Slov Vet Res 2007; 44 (1/2) - Slovenian veterinary research

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B. Podpečan, A. Pengov, S. Vadnjal
Molecular test, Random Amplified Polymorphic
DNA (RAPD-PCR), is a useful method for differentiation
of strains of S. aureus. Several authors reported
that a strain of S. aureus isolated from cows with
mastitis can be differentiated using a PCR method
with random primers. This method is preferable
over biotyping, as it is a much more specific method
(1). Molecular methods such as PCR-based DNA
fingerprinting may be useful for epidemiological
purposes (2).
Material and methods
Collection of samples
Swabs: Swabs were taken with sterile cotton
sticks in mannitol salt broth (Mannitol salt broth-
Biolife Italiana S.r.l.) from a surface of 25 cm 2 . We
limited the area with a sterile, paper model.
Swabs were taken from surface of equipment
on the slaughter line and in the cutting room, from
hands of workers on the slaughter line and in the
cutting room and from the surface of carcasses on
five different areas on the carcass; thigh, abdomen
wall, thorax, front leg and neck.
Cough up air samples: Samples were taken directly
on Baird –Parker broth during coughing of
workers.
Ground meat samples: Samples were taken in a
sterile plastic bag immediately after grinding.
Isolation and identification of S. aureus
Swabs were incubated in mannitol salt broth
(Mannitol salt broth- Biolife Italiana S.r.l.) at a temperature
of 37°C for 24 hours. 1g of ground meat is
diluted in 9 ml of mannitol salt broth.
Mannitol salt broth was spread on Baird –Parker
agar (Baird –Parker agar Staphylococcus Selective
Agar base acc. to Baird –Parker) and incubated
at 37°C for up to 48 hours. Colonies which exhibited
typical morphology ( grey-black shiny convex
colonies, 1-1.5 mm in diameter with a narrow white
entire margin surrounded by a zone of clearing, 2-
5mm) were spread on blood agar
( Blood Agar Base N°2+5% blood) and incubated
at 37°C for up to 48 hours. Colonies were tested for
coagulase production using rabbit blood plasma
(BBL coagulase plasma, rabbit with EDTA). Additional
phenotypic traits were used with a commercial
kit (BBL CRYSTAL GP Identification System,
Gram-Positive ID Kit).
DNA extraction
DNA from bacterial cultures was extracted by
the PROMEGA method. (Wizard ® Genomic DNA
Purification Kit) according to the manufacturer instructions.
For destruction of the bacteria cell wall
lisozim and lisostafin were used.
DNA amplification
Amplification was performed with RAPD-PCR.
For differentiation of separate strains of bacteria
S. aureus we used the RAPD-PCR method and four
different oligo-nucleotide primers OPJ5, OPJ6, E7
and E8 (2).
Results
For establishing the hygiene status of the establishment
140 swabs were taken from surfaces
of equipment. Established contamination (> 100
micro-organisms/cm 2 ) was 6.42% (9/140) of specimens.
For establishing contamination before the
beginning of work with S. aureus on surfaces of
equipment we took 40 specimens. S. aureus was
isolated from 5% (2/40) of specimens. In addition we
took 41 specimens from equipment after the handling
of five beef carcasses. S. aureus was isolated
from 46.34% (19/41) of specimens.
For establishing contamination of workers
hands, 24 swabs were taken before the beginning
of the work and 24 swabs after handling five beef
carcasses. S. aureus was isolated from 50% (12/24)
of specimens and 58.33% (14/24) of specimens, respectively.
From 10 specimens of coughing up air of
workers, S. aureus was recovered from 10% (1/10) of
specimens.
From the surface of beef carcasses samples
were taken from five different areas on the carcass.
S. aureus was isolated from 78% (39/50) of specimens
taken from the thorax area, 62% (31/50) of
specimens taken on the front leg, 58% (29/50) of
specimens taken on the abdomen wall, 14% (7/50)
of specimens taken on the thigh and 10% (5/50) of
specimens taken from the area of the neck. Altogether,
S. aureus was isolated from 44.4% (111/250)
of specimens taken from surfaces of beef carcasses.
From ground meat specimens S. aureus was
recovered from 62.5% (10/16) specimens. Results
of genotyping of S. aureus, isolated from specimens
taken in slaughterhouse by the RAPD-PCR method.