleotide sequence of each primer is shown in Table 1. The PCR was performed under the
following parameters: the reaction mixture consisted of 2.5 µl of
10× reaction buffer without MgCl2 (Promega Corp.); 400 M
dNTP; 3 mM MgCl2; 7.5% dimethyl sulfoxide; 50 pmol of
primers for sea, seb, sec,
sed, and see; 100 pmol of primers for
tst; and 100 ng of template DNA; and brought up to a 25-µl
final volume with distilled water. Reactions were hot started for 5 min
at 95°C and placed on ice, and 1 U of Taq polymerase (Promega Corp.) was added. Each sample was subjected to 30 PCR cycles,
consisting of 95°C for 1 min, 2 min at 56°C (for the combination of
primer sets for SEA, SEC, and SED) or 50°C (for SEB, SEE, and TSST-1), and 1 min at 72°C. PCR products were separated on a 1.5% agarose gel and visualized under UV illumination.
Statistical analysis.
Discriminatory powers of both
coagulase and agr genotyping were evaluated as described by
Hunter (14). Concordance analysis (10) of
agr genotype, coagulase genotype, and enterotoxin production capabilities was conducted by using the Minitab (State College, Pa.)
statistical program to determine matches and mismatches among isolates
within the same and different groups. Pairwise comparisons (number of
combinations) were determined with the following formula: Pk,n/k! = n!/k!(n k)!, where n
= number of samples and k = number of combinations
chosen. The G test of independence using Yate's correction for
continuity (31) was used to evaluate statistical significance.
Bovine blood neutrophil bactericidal assay.
The functional
capabilities of bovine neutrophils are a major factor which determines
the establishment of new intramammary infections. For this reason, a
series of in vitro assays were conducted to assess the relative
abilities of certain strains to resist this important host defense
mechanism. Bovine neutrophils were isolated from four lactating
Holstein cows free of intramammary infection as determined by
microbiological analyses of milk samples. Neutrophils were isolated as
previously described (2). For the purpose of opsonization,
bovine antiserum was collected from cows diagnosed with S. aureus mastitis.
Ten
S. aureus isolates were selected from both predominant
(
n = 5) and rare (
n = 5) genotypes to
evaluate differences in capabilities
of prevalence groups to evade
neutrophil killing. Additionally,
eight
S. aureus isolates
were selected from predominant genotypes
with enterotoxin genes
(
n = 4) and without enterotoxin genes (
n = 4) to evaluate the effect of enterotoxin genes on neutrophil
killing capabilities. Bacteria were prepared by initial culturing
in 30 ml of assay medium (RPMI-5% fetal bovine serum-1%
L-glutamine)
at 37°C for 6 to 12 h. After incubation
bacterial concentration
was determined via serial dilutions. Bacterial
inocula were stored
at 4°C, while concentrations were determined and
final concentrations
were adjusted to 10
7 CFU/ml in assay
medium. The resistance of bacteria to bovine
neutrophil bactericidal
activities was evaluated by the bactericidal
assay as previously
described (
2). In brief, bacteria were
opsonized in 6.25%
bovine antiserum for 30 min at 37°C. In a 96-well
plate, 100 µl of
bacteria (10
7 CFU/ml) was combined with 100 µl of
neutrophils (10
8/ml) and incubated at 37°C for 1 h.
Following incubation, neutrophils
were lysed with the addition of 0.2%
Saponin (Sigma) followed
by the addition of MTT (1 mg/ml; Sigma). Upon
color development,
the addition of extraction buffer lysed bacterial
cells. Plates
were read at a wavelength of 595 nm on a microplate
reader (Bio-Rad,
Hercules, Calif.).
Additional assays were conducted to determine if SEA, the enterotoxin
gene type observed most frequently in field isolates,
could affect
neutrophil bactericidal activity. For experiments
involving SEA
(Sigma), neutrophils were preincubated with various
concentrations of
enterotoxin A for 15 min at 37°C in order to
determine the direct
effect of this enterotoxin on neutrophil
killing capabilities. The
dessicated SEA contained 10% protein
and 90% sodium phosphate buffer
by weight; therefore, neutrophils
were preincubated with 4 mM sodium
phosphate, the highest concentration
found when 20 µg SEA was tested,
to serve as control. Bacterial
strain Newbould 305 was used to
determine the effect of SEA on
neutrophil bactericidal capability.
Bacteria were inoculated as
described above and washed three times in
phosphate-buffered saline
to remove any secreted enterotoxin produced
during growth. Bacteria
were then resuspended in assay medium and
counted as described
above. Student's
t test was used to
compare percentages of bacteria
killed for strains within each
prevalence or treatment
group.
Detection of enterotoxin protein.
S. aureus strains
used in the neutrophil bactericidal assays for toxin evaluation were
further studied to determine protein production of enterotoxins under
assay conditions. Bacteria were prepared by initial culturing in 30 ml
of assay medium (RPMI-5% fetal bovine serum-1%
L-glutamine) at 37°C for 6 to 12 h. After incubation, bacterial cultures were centrifuged at 3,500 × g for 5 min at 15°C. Supernatants were then filter sterilized
and used in an enzyme immunoassay for the detection of enterotoxins A, B, C, D, and E (RIDASCREEN Set A,B,C,D,E; r-biopharm, Darmstadt, Germany).
| |
RESULTS |
agr genotyping.
Available agr sequences
were analyzed in order to determine which region of the gene had the
largest diversity to ensure that a variety of restriction fragment
length polymorphism profiles would be attained (Table
2). DNA sequence analysis of several S. aureus strains showed highly conserved regions at
the 5' end of the agrB gene and the 3' end of the
agrC gene (GenBank sequence accession numbers
AF001782, AF001783, U85095, and X52543). These conserved sequences were
used to design primers to amplify agr gene fragments
displaying areas of high divergence found within the stable regions.
Specificity of the designed primers was confirmed using
Escherichia coli and an agr S. aureus mutant
RN6391 (Fig. 1). Sequencing of PCR
products from S. aureus ATCC 27217 and S. aureus
RN6390 confirmed that the PCR product was the targeted region of the
agr locus an expected product of approximately 1,386 bp.
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FIG. 1.
Nested PCR products of S. aureus ATCC 27217 (lane 1), RN6390 (lane 2), agr mutant RN6391 (lane 4), and
E. coli (lane 3).
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S. aureus strains collected from clinical bovine milk
samples from Czech Republic, France, Korea, and several states in the
United States were genotyped by
agr gene polymorphism.
Twenty
genotypes have been identified, and an example of an agarose gel
electrophoresis is shown in Fig.
2.
Genotype profiles were designated
by the total number of major bands
followed by the estimated molecular
weight (in thousands) of each band.
The most prevalent type was
found to have eight major bands with
molecular weights of 480,
385, 310, 257, 168, 156, 104, and 75 and was
therefore designated
8:480, 385, 310, 257, 168, 156, 104, 75. The
frequencies of all
the identified
agr genotypes within the
tested samples are shown
in Table
2. From a total of 255 strains
tested, seven samples
were nontypeable using
agr genotyping
methods.
agr genotyping
methodology had a discriminatory
power (
D) of 0.7217.
Coagulase genotyping.
The 255 available isolates were
subdivided into 40 different coagulase types according to previously
designated typing patterns (1). There were four samples
that were nontypeable using the coagulase genotyping method. Coagulase
typing had a very high D of 0.938. As shown previously, only
a few genotypes predominate in each country (32). However,
comparisons among countries indicated that predominant types could be
distributed in different geographical locations (32).
Detection of enterotoxin genes by multiplex PCR.
The presence
of enterotoxin genes was assessed in 211 isolates randomly chosen from
the available 255 isolates from various locations. Of the isolates
tested, 118 isolates tested negative for enterotoxin genes (56%), and
93 isolates were positive for enterotoxin genes (44%). Of the isolates
positive for enterotoxin genes 58 were positive for SEA (62.4%) only,
3 were positive for SEB (3.2%) only, 5 were positive for SEC (5.4%)
only, 3 were positive for SED (3.2%) only, and 4 were positive for
TSST (4.3%) only. In addition, 20 of the isolates tested positive for
more than one enterotoxin gene: 4 isolates were positive for both SEA
and SEC (4.3%); 2 isolates were positive for SEA, SEC, and TSST
(2.15%); 2 isolates were positive for SEA and TSST (2.15%); 3 isolates tested positive for SEC, SED, and TSST (3.2%); and 9 isolates possessed SEC and TSST (9.7%) genes. Selected strains of S. aureus used in the bactericidal assays also were evaluated
for expression of enterotoxin genes. Strains with enterotoxin
genes were found to produce the respective enterotoxin protein as
evaluated through an enzyme immunoassay. Additionally, strains
that tested negative for enterotoxin genes did not test positive for
any enterotoxin protein production under assay conditions (data not shown).
Concordance analyses of typing techniques.
All possible pairs
of agr genotyping and coagulase genotyping were compared
among the 248 samples that were typeable using the two techniques.
Pairs of isolates were classified by whether they possessed the same or
different agr genotypes and whether they matched or
mismatched in coagulase genotypes (Table
3). A total of 30,628 pairwise
comparisons for the 248 isolates were possible. For the isolates with
the same agr type, 4% had matching and 23% had mismatching
coagulase genotypes. For the isolates with different
agr types, 2% had matching and 71% had mismatching coagulase genotypes. The overall concordance percentage for
coagulase and agr genotypes was 75.2% (simple
matching coefficient [S] = 0.752) and had a significance of
P < 0.001 (G test of independence, G = 1621.4; df = 1).
Concordance analysis of genotyping methods and enterotoxin
typing.
Of the 211 isolates tested for enterotoxin type, 6 isolates had unidentifiable agr genotypes and 3 isolates had
unidentifiable coagulase types. Pairs of isolates were identified by
whether they had the same or different enterotoxin type and whether
they matched or mismatched in either agr genotype or
coagulase genotype (Table 4). Of the
total of 21,910 possible pairwise comparisons between enterotoxin type
and agr genotype (205 isolates), 10% of the same
enterotoxin type had matching agr genotypes and 29% had
mismatching agr genotypes. For the isolates with different enterotoxin types, 17% had matching and 44% had mismatching
agr genotypes. The overall concordance of enterotoxin type
with agr genotype was 54.3% (simple matching coefficient,
S = 0.543) and had a significance of P < 0.001 (G test of independence, G = 14.2; df = 1).
In the total of 21,528 possible pairwise comparisons between
enterotoxin type and coagulase genotype (208 isolates), 3% of
same
enterotoxin type had a matching coagulase genotype and 36%
had a
mismatching coagulase genotypes. For the isolates with different
enterotoxin types, 3% had matching and 58% had mismatching coagulase
genotypes. The overall concordance of enterotoxin type with coagulase
type was 61.2% (simple matching coefficient,
S = 0.612) and had
a significance of
P < 0.001
(
G test of independence,
G = 64.4;
df =
1).
Bovine blood neutrophil bactericidal assay.
The results of
bactericidal assays are shown in Fig. 3 to
5. The mean percentage
of killing was 41% (standard error
[SE] = 1.4) for the five high prevalence S. aureus strains
and 59% (SE = 0.9) for the five
low-prevalence strains evaluated. There was a significant difference in
bactericidal effects (P = 0.004). Additionally, the
mean percentage of killing was 35% (SE = 1.6) for four
high-prevalence strains with enterotoxin genes and 54% (SE = 1.2)
for strains without enterotoxin genes. A significant difference was
found in bactericidal effects (P = 0.005) between those
strains with and without enterotoxin genes. Strains with enterotoxin
genes were found to produce the respective enterotoxin protein as
evaluated through an enzyme immunoassay (data not shown). Additionally,
strains that tested negative for enterotoxin genes did not test
positive for any enterotoxin protein production under assay conditions.
Addition of exogenous SEA to the bactericidal assay resulted in a
significant decrease in neutrophil killing capabilities (P < 0.05) at concentrations of 5, 15, and 20 µg.
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FIG. 3.
Bovine blood neutrophil bactericidal activity against
either high- or low-prevalence S. aureus strains (*,
P < 0.005). Error bars, SE.
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FIG. 4.
Bovine blood neutrophil bactericidal activity against
high-prevalence S. aureus strain with or without enterotoxin
genes. *, P = 0.005; error bars, SE.
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FIG. 5.
Bovine blood neutrophil bactericidal activity against
S. aureus strain Newbould 305 in the presence of enterotoxin
A. Bars
labeled with different letters indicate that values differ
significantly (P < 0.05). Error bars, SE.
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DISCUSSION |
Previous work in our laboratory has used the hypervariable region
of the coagulase gene to type S. aureus strains
(32). The coagulase gene was chosen due to its ubiquitous
presence among S. aureus strains, and this gene proved to
create a very powerful typing method. In this study, the agr
gene also was used to further type strains and provided a means of
grouping S. aureus isolates based on a factor that controls
virulence-related gene production. The agr gene was used
successfully to subtype S. aureus strains, and when the
information attained from genotyping of the coagulase gene and the
agr gene was combined, the discriminatory power of each
method was greatly empowered. The ability to group pathogens based on
field prevalence may provide important information pertaining to the
coevolution of hosts and pathogens which will influence the genetic
diversity of disease-causing microorganisms (13, 22).
Selection within the pathogen population will favor mechanisms to avoid
host defense and to colonize the host. The pathogens that are most
efficient at avoiding the host's defense mechanisms will be the most
prevalent type found in the microenvironment of interest. The argument
for this phenomenon is supported by the findings in this study in which
the abilities of neutrophils to kill different S. aureus
genotypes varied with respect to their prevalence in cases of mastitis.
We discovered that the most common genotype of S. aureus
also was the type against which the neutrophils, and thereby the
host's initial defense mechanism, were least efficient. In contrast,
the neutrophils were highly efficient against the rarely found
genotypes. These results are consistent with our previous reports
(2, 32) and suggest that types found in high prevalence
have unique characteristics, which in contrast to the rare types, endow
them with the superior ability to suppress or resist killing by
neutrophils. In the past, in vitro studies of host-pathogen
interactions have focused on the use of few selective bacterial strains
studied routinely in a laboratory environment (3, 4, 12).
However, statistical analysis of a large database of pathogenic strains
may provide critical information concerning key virulence-related genes
in a prevalent group of disease-causing bacteria that can be evaluated
relative to the interaction between pathogen and host in a specific
disease model.
Bacterium-host interactions depend on several factors, including the
efficiency of the host's defense, growth rate of the bacteria, and
production of virulence factors. The neutrophils used in the assays
were obtained from the same group of animals, so differences in killing
ability cannot be attributed to variations in host defense. In
addition, no differences in the growth potential were observed among
the strains used in the bactericidal assays. One possible explanation
for the variation in killing efficiency may be a consequence of the
expression of certain bacterially produced factors. S. aureus has the capacity to synthesize a repertoire of known
virulence factors associated with mastitis, including capsular
polysaccharide, cell-surface associated proteins, and several hemolytic
toxins (33). However, the potential role of enterotoxins
in the pathogenesis of S. aureus mastitis is uncertain and
has been the topic of several conflicting studies (9, 19). In this study, the use of multiplex PCR technology of the enterotoxin gene was conducted to evaluate the significance of this group of
potential virulence factors associated with mastitis-causing S. aureus strains. Use of this technique to evaluate enterotoxin genes eliminated the variations previously observed for enterotoxin detection as a result of various growth conditions influencing the cell
density sensing system that controls virulence factor production in
S. aureus (16). We found that 44% of S. aureus isolates tested contained enterotoxin genes, and these
values are consistent with previous published findings using similar methodologies (20). The significant relationship between
enterotoxin types and agr genotypes reported here is
supported by previous work in which differences were observed in toxin
production due to mutations in the agr gene (11,
25).
It was interesting to further delineate if the ability of prevalent
S. aureus strains to evade host defense mechanisms was related, at least in part, to enterotoxin production capabilities. We
showed that strains with enterotoxin genes were significantly (P = 0.005) better at evading this nonspecific cellular
defense mechanism of the host compared to strains without these genes. S. aureus enterotoxins have been shown to activate T-cell
subsets (34) as well as provide protection against
neutrophil apoptosis (23). However, very limited
information is available as to the effect of enterotoxins on neutrophil
bactericidal activities. In order to establish a direct link between
status of the agr gene, enterotoxin genes, and neutrophil
killing capabilities, specific S. aureus enterotoxins were
studied for the ability to alter bactericidal activities of
neutrophils. We assessed the direct effects of SEA on neutrophil
bactericidal capabilities since this enterotoxin gene was most commonly
observed among the most prevalent agr genotypes. The
addition of exogenous SEA was shown to significantly reduce the killing
abilities of neutrophils under our assay conditions. Previously, work
by Berger et al. (8) observed no effect on SEA on
neutrophil bactericidal activity. However, the preincubation of
neutrophils with enterotoxin prior to evaluation of killing abilities,
as carried out in the present study, may be the deciding factor in the
varying results observed. Though there are several putative virulence
factors that can account for the effect on neutrophil killing ability
observed in Fig. 3, singling out SEA, the most-common enterotoxin gene
possessed by the isolates in this study provides a direct correlation
between variations in the agr gene sequences, enterotoxin
production, and ability to evade neutrophil killing.
The epidemiological study of a large database of disease-causing
bacteria can be used to identify uniquely expressed virulence factors
that are associated with the ability of pathogens to evade important
host defense mechanisms. Using this approach, we showed for the first
time that staphylococcus enterotoxins can contribute to the
pathogenesis of S. aureus mastitis as suggested by the agr genotype field prevalence data. We also showed that one
possible mechanism by which SEA may affect virulence is through its
ability to directly hinder neutrophil bactericidal activities. A better understanding of important host-pathogen interactions that contribute to field prevalence of a specific disease may provide greater insight
into the development of effective intervention strategies.
| |
ACKNOWLEDGMENTS |
This research was supported in part by agricultural research
funds administered by the Pennsylvania Department of Agriculture (ME445126) and the USDA-BARD Grants Program (US-2648-95).
We appreciate B. Poutrel for providing S. aureus isolates
from France and R. P. Novick for providing S. aureus
isolates RN6390B and RN6911.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Science, Center for Mastitis Research, The Pennsylvania
State University, 115 Henning Building, University Park, PA 16802-3500. Phone: (814) 863-2165. Fax: (814) 863-6140. E-mail:
LMS10{at}psu.edu.
Editor:
J. T. Barbieri
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Infection and Immunity, January 2001, p. 45-51, Vol. 69, No. 1
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.1.45-51.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
