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JOURNAL OF CLINICAL MICROBIOLOGY. July 1989. p. 1631-1635 0095-1137/89/071631-05$02.00/0 Copyright (1989. American Society for Microbiology Staphylococcus aureus ...

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JOURNAL OF CLINICAL MICROBIOLOGY. July 1989. p. 1631-1635 0095-1137/89/071631-05$02.00/0 Copyright (1989. American Society for Microbiology Staphylococcus aureus ...

JOURNAL OF CLINICAL MICROBIOLOGY. July 1989. p. 1631-1635 Vol. 27, No. 7
0095-1137/89/07163 1-05$02 .00/0
Copyright ( 1989. American Society for Microbiology

Staphylococcus aureus Exopolysaccharide In Vivo Demonstrated by Downloaded from on January 29, 2016 by guest
Immunomagnetic Separation and Electron Microscopy


National Veterinarv Institute, Oslo,' and Deparutnent of Microhiology & Imrnunology, Univ'ersity of Bergen,

Bergen,l Norwt'ay

Received 9 December 1988/Accepted 10 April 1989

Staphylococcus aureus strains were separated from mastitis milk samples without cultivation by using
monodisperse magnetic polymer particles coated with polyclonal antiserum against an encapsulated S. aureus
strain. Exopolysaccharide was verified by transmission electron microscopy and the serum soft-agar culture
technique. Capsular polysaccharide was found on virtually all clinical isolates. Surface protein A and S.
aureus-specific cell wall components were masked when the strains were cultured on an exopolysaccharide-
promoting medium. Masking of surface determinants was dependent on their concentration on the bacterial
surface as well as on exopolysaccharide abundance. The polysaccharide layer on in vivo bacteria was reduced
markedly after just one transfer from milk to blood agar plates but was reexpressed after culturing was done
on a capsule-generating medium.

Staphylococcus aureus is a major pathogen in bovine Determination of strains with exopolysaccharide. The se-
mastitis, and the importance of bacterial cell surface prop- rum soft-agar technique (19) was used to determine produc-
erties as virulence factors has gained considerable attention tion of capsular polysaccharide in freshly isolated S. aureus
(9, 18, 19). The involvement of capsular material in the strains. Extracellular bacterial sugars were demonstrated as
invasiveness of bacteria is well established (7, 16, 20, 27).
The findings of different in vitro studies differ as to the follows. Bacteria grown on S-110 agar were scraped off,
nature, occurrence, and pathogenic importance of capsular washed with distilled water, and pelleted. The supernatant
structures in S. aureus from bovine mastitis (19, 22, 23. 25). was sterilized by filtration, lyophilized, and subjected to
However, exopolysaccharides in themselves may mask im-
portant structures on the bacterial surface and thus influence methanolysis and gas chromatography by the method of
pathogenicity. Furthermore, capsular polysaccharides are Bryn and Jantzen (1). Lyophilized S-110 medium was used
important immunogenic structures (15) and mediators of
bacterial adherence (21, 24). as a control.

The present study was conducted to investigate the occur- Antiserum. New Zealand White rabbits were immunized
rence of exopolysaccharide in vivo on S. aureus isolated with an exopolysaccharide-positive S. aureus field strain
from bovine mastitis milk by immunomagnetic separation (K-12) isolated from bovine mastitis milk and grown on
without cultivation. Masking of bacterial cell wall determi- S-110 agar at 37°C for 24 h (19). Bacteria were suspended in
nants was studied under different culture conditions. phosphate-buffered saline (PBS) with 0.3% Formalin. and
the concentration was adjusted to 10% transmission at 525
(Preliminary data from this study were presented orally at nm. Injection of 0.2 ml into the ear veins of the rabbits was
the Annual Meeting of the American Society for Microbiol- performed three times per week for 3 weeks, with 1-week
ogy, Miami Beach, Fla.. 8 to 13 May 1988.) intervals. The rabbits were bled 1 week after the final
injection, and the antiserum was stored at -20°C in aliquots

Bacterial strains. Staphylococcus aureus (36 strains), 2 Immunomagnetic separation. Magnetic monodisperse poly-
strains of coagulase-negative staphylococci, 3 Esclerichia mer particles (Dynabeads M-450; Dynal A. S., Oslo, Nor-
coli strains, 1 strain of Streptococcus uberis, and 2 strains of way) (8, 28) precoated with sheep anti-rabbit immunoglobu-
Streptococcuis dysgalactiae were cultivated from clinical lin were further coated with the polyclonal rabbit antiserum
mastitis milk samples submitted to our laboratory during the against strain K-12 by incubating 0.5 ml of the bead suspen-
period September to December 1987. Strains were identified sion (3.8 x 108 Dynabeads per ml) in 3 ml of antiserum at 4°C
by recommended methods (14). Reference strains through- with slow tilting and rotation for 2.5 h. The beads were
out the study were Cowan 1 (ATCC 12598), Wood 46 (ATCC separated from the serum by a magnetic separator (Dynal
10832), and Smith diffuse (ATCC 19636). A.S.), washed twice with 0.02 M PBS containing 0.1%,r
bovine serum albumin (Sigma Chemical Co., St. Louis,
Growth conditions. Bacteria were cultured on bovine Mo.), and suspended in the same buffer at a concentration of
blood agar or on Staphylococc,îs medium 110 (S-110) (Difco 0.6 x 108 Dynabeads per mi.
Laboratories, Detroit, Mich.) agar (1.5%) plates either di-
rectly from the milk sample or after immunomagnetic sepa- A 0.25-ml sample was taken from each mastitis milk
ration of bacteria from the milk and were incubated for 24 h sample, and 300 ,ul of PBS and 8 pu of bead suspension (0.6
in an aerobic atmosphere at 37°C. x 108 Dynabeads per ml) were added. The samples were
incubated in a Heidolph "end-over-end" mixer at slow
* Corresponding author. speed overnight at 4°C. The samples were transferred to test
T Present address: Nycomed A.S., P.O. Box 4220 Thorshov. 0401 tubes, and 3 ml of PBS was added; the Dynabeads with
Oslo 4, Norway. bacteria were separated after 20 s with the magnetic separa-
tor, washed once carefully, and suspended in 100 of PBS
with 0.1% bovine serum albumin. The purity of the isolated



TABLE 1. Percent distribution of S. aureus clinical isolates
with surface expression of cell wall determinants after

one transfer in vitro on blood or S-110 agar

Surface % of S. iaureus clinical isolates with surface expression of
the following cell wall determinant after one transfer
score" in vitro on blood or S-110 agar'

*fi Protein A" hl-Agglutinogen" (gly)5"
(n = 36) (n = 20)
FIG. 1. Phase-contrast micrograph of S. aureus on Dynabeads (n = 22)
(black spheres) separated from a mastitis milk sample. Bead diam-
eter, 4.5 ,um. Blood S-110 Blood S-110 Blood S-110
agar agar agar agar agar agar
bacteria was tested after cultivation on blood agar plates for
24 h at 370C. - 8 69 18 77 5 55

Microscopic examination of beads with bacteria was per- + 8 il 9 14 10 35
formed as follows. Acridine orange-ethidium bromide solu-
tion was added to the bead pellet and mixed carefully. The ++ 84 20 73 9 85 10
bead suspension was placed on a microscopic slide (8-field
multislide; Falcon Plastics Co., Los Angeles, Calif.) under a " -, No expression; +. expression; ++, abundant expression of cell wall Downloaded from on January 29, 2016 by guest
cover slip. Duplicate preparations were investigated by determinant.
using a double-blind method in a Leitz inverted microscope
by phase-contrast or fluorescent light microscopy. " Reference strain Cowan 1 produced ail three ceil wall components on

Protein A assay. Expression of surface protein A on the blood agar but did not express surface protein A on S-110 agar. Reference
bacteria was investigated in a previously described Dyna- strain Smith produced al three cell wall components on blood agar but
beads agglutination assay (8). expressed none of the three cell wall components on S-110 agar. Reference
strain Wood did not produce protein A or hl-agglutinogen on either type of
Sait aggregation test. The salt aggregation test was per-
formed as described by Jonsson and Wadstrom (10) and agar tested.
modified by Rozgonyi et al. (26).
' Determined by Dynabeads agglutination assay.
Monoclonal antibodies. S. aureus-specific mouse monoclo- " Determined by indirect immunofluorescence with monoclonal antibodies.
nal antibodies against cell wall determinants were produced
by immunization with purified protein antigen for the hl- RESULTS
agglutinogen (4) and with a synthetic (gly)5 peptide for the
peptidoglycan determinant (29). S. auireuis was separated without cultivation from mastitis
milk with Dynabeads M-450 coated with rabbit polyclonal
Immunofluorescence. Expression on the bacterial surface antibody against S. aureus K-12 (Fig. 1). These beads bound
of the cell wall determinants hl-agglutinogen and (gly)5 was S. aureus and coagulase-negative staphylococci and some
determined by indirect immunofluorescence. Bacteria were strains of streptococci and E. coli. Thus, the separation was
grown on agar plates (blood agar or S-110 agar) for 24 h at not species specific, although S. auireuis was isolated from all
37°C and heat fixed on microscope slides. Ascitic fluids the S. aiireiis-positive milk samples evaluated by the cultur-
containing the respective monoclonal antibodies were di- ing of bead extracts. Control beads coated with sheep
luted 1:50 in PBS, pH 7.3, with 0.1% bovine serum albumin anti-rabbit immunoglobulin or sheep anti-mouse immuno-
(Sigma) before application. Bacteria with monoclonal anti- globulin only were not able to bind staphylococci from the
body solution were incubated for 25 min at room tempera- mastitis milk, although beads coated with fetal calf serum did
ture in 100% humidity and then washed twice with distilled bind some staphylococci.
water and once with PBS. Fluorescein isothiocyanate-con-
jugated goat-anti-mouse immunoglobulin G and immuno- Attachment of bacteria to the beads was visualized by
globulin M F(ab)2 fraction (Tago Inc., Burlingame, Calif.) phase-contrast microscopy in 32 of 36 investigated S. au-
was diluted 1:50 in PBS containing 0.1% bovine serum
albumin. The antibody solution was applied, and the slides reits-positive milk samples (Fig. 1). Bead aggregation with-
were incubated for 25 min at room temperature in 100% out visible bacteria, possibly due to soluble exopolysaccha-
humidity. The preparations were washed three times in
distilled water, air dried, and mounted in 50% glycerol in ride, was observed. Most strains had an aggregate size of 15
PBS under cover slips. Fluorescence was read in a Leitz to 40 beads (Fig. 1).
microscope through a x40 objective.
Production of exopolysaccharide, demonstrated in the
Electron microscopy. Reference strains were grown on serum soft-agar test, was confirmed by masking of the cell
blood agar or S-110 agar, and S. aureus strains were isolated wall determinants [i.e., protein A, hl-agglutinogen, and
from mastitis milk samples by immunomagnetic separation (gly)5] in bacteria grown on S-110 agar but not on blood agar
as described above. Bacteria were prepared essentially as (Table 1). In the case of reference strain Cowan 1, protein A
described by Costerton et al. (2). The preparations were
subjected to ultrathin sectioning and electron microscopic A

examination. FIG. 2. Immunofluorescence of hl-agglutinogen expression on
S. aureus Cowan 1 (A) and S. aureus from acute mastitis (B). Both

strains were subcultured twice on blood agar.


-36 B

D; e». Downloaded from on January 29, 2016 by guest

A I.;` ,. * ..Y




t l' 't

FIG. 3. (A) Transmission electron micrograph of S. aureus separated from mastitis milk by immunomagnetic separation with Dynabeads.

S-11O(B) Enlarged bacteria with abundant exopolysaccharide. (C) Nonencapsulated control strain Wood-46 grown on agar. (D) Encapsulated

control strain Smith grown on S-l10 agar. D, Dynabead; B, bacteria; E, exopolysaccharide.


could not be demonstrated after culturing on S-110 agar, also agrees with the results previously presented by Johne Downloaded from on January 29, 2016 by guest
whereas the other two cell wall components could be shown and Jarp (8). In the protein A-negative strain Wood 46 no
hl-agglutinogen could be demonstrated, possibly because
after both culturing methods. In the case of strain Wood 46, this structure might be a protein antigen closely related to
protein A and thus absent in this strain. The reference strains
neither protein A nor hl-agglutinogen could be demon- cultured on S-110 agar represent a strong capsule producer,
strated. Protein A masked by exopolysaccharide was par- Smith, and the two weaker capsule producers, Cowan 1 and
tially expressed after one transfer of bacteria from milk to Wood 46. This supports the variation in masking of ce!l wall
blood agar and was wholly expressed after two transfers in structures seen in the mastitis strains, with the majority
vitro, as was also seen with the other investigated cell wall being exopolysaccharide producers.

determinants. The abundance of bacterial capsular material in the S.
aiureuis-containing milk demonstrated by the present study
Extracellular bacterium-specific sugars were demon- may suggest biological significance regarding pathogenic
strated by gas chromatography (1) in S. aureus obtained mechanisms (3, 17, 30, 31) and may be of importance in S.
from clinical mastitis and grown on S-110 agar. Altogether,
88% of the investigated strains produced exopolysaccharide, auireuis immunoprophylaxis and diagnosis (12, 23).
as evaluated by one or more of the above methods.
Of the 36 investigated strains, 32 gave a positive serum
soft-agar test which could be inhibited with the K-12 antise- The skilled technical assistance of Anita S0rensen is highly
rum. In the salt aggregation test, 9 of 13 investigated strains appreciated. Electron microscopy was carried out by Renate Hars,
showed higher hydrophobicity on blood agar than on S-110 Department of Anatomy, Dental Faculty, University of Oslo, Oslo,
agar. After five transfers on blood agar, exopolysaccharide Norway. Gas chromatography was performed by Klaus Bryn,
was reexpressed on S-110 agar, as evaluated by surface National Institute of Public Health, Oslo, Norway.
expression of cell wall determinants. Figure 2 shows indirect
immunofluorescence of hl-agglutinogen expression on S. This work was financially supported by Apothekernes Laborato-
aureus Cowan 1 and S. aureus obtained from acute mastitis rium A.S., Oslo, Norway, and by the Agricultural Research Council
milk and subcultured twice on blood agar. of Norway, Oslo.

Transmission electron microscopy of ruthenium red- LITERATURE CITED
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2. Costerton, J. W., D. W. Lambe, K.-J. Mayberry-Carson, and B.
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stained for polysaccharide indicated that extracellular the hl agglutinogen from Staphylococcus aureus 17A. Serolog-
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