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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, September 2001, p. 959-964, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.959-964.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Toxin Genes and Other Characteristics of
Staphylococcus aureus Isolates from Milk of Cows
with Mastitis
Ö.
Akineden,1
C.
Annemüller,1
A. A.
Hassan,1
C.
Lämmler,1,*
W.
Wolter,2 and
M.
Zschöck2
Institut für Tierärztliche
Nahrungsmittelkunde, Professur für Milchwissenschaften der
Justus-Liebig-Universität Gießen, 35392 Gießen,1 and Staatliches Medizinal-,
Lebensmittel- und Veterinäruntersuchungsamt Mittelhessen,
35396 Gießen,2 Germany
Received 28 February 2001/Returned for modification 9 May
2001/Accepted 5 June 2001
 |
ABSTRACT |
In the present study, 103 Staphylococcus aureus
strains isolated from milk samples from 60 cows with mastitis from
eight different farms in seven different locations in one region of
Germany were compared pheno- and genotypically and by
identification of various toxins. On the basis of culture and
hemolytic properties and by determination of the tube coagulase
reaction, all of the isolates could be identified as S.
aureus. This could be confirmed by PCR amplification of
species-specific parts of the gene encoding the 23S rRNA. In addition,
all of the S. aureus isolates harbored the genes
encoding staphylococcal coagulase and clumping factor and the genes
encoding the X region and the immunoglobulin G binding region of
protein A. These four genes displayed size polymorphisms. By PCR
amplification, the genes for the toxins staphylococcal enterotoxin
A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for
SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be
detected. To analyze the epidemiological relationships, the isolates
were subjected to DNA fingerprinting by macrorestriction analysis of
their chromosomal DNAs. According to the observed gene polymorphisms,
the toxin patterns, and the information given by macrorestriction
analysis of the isolates by pulsed-field gel electrophoresis, a limited
number of clones seemed to be responsible for the cases of bovine
mastitis on the various farms.
| |
INTRODUCTION |
Staphylococcus aureus is
recognized worldwide as a frequent cause of subclinical intramammary
infections in dairy cows. The main reservoir of S. aureus
seems to be the infected quarter, and transmission between cows
usually occurs during milking.
S. aureus produces a spectrum of extracellular protein
toxins and virulence factors which are thought to contribute to the pathogenicity of the organism. The staphylococcal enterotoxins (SEs)
are recognized agents of the staphylococcal food poisoning syndrome and
may be involved in other types of infections with sequelae of shock in
humans and animals (4, 25).
Nine major antigenic types of SEs have been recognized and designated
SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, and SEJ (4, 5, 30, 31,
43, 50). All these toxins exhibit superantigenic activity by
interacting with antigen-presenting cells and T lymphocytes without
regard for the antigen specificity of the cells. This induces cellular
proliferation and a high level of cytokine expression (9).
A distantly related protein, toxic shock syndrome toxin 1 (TSST-1),
also produced by S. aureus, was the first toxin shown to be
involved in toxic shock syndrome, in both menstrual and nonmenstrual
cases (3, 33). However, no immunological identity and
little amino acid homology between TSST-1 and the staphylococcal enterotoxins exist (6). Some strains of S. aureus produce one or both of two immunologically distinct
exfoliative toxins, exfoliative toxin A (ETA) or ETB (23,
25). These toxins have been associated with impetiginous
staphylococcal diseases referred to as staphylococcal scaled skin syndrome.
At present little is known about the occurrence of these toxins among
S. aureus isolates from cattle with bovine mastitis. The
present study was designed to investigate S. aureus isolates from cattle with bovine subclinical mastitis from one region of Germany
phenotypically, genotypically and by the identification of various toxins.
| |
MATERIALS AND METHODS |
Sample collection and identification.
A total of 103 S. aureus isolates were collected from milk samples from 60 cows with mastitis from eight different farms in seven different
locations in one region of Germany. All of the isolates were identified
by culture properties, by the detection of hemolysis (38),
and by the tube coagulase reaction.
The isolates were additionally investigated by PCR amplification of
species-specific parts of the gene encoding the 23S rRNA with the
oligonucleotide primers shown in Table 1.
For PCR amplification, the reaction mixture (30 µl) contained 1 µl
of primer 1 (10 pmol/µl), 1 µl of primer 2 (10 pmol/µl), 0.6 µl
of deoxynucleoside triphosphate (10 mmol/liter; MBI Fermentas, St.
Leon-Rot, Germany), 3.0 µl of 10× thermophilic buffer (Promega,
Mannheim, Germany), 1.8 µl of MgCl2 (25 mmol/liter; Promega), 0.1 µl of Taq DNA polymerase (5 U/µl; Promega/Boehringer), and 20.0 µl of distilled water. Finally, 2.5 µl of DNA preparation was added to each 0.2-ml reaction tube. The tubes were subjected to thermal cycling (Techne-Progene; Thermodux, Wertheim, Germany) with the program shown in Table 1. For
DNA preparation, 5 to 10 colonies of the bacteria were incubated in 100 µl of TE buffer (10 mmol of Tris-HCl/liter, 1 mmol of EDTA/liter, pH
8.0) containing 5 µl of lysostaphin (1.8 U/µl; Sigma, Deisenhofen,
Germany), for 1 h at 37°C and subsequently treated with
proteinase K (14.0 mg/ml; Boehringer, Mannheim, Germany) for 120 min at
56°C. To inactivate the proteinase K, the suspension was heated for
10 min at 100°C and centrifuged at 10,000 × g for 20 s. Ninety microliters of the supernatant was treated with 10 µl of 5-mol/liter NaClO4 and 50 µl of
isopropanol (99.7%; Roth, Karlsruhe, Germany), mixed, placed on an ice
block for 10 min, and centrifuged for 30 min at 13,000 rpm. The
supernatant was discarded, 250 µl of ethanol (70%) was added, and
the tube was again centrifuged for 5 min at 13,000 rpm. The
supernatant was again discarded, and the pellet was dried in a
desiccator for 5 min. After the addition of 50 µl of sterilized aqua
dest, the tubes were cooled until they were used. The presence of
PCR products was determined by electrophoresis of 12 µl of the
reaction product in a 2% agarose gel with Tris-acetate-electrophoresis
buffer (0.04 mol of Tris/liter, 1 mmol of EDTA/liter, pH 8) and a
100-bp DNA ladder (Gibco BRL, Eggenstein, Germany) as a molecular
marker.
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TABLE 1.
Oligonucleotide primers and PCR programs for
amplification of the genes encoding staphylococcal 23S rRNA and
staphylococcal proteins including various toxins
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PCR amplification of genes encoding staphylococcal proteins and
toxins.
A PCR amplification was performed for the genes encoding
staphylococcal coagulase (coa), clumping factor
(clfA), protein A (spa), SEA (sea),
SEB (seb), SEC (sec), SED (sed), SEE
(see), SEG (seg), SEH (seh), SEI
(sei), SEJ (sej), TSST-1 (tst), ETA (eta), and ETB (etb). The sequences of the
oligonucleotide primers, the thermocycler programs, and the references
are summarized in Table 1.
Macrorestriction analysis by PFGE.
The isolates were also
investigated by digestion of their chromosomal DNAs with the
restriction enzyme SmaI and subsequent separation of the
fragments by pulsed-field gel electrophoresis (PFGE) using the Chef-Dr
II pulsed-field electrophoresis system (Bio-Rad, Munich,
Germany). The preparation of the DNA and the program for PFGE were
described previously (46). In accordance with the method
of Tenover et al. (45), restriction patterns with no
fragment difference were recorded as indistinguishable, restriction
patterns with two to three fragments difference were recorded as
closely related, and those with four and more differences were recorded
as not related.
| |
RESULTS |
According to culture and hemolytic properties and a
positive tube coagulase test, all 103 isolates used in the present
study could be identified as S. aureus. Among the 103 cultures, 25 cultures showed alpha-hemolysis, 50 cultures showed
beta-hemolysis, and 28 cultures were nonhemolytic (Table
2). The identification of the isolates as
S. aureus could be confirmed by PCR amplification of the
genes encoding the 23S rRNA, coagulase, and clumping factor and the
genes encoding the X region and the immunoglobulin G (IgG) binding
region of protein A. The amplicon of the 23S rRNA gene had a uniform
size of 1,250 bp; all the other genes displayed polymorphisms.
Typical polymorphisms of the genes encoding staphylococcal coagulase
and the X region and the IgG binding region of protein A are shown in
Fig. 1, 2, and 3. These results, together
with the origins and hemolytic properties of
the isolates, are summarized in Table 2.
Among the 103 S. aureus
cultures investigated, 17 cultures
contained the gene encoding SEI, 21 cultures contained the genes
for SEG and SEI, 21 cultures contained the genes for SED and SEJ, 15 cultures contained the genes for SEC, SEG, SEI, and TSST-1, and 1 culture contained the genes for SEA, SEC, and TSST-1. All isolates
containing SEC genes were simultaneously positive for TSST-1, and all
isolates containing the gene for SED were positive for SEJ. With the
available oligonucleotide primers, no toxin formation could be detected
for the 28 S. aureus isolates from farm 1. The distribution
of toxin formation among the S. aureus isolates from the
various farms is summarized in Table 2. None of the strains harbored
the genes encoding SEB, SEE, SEH, ETA, or ETB.
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TABLE 2.
Origin, PFGE patterns, and hemolysis and other
characteristics of S. aureus isolates from milk samples from
cows with bovine mastitis
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FIG. 1.
Polymorphisms of the gene encoding staphylococcal
coagulase. Lane 1, 840 bp; lanes 2, 3, 4, and 5, 600 bp; lane 6, 440 bp; M, 100-bp ladder serving as a size marker.
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FIG. 2.
Amplicons of the gene encoding the X region of protein
A. Lane 1, 110 bp, 3 repeats; lane 2, 140 bp, 4 repeats; lane 3, 220 bp, 7 repeats; lane 4, 240 bp, 8 repeats; lane 5, 270 bp, 9 repeats;
lane 6, 290 bp, 10 repeats; lane 7, 320 bp, 11 repeats; M, 100-bp
ladder.
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FIG. 3.
Amplicons of the gene encoding the IgG binding region of
protein A. Lanes 1 and 2, 390 bp; lanes 3 and 4, 920 bp; M, 100-bp
ladder.
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|
Digestion of the chromosomal DNAs of the isolates with the restriction
enzyme SmaI revealed 12 different restriction patterns, with
mostly identical restriction patterns for the isolates from the eight
farms. A typical uniform restriction pattern of the S. aureus isolates from farm H7 and restriction patterns of S. aureus isolates representing nine different restriction patterns are shown in Fig. 4 and
5, respectively. The restriction patterns of all 103 S. aureus isolates and the above-mentioned
results are summarized in Table 2. A further analysis of the 12 restriction patterns revealed that the bacterial clones with PFGE
patterns Ia and Ib, IIIa and IIIb, and IVa, IVb, and IVc (all one- or
two-fragment differences) and IIIa and IVb and IIIa and IVc (two- or
three-fragment differences) are closely related; the remaining
bacterial clones with PFGE patterns I to XII are not related (Table 2).
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FIG. 4.
Pulsed-field electrophoretic restriction patterns of
chromosomal DNAs of nine S. aureus isolates from farm H7
with DNA restriction pattern XI. A 0.1- to 200-kb ladder (Low Range PFG
Marker; BioLabs, Schwalbach, Germany) and a 50- to 1,000-kb ladder
(Lambda Ladder PFG Marker, BioLabs) served as size markers.
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FIG. 5.
Pulsed-field electrophoretic restriction patterns of
chromosomal DNAs of S. aureus isolates with DNA
restriction patterns Ib (lane 1), Ia (lane 2), XII (lane 3), IIIa (lane
4), IIIb (lane 5), X (lane 6), IVa (lane 7), IVb (lane 8), and IVc
(lane 9). A 0.1- to 200-kb ladder (Low Range PFG Marker; BioLabs,
Schwalbach, Germany) and a 50- to 1,000-kb ladder (Lambda Ladder PFG
Marker, BioLabs) served as size markers.
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| |
DISCUSSION |
The identification of the 103 S. aureus isolates of the
present study could be performed by conventional methods and by PCR technology. The latter uses oligonucleotide primers targeted to species-specific parts of the gene encoding the 23S rRNA and the genes
encoding coagulase, clumping factor, and protein A. Comparable PCR-based systems for identification of S. aureus isolates
from various origins have been used by numerous authors (1, 7, 13, 26, 40, 41). All these target genes allowed a rapid identification of this species with high sensitivity and specificity. As was found by Straub et al. (41), the amplification of
the gene encoding an S. aureus-specific part of the 23S rRNA
revealed an amplicon with a size of 1,250 bp for all S. aureus isolates investigated.
The PCR products of the genes encoding staphylococcal coagulase,
clumping factor, and protein A displayed gene polymorphisms and allowed
a genotypic characterization of the bacteria. Length and sequence
polymorphisms of the coagulase gene and its use for genotypic
characterization of S. aureus had been already shown (1, 16, 18, 37, 40, 42).
As with previous studies (40), the amplification of the
clumping factor (clfA) gene resulted in a single amplicon
with a size of approximately 1,000 bp, indicating no size polymorphisms of this gene. However, the clumping factor genes of eight isolates investigated in the present study had a size of 950 bp. At present, no
information is available about the sequence variation of these strains.
Amplification of the X region of the protein A (spa) gene
yielded a single amplicon for all 103 isolates. Ten different-sized amplicons of approximately 90, 110, 140, 170, 190, 220, 240, 270, 290, and 320 bp and calculated numbers of 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 repeats, respectively, were observed. Comparable differences in the
number of repeats of the X region of the protein A (spa)
gene had already been used for genotyping isolates of this species
(14, 15, 46, 48).
The PCR amplification of the gene encoding the IgG binding region of
protein A revealed a size of 920 bp for most of the isolates investigated. However, the genes of five strains showed an amplicon size of 390 bp. Because in these five strains the PCR products were 390 bp smaller and because 170 bp is the fragment size that is required to
encode one IgG binding domain, a lack of two domains is assumed for
these strains. Comparable spa gene polymorphisms were
observed by Schwarzkopf et al. (34) and Stephan et al. (40).
Investigating the S. aureus isolates for toxin formation
revealed that, besides enterotoxins A, C, and D and TSST-1, the newly described enterotoxins G, I, and J seemed to be the predominant enterotoxins of S. aureus isolated from cattle with bovine
mastitis. The enterotoxin studies were done by PCR amplification of the respective genes. According to Becker et al. (2), Mc
Lauchlin et al. (29), and Sharma et al. (36),
there is an excellent correlation between PCR results and the detection
of enterotoxins by commercial reverse passive latex agglutination
assays. The involvement of enterotoxin G- and I-producing S. aureus strains had been previously demonstrated for S. aureus isolates from humans with staphylococcal toxic shock
syndrome and staphylococcal scarlet fever (19). Production
of SEA, SEB, SED, SEE, and TSST-1 by S. aureus strains
associated with bovine mastitis has been described by numerous authors
(8, 24, 27, 39, 40, 44). It was interesting that all SEC
producers of the present study were TSST-1 positive and all SED
producers were SEJ positive. A comparable relationship between the
detection of SEC and TSST-1 has been reported in the literature
(11, 27, 32, 40, 51). The enterotoxins D and J are encoded
by a plasmid and separated from each other by an intergenic region
(50). None of the strains harbored any of the genes
seb, see, seh, eta, and
etb. As shown in the studies of Hayakawa et al.
(17), the production of exfoliative toxins among S. aureus isolates from cattle with bovine mastitis seems to be rare.
According to the present results, the formation of enterotoxins,
including the newly described enterotoxins G, I, and J, appears to be
widely distributed among S. aureus isolates from the milk of
mastitic cows. In the studies of Larsen et al. (22), only 1 of 414 S. aureus isolates from cattle with bovine mastitis
in Denmark carried a toxin gene. However, these authors did not
investigate their strains for the newly described enterotoxins SEG,
SEI, and SEJ.
The importance of toxin formation by S. aureus for udder
pathogenesis remains unclear. According to Ferens et al.
(10), the superantigenic toxins seem to induce
immunosuppression in dairy animals.
The 103 S. aureus isolates of the present study were further
analyzed for epidemiological relationships by macrorestriction analysis
of their chromosomal DNA by PFGE. Comparable studies had already been
successfully used for investigating mastitis isolates of this species
(1, 21, 40). By means of SmaI macrorestriction
analysis, the isolates of the present investigation yielded 12 different PFGE patterns. The PFGE patterns of bacteria from single
farms were mostly identical. Heterogeneity in other pheno- or genotypic
properties among isolates of a single PFGE pattern may be due to
evolutionary processes. Some of the PFGE patterns differed from each
other in only a few fragments (Ia and Ib, IIIa and IIIb, and IVa, -b,
and -c) and thus displayed a clonal relationship. The PFGE patterns,
the size polymorphisms of the coagulase, clumping factor, and protein A
genes, and the formation of the various toxins again substantiates the
existence of a limited number of S. aureus clones
responsible for the observed cases of bovine mastitis on the various
farms. However, besides PFGE patterns Ia and Ib and IIIa and IIIb, the
S. aureus clones of the present study could be found only in
single farms and did not show a broad geographic distribution. This is
partly in contrast to previous studies where identical or closely
related S. aureus clones were responsible for the cases of
bovine mastitis within herds and also between herds occurring in
different regions of the world, (1, 12, 21, 28, 40, 49).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Tierärztliche Nahrungsmittelkunde, Professur für
Milchwissenschaften der Justus-Liebig-Universität Gie
en,
Frankfurter Str. 107, 35392 Gieen, Germany. Phone: 49 641 99 37663. Fax: 49 641 99 38389. E-mail:
christoph.laemmler{at}vetmed.uni-giessen.de.
| |
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Clinical and Diagnostic Laboratory Immunology, September 2001, p. 959-964, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.959-964.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
