EVALUATION OF THREE RAPID TESTS FOR
IDENTIFICATION OF STAPHYLOCOCCUS
AUREUS ISOLATED IN BOVINE MILK
B. Poutrel, M. Ducelliez
To cite this version:
B. Poutrel, M. Ducelliez. EVALUATION OF THREE RAPID TESTS FOR IDENTIFICATION OF STAPHYLOCOCCUS AUREUS ISOLATED IN BOVINE MILK. Annales de
Recherches Vétérinaires, INRA Editions, 1979, 10 (1), pp.125-129. <hal-00901106>
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EVALUATION OF THREE RAPID TESTS FOR IDENTIFICATION OF
STAPHYLOCOCCUS AUREUS ISOLATED IN BOVINE MILK
B. POUTREL
lnstitut National de la Recherche
M. DUCELLIEZ
de
France
Agronomique, Station
Pathologie
de la
Reproduction,
3738n Nouzilly,
Résumé
VALEUR DE TROIS TESTS RAPIDES D’IDENTIFICATION DE STAPHYLOCOCCUS AUREUS
D’ORIGINE BOVINE ISOLE DU LAIT. - Afin de disposer d’une méthode de routine, de diagnostic rapide des souches bovines de Staphylococcus aureus isolées du lait, nous avons cherché à
établir la valeur des trois caractères suivants : hémolysine fi, clumping-factor et protéine A. Les
tests ont été réalisés sur 200 souches d’origines diverses, à partir des colonies présentes sur le
milieu d’isolement. Le clumping-factor a été retrouvé sur 93,5 % des souches, l’hémolysine
(3
chez 76 p. cent et la protéine A chez 56 p. cent. L’analyse de la fréquence d’association de ces
caractères montre qu’aucune des souches n’est négative pour l’ensemble des trois caractères.
Un schéma de diagnostic rapide de S. aureus est proposé.
Although a certain number of factors can
affect the results of a reaction (Sperber and
Tatini, 1975) the free coagulase test is recognized as one of the more reliable for the identification of Staphylococcus aureus (Subcommittee on Taxonomy of Staphylococci and
Micrococci, 1965 ; Zarzour and Belle, 1978).
This test, however, can only be carried out
with a culture in a broth medium, which is
itself preceded by isolation in a solid medium.
It is, moreover, necessary at times to wait 24
hours before reading the reaction. The delay
thus imperative to the identification does not
allow the practitioner to efficiently intervene as
quickly as he would like. The immediate identification with the isolation medium is sometimes possible for bovine strains of S. aureus
isolated from the milk, which produce a
hemolysin of the type (1, when the milk sample
taken asepticaly is cultured on a nutritive agar
medium with sheep blood (Plommet, 1962).
This characteristic, though widespread (Live,
1972), does not, however, allow all the S.
aureus strains responsible for mammary infections to be identified. Two other characteristics, specific to the S. aureus strain : the clumping factor and protein A (Bergey’s, 1974) are
found fairly frequently (Hajek and Marsalek,
1969 ; Hajek and Marsalek, 1976). They present the advantage of being able to be researched and defined in a few minutes by simple
tests carried out directly on suspected colonies present in the initial isolation medium.
In this work, we have sought to evaluate
these three characteristics : hemolysin (3,
clumping factor and protein A, in order to
identify rapidly strains of S. aureus isolated in
bovine milk.
Material and methods
Bacterial strains and culture
aureus strains of bovine
These are either strains
collected from diverse origins (Station de
Pathologie de la Reproduction at Nouzilly &horbar;
Institut Mérieux at Lyonl, or are strains sent to
us by the Laboratoires Départementaux Vétérinaires. Some of them (137) were isolated
after milk samples were aseptically taken in the
udder ; the others (37) come from bulk milk
and were isolated on a selective medium,
T.G.C. (Tellurite, Glycocoll, Camp-factor)
(Plommet, 1962). All these strains were found
to be Gram positive Cocci, catalase positive.
They were also found to be free coagulase
positive in 24 hours, using the technique described here.
In order to obtain normal diagnostic conditions, the strains were cultivated on a nutritive
sheep blood agar medium with aesculin
(Tryptose blood agar base 33 g ; aesculin 1 g ;
iron citrate 1 %, 5 ml ; 60 ml of sheep blood ;
1 000 ml of distilled water) and incubated 16
hours at 37 °C.
Altogether, 200 S.
origin were utilised.
Staining procedure and catalase test
The staining on the colony to be tested need
not a complete Gram stain since we are mostly
interested in morphology.
The catalase test is carried out by mixing on
a glass slide of the colony presumed to be S.
aureus with a drop of 3 % hydrogen peroxide
solution.
Free coagulase test
To a 0,5 ml special 16 hours 37 °C culture
medium (lnstitut Pasteur), 0.5 ml of rabbit
plasma is added (lnstitut Pasteur). After
mixing, the mixture is incubated at 37 °C for 4
hours, then eventually left for about 20 hours
at room temperature, according to the speed
at which the plasma clotting occurs. The reading is carried out at 2,4 and 24 hours later.
Characferisation of hemolysin
(3
After 16 hours of incubation at 37 °C on a
nutritive sheep blood agar, the S. aureus colo-
nies, producing a type(3 hemolysin, are surrounded by an area of partial hemolysis with a
characteristic distinct border. It’s no necessary
use the hot-cold lysin test for this characterisation. The utilisation of a reference strain, a
good producer of hemolysin (3, enables the
quality of the sheep blood utilised to be checked, the presence of antibodies being capable
of inhibiting this hemolysis.
to
Clumping factor test
The colony to be tested
is mixed
on a
glass
slide with a drop of rabbit plasma (lnstitut Pasteur). A positive reaction is interpreted by the
formation of agglutinats, the back ground of
the preparation remaining clear. When the
reaction is negative, the suspension remains
homogeneous with a milky appearance.
Protein A test
The colony to be tested is mixed
on a
glass
slide with a drop of a suspension at 3 % (V/V)
of a sheep red blood cells, sensitised by a rabbit serum (hemolytic serum from the Institut
Pasteur at 1/100) and stabilised by glutaraldehyde at 0.0075 % (V/V) (Poutrel, 1978). A
positive reaction is interpreted by a hemagglutination occuring in less than thirty seconds.
For each test only 2 types of response, positive or negative, were noted, the suspect
reactions being classed as negative. This enabled the tests to be evaluated in the simplest
and most strict conditions.
Results and discussion
The relative frequencies of the three characteristics studied are comparable to those
already reported (Table 1The isolated strains
in the bulk milk, having been distinguished in
the T.G.C. medium by the production of a
type(i hemolysin have a slighty over estimated
frequency (81,5 %) if one takes into consideration all the strains tested, compared with the
frequency of the strains isolated from individual samples (76 %). Elek (1959) reported
that about 88 % of coagulase positive animal
strains produced(3 hemolysin and Hajek and
Marsalek (1969) found 100 % of strains isolated from bovine milk were positive. In the present
investigation, clumping-factor
was
posi-
tive for about 94 % of the strains ; Hajek and
Marsalek (1969, 1976) found similar results
(96 %1. We found 56 % of strains positive for
the protein A. This corresponds with previous
work carried out by Kronvall et al. (1972)
(54 %), Holmberg (1973) (43 %) and Hajek
and Marsalek (1976) (40 %)for strains isolated
from bovine milk.
The(i hemolysin is nearly always associated
with one of the other two characteristics (table
2). On the other hand, identification is only
ensured by the positive clumping factor test
for 6 % of the strains and only by the positive
protein A test for 2 % of the strains.
The analysis of the association frequency of
the three characteristics (table 2) shows that
none of the strains tested was negative for all
the three characteristics. Thus it seems that
the rapid and routine identification of S.
isolated from bovine milk can be established according the scheme given in table 3.
In fact, our data reveal that only about
0.5 % (0.185 x 0.065 x 0.44) of the isolates
tested would be expected to be negative for
the three characteristics. We did never find
non-S. aureus strains protein A positive (Poutrel, 1978) with the reagent we used for the
test. Holmberg (1973) reported 0.5 % fo S.
epidermidis strains isolated from bovine milk
were positive for the protein A, but all the S.
epidermidis strains are negative for(3 hemolysin and clumping factor characteristics. This
shouldn’t be detrimental to the proposed
scheme, since it would result in only 0.5 % of
false negative identifications and 0.5 % of
false-positive identifications.
The bacteriological examinations for persistant staphylococcic mammary infections,
experimental (Postle et al., 1978 ; Poutrel and
Lerondelle, 1978) or natural (unpublished
results) show that these characteristics are
permanent for a given strain. They can therefore be considered as markers of strain, easy
to characterise and can be useful in developing
sophisticated techniques, such as serology or
phage typing, either used in epidemiological
investigations or in experiments for treatments
of mastitis.
aureus
Accepted for publication December2lsi 1978.
Acknowledgements
We would like to thank Mrs Le Menec, Mr
and Protin respectively of the Labora-
Mi6ge
toires Départementaux Vétérinaires at SaintBrieuc, Annecy and Nantes, as well as Mr
Desmettre of the Institut Mérieux, for having
sent us strains.
Summary
In order to develop a routine and rapid method for the identification of Staphylococcus aureus
isolated from bovine milk, three tests were evaluated with 200 strains isolated from different
regions. Tests were performed with colonies isolated from the initial agar plate medium. Clumping factor reations were positive with 93.5 % of the strains,(3 hemolysin with 76 % and protein
A with 56 % of them. It was found the three tests were never all negative for any particular
strain. A scheme for rapid identification is proposed.
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