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John Hwa Lee*
Veterinary Public Health, College of Veterinary Medicine, Chonbuk
National University, Chonju, Republic of Korea
*Corresponding author. Mailing address: College of Veterinary
Medicine, Chonbuk National University, Chonju 561-756, Republic of
Korea. Phone: 82 63 270 2553. Fax: 82 63 270 3780. E-mail:
johnhlee@chonbuk.ac.kr.
Received June 24, 2003; Accepted August 26, 2003.
 This article has been cited by other articles in PMC. 
Top 
Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
REFERENCES  AbstractFrom May 2001 to April 2003, various types of
specimens from cattle, pigs, and chickens were collected and examined
for the presence of methicillin (oxacillin)-resistant Staphylococcus
aureus (MRSA). S. aureus was isolated and positively identified by
using Gram staining, colony morphology, tests for coagulase and urease
activities, and an API Staph Ident system. Among 1,913 specimens
collected from the animals, 421 contained S. aureus; of these, 28
contained S. aureus resistant to concentrations of oxacillin higher
than 2 µg/ml. Isolates from 15 of the 28 specimens were positive by
PCR for the mecA gene. Of the 15 mecA-positive MRSA isolates, 12 were
from dairy cows and 3 were from chickens. Antimicrobial susceptibility
tests of mecA-positive MRSA strains were performed by the disk
diffusion method. All isolates were resistant to members of the
penicillin family, such as ampicillin, oxacillin, and penicillin. All
isolates were also susceptible to amikacin, vancomycin, and
trimethoprim-sulfamethoxazole. To determine molecular epidemiological
relatedness of these 15 animal MRSA isolates to isolates from humans,
random amplified polymorphic DNA (RAPD) patterns were generated by
arbitrarily primed PCR. The RAPD patterns of six of the isolates from
animals were identical to the patterns of certain isolates from
humans. The antibiotypes of the six animal isolates revealed types
similar to those of the human isolates. These data suggested that the
genomes of the six animal MRSA isolates were very closely related to
those of some human MRSA isolates and were a possible source of human
infections caused by consuming contaminated food products made from
these animals. 
Top 
Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
REFERENCES   Staphylococcus aureus causes severe animal diseases, such
as suppurative disease, mastitis, arthritis, and urinary tract
infection, that are associated with numerous virulence factors, such
as the production of extracellular toxins and enzymes (5, 33). For
humans, this organism is an important cause of food poisoning,
pneumonia, postoperative wound infections, and nosocomial bacteremia
(8). Human isolates of S. aureus, unlike animal isolates, are
frequently resistant to the penicillinase-resistant penicillins (12,
23, 28). An organism exhibiting this type of resistance is referred to
as methicillin (oxacillin)-resistant S. aureus (MRSA). Such organisms
are also frequently resistant to most of the commonly used
antimicrobial agents, including the aminoglycosides, macrolides,
chloramphenicol, tetracycline, and fluoroquinolones (14). In addition,
MRSA strains should be considered to be resistant to all
cephalosporins, cephems, and other ß-lactams (such as
ampicillin-sulbactam, amoxicillin-clavulanic acid,
ticarcillin-clavulanic acid, piperacillin-tazobactam, and the
carbapenems) regardless of the in vitro test results obtained with
those agents (19).
MRSA is known to be one of the most prevalent nosocomial pathogens
throughout the world and to be capable of causing a wide range of
hospital-linked infections. MRSA was first reported in the United
Kingdom in 1961 (soon after the introduction of methicillin) and by
the mid-1970s had become endemic in many countries (32). Some strains
of MRSA have been designated epidemic strains; these are associated
with a higher prevalence and have been shown to spread within
hospitals, between hospitals, and between countries (1, 11, 17, 25,
26). MRSA has become a widespread problem in Korea. The rate of
methicillin resistance among human S. aureus isolates in Korea is over
50% (13). MRSA has now become established outside the hospital
environment and is appearing in community populations without
identifiable risk factors (7). To control the spread of the
infections, sources of contamination and mechanisms of transmission
must be identified. Transmission of MRSA is thought to occur primarily
from colonized or infected persons to other persons (3, 16, 22). While
the environment contributes to MRSA transmission (31), transmission
through food products has not been thoroughly investigated.

There is a limited number of publications on the epidemiological
aspects of MRSA infections in animals; a few veterinary reports have
been published on MRSA infections in dairy herds with mastitis and in
companion animals (2, 4, 27, 30). The present report provides data on
the isolation of MRSA from 12 dairy cow and 3 chicken specimens
collected over a 2-year period. To investigate food animal MRSA
isolates as a possible source of human infections, genetic relatedness
among the isolates from food animals and humans was determined by
random amplified polymorphic DNA (RAPD) patterns generated with
arbitrarily primed PCR (AP-PCR).

 
Top 
Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
REFERENCES  MATERIALS AND METHODSIsolation and processing of MRSA.
Feces, milk, feed material, joint, trachea, uterus, and meat specimens
of beef cattle, dairy cattle, pigs, and chickens were collected
between May 2001 and April 2003 at monthly intervals from 15
slaughterhouses, seven meat processing facilities, 58 feedlots, and 11
food stores located throughout Korea, including the provinces of
Chungcheong, Gyeongsang, and Jeolra. One specimen per animal was
collected from the various sites. For joint, trachea, and uterus
specimens, surface areas of at least 10 by 10 cm were swabbed with
staphylococcus broth (Difco, Detroit, Mich.). The total number of
specimens collected was 1,913 (Table 1). During each sampling
occasion, two to five randomly selected samples were collected per
feedlot and food store and 5 to 15 samples were collected per
slaughterhouse and meat processing facility. All samples were
immediately transported to the laboratory in ice-cooled containers.
Approximately 10 g of each specimen of feces, feed material, and
homogenized meat and 10 ml of each specimen of milk and of the swabbed
specimens of joint, trachea, and uterus were inoculated into 100 ml of
staphylococcus broth or Trypticase soy broth (Difco) with 70 mg of
NaCl/ml and incubated at 35°C for 20 h with shaking. The inoculum was
spread onto Baird-Parker agar and incubated at 35°C for 24 to 48 h.
The colonies were tested (using conventional methods that included
Gram staining, colonial morphology, and coagulase and urease assays)
for S. aureus levels. They were also tested with an API Staph Ident
system (Biomerieux, Lyon, France). Phenotypic oxacillin resistance of
S. aureus was determined by an agar screen test performed according to
the recommendations of the National Committee for Clinical Laboratory
Standards (NCCLS) (20, 34) with Mueller-Hinton agar (Difco) containing
4% NaCl and 2, 4, or 8 µg of oxacillin (Sigma, St. Louis, Mo.) per ml.
Oxacillin-resistant colonies were stored at -70°C in freezer vials
pending further analysis.

 TABLE 1.
Results for MRSA isolates from major food animals (sampled during May
2001 to April 2003) 

To examine the genetic relatedness of the animal MRSA isolates to
human isolates and the possibility of transmission, during the
investigation period a total of 38 MRSA isolates originating from
humans were collected from several hospitals located throughout the
nation and examined. Resistance to oxacillin and the presence of the
mecA gene were confirmed for these isolates.


Preparation of whole-cell DNA for PCR and RAPD. A previously described
method (15) was used (with modifications) for whole-cell DNA
extraction. Cells grown in 1.5 ml of Trypticase soy broth at 35°C for
20 h were harvested and centrifuged at 16,000 × g for 3 min. The
pellet was washed with 1.0 ml of sterile distilled water, resuspended
in 50 µl of Triton X-100 lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH
8], 1 mM EDTA [pH 9], 1% Triton X-100), boiled for 10 min, and then
centrifuged at 16,000 × g for 3 min. The suspension was cooled at room
temperature for 5 min and centrifuged at 16,000 × g for 3 min. A total
of 2 ml of the supernatant was used as the template.

Amplification of the mecA gene. The presence of the mecA gene was
verified for the oxacillin-resistant isolates by means of PCR.
Amplification of the mecA gene was performed using the primers mecA1
(5'-AAAATCGATGGTAAAGGTTGGC) and mecA2 (5'-AGTTCTGCAGTACCGGATTTGC),
yielding a PCR product of 533 bp (18). PCR was performed in a 25-µl
volume with a PCR buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl2, a 200 µM concentration of each deoxynucleoside
triphosphate (Promega, Madison, Wis.), 2.5 U of Taq polymerase
(Promega), and a 0.2 µM concentration of each primer. Amplification
was carried out using 40 cycles of amplification at 94°C for 30 s,
55°C for 30 s, and 72°C for 1 min; this reaction was followed by 5 min
of an additional extension at 72°C. PCR products were electrophoresed
on a 1.5% agarose gel. A positive result was inferred by detection of
a 533-bp band, which represented a part of the mecA gene.

Antimicrobial susceptibility testing. The susceptibilities of all
mecA-positive MRSA isolates to different antimicrobial agents were
tested by the disk agar method as standardized by the NCCLS (21). The
isolates were tested with a panel of 15 antibiotics (amikacin,
ampicillin, cefoxitin, cephalothin, chloramphenicol, ciprofloxacin,
clindamycin, erythromycin, gentamicin, imipenem, kanamycin,
norfloxacin, ofloxacin, tetracycline, and
trimethoprim-sulfamethoxazole) (Biomerieux). The results were recorded
after 24 h of incubation at 35°C and interpreted according to the
guidelines of the NCCLS. Antibiotypes were considered identical when
the results of susceptibility tests with all agents tested were
identical. The MICs of oxacillin and vancomycin were also examined
according to NCCLS recommendations (20) by an agar dilution method
using an inoculum of 104 CFU/spot on Mueller-Hinton agar containing 2%
NaCl as well as oxacillin and vancomycin in concentrations ranging
from 0.5 to 128 and 2 to 128 µg/ml, respectively.

AP-PCR. The RAPD patterns generated by AP-PCR of the MRSA isolates
were determined. Approximately 5 ng of DNA was included per PCR
mixture. The PCR mixture consisted of 10 mM Tris-HCl (pH 9.0), 50 mM
KCl, 2.5 M MgCl2, 0.01% gelatin, and 0.1% Triton X-100.
Deoxyribonucleotide triphosphate (0.2 mM) and Taq DNA polymerase (0.5
U) were present. A 0.2 µM concentration of each of two arbitrary
oligonucleotide primers, M13 (5'-GAGGGTGGCGGTTCT-3') and H12
(5'-ACGCGCATGT-3'), was used (10). The amplification was performed
using a program consisting of 40 repeated cycles of 1 min at 94°C, 1
min at 35°C, and 2 min at 72°C. Prior to cycling, the mixture was
denatured at 94°C for 3 min; postcycling, the mixture was incubated at
72°C for an additional 10 min. PCR products were analyzed by 2%
agarose gel electrophoresis. The AP-PCR analyses of these isolates
were performed in duplicate.

RAPD patterns were analyzed both visually and by computer-aided
methods. Visual interpretation of banding patterns was done according
to a previously reported guideline (29). The results for each primer
were indexed by numbering, thereby defining the number of different
patterns. Isolates with one or two minor band differences between two
patterns derived from different isolates were assigned the same type.
The overall types were defined by a combination of the results
obtained with the two primers. Patterns were digitized with a
Hewlett-Packard Scanjet IIcx/T scanner and stored as TIFF files.
Banding patterns generated with each primer were integrated and
analyzed using GelCompar software (version 4.0; Applied Maths,
Kortrijk, Belgium) to calculate Dice coefficients of correlation and
to generate a dendrogram by the unweighted pair group method using
arithmetic average clustering.

 
Top 
Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
REFERENCES  RESULTSIsolation of MRSA from food animal specimens. S.
aureus from food animal specimens was identified by conventional
methods and the API Staph Ident system as described above. Of 1,913
animal specimens collected, S. aureus was isolated from 421 (Table 1).
The majority of the isolates identified were from milk specimens. The
presence of MRSA isolates was determined by an agar screen test with
various concentrations of oxacillin (i.e., 2, 4, and 8 µg/ml). S.
aureus isolates from 28 specimens were resistant to levels of
oxacillin higher than 2 µg/ml. Many isolates were resistant to 2 to 8
µg of oxacillin/ml in the initial culture and became sensitive to the
oxacillin after multiple subculturing. Isolates from the 28 specimens
were tested for the presence of the mecA gene. Isolates from 15
specimens were positive for mecA and were consistently resistant to
oxacillin. Among the 15 mecA-positive MRSA isolates, 12 were from
cattle and 3 were from chicken specimens. All 12 cattle isolates
originated from milk. Nine of the milk specimens that harbored MRSA
were from cows that showed signs of mastitis, as indicated by results
of the somatic cell count assays (data not shown). Among three MRSA
strains from chickens, one was isolated from a suppurative region in
chicken meat. The other two isolates were from joints of the chicken,
which had signs of arthritis. No mecA-positive MRSA strains were
isolated from any specimens of pigs during this investigation.

The MRSA isolates were detected from March to October during the study
period (Table 2).

 TABLE 2.
Summary of results for mecA-positive MRSA isolates from major food
animals 


Antimicrobial susceptibility. The mecA-positive MRSA strains isolated
in this study showed the following frequencies of resistance to the 15
antibiotics (Table 3). All MRSA isolates from the animals were
resistant to penicillin and ampicillin. Among the 15 isolates, 11, 10,
10, 8, 6, 8, 10, 4, 3, 2, and 1 were resistant to erythromycin,
gentamicin, kanamycin, clindamycin, tetracycline, cefoxitin,
cephalothin, imipenem, ciprofloxacin, ofloxacin, and norfloxacin,
respectively. All isolates were susceptible to amikacin and
trimethoprim-sulfamethoxazole. All MRSA strains showed a multidrug
resistance phenotype with increased susceptibility to
fluoroquinolones. The results from the susceptibility profiles
indicated that the animal isolates from this study were within nine
different antibiotypes. Oxacillin resistance was confirmed by
determining the MICs for all MRSA isolates. The range of the oxacillin
MICs for the animal MRSA isolates was between 4 and greater than 128
µg/ml (Table 2). The MICs of vancomycin for the MRSA isolates were all
less than 2 µg/ml.

 TABLE 3.
Antibiotic susceptibility profiles determined by the agar diffusion
method for mecA-positive MRSA isolates 


RAPD patterns. To determine the genetic relatedness of the animal MRSA
isolates to human isolates, RAPD patterns were generated. Among the 15
animal MRSA isolates, eight visually different RAPD patterns (types I
to VIII) were generated by AP-PCR based on combinations of primers M13
and H12 (Fig. 1 and Table 2). Among all 38 human MRSA isolates during
this study, four different patterns were visually identified. A total
of 4 isolates were selected from the 38 human MRSA isolates. Each of
the four isolates was representative of one of the four different RAPD
patterns determined for the human MRSA isolates. These isolates were
designated isolates 16, 17, 18, and 19 (Fig. 1 and Table 2). Among the
MRSA isolates from dairy cattle the most frequent pattern was RAPD
type II, which was characteristic of five isolates (41.7%), including
isolates 2, 5, 10, 11, and 12. One of the human isolates, isolate 19,
showed a RAPD pattern (type II) identical to that of these isolates.
RAPD type II, which was shared by 14 of the 38 human MRSA isolates
collected in this study, was also the most prevalent pattern in human
isolates. In the results of a GelCompar analysis, the isolates showing
a RAPD type II pattern grouped together with 95% genetic similarity
(Fig. 2). Isolates 9 and 13, which possessed type VI RAPD patterns,
shared 91% similarity with the isolates of type II. One of the dairy
cattle isolates (isolate 7) showed a pattern identical to that of a
human isolate (isolate 17) that exhibited type IV characteristics.
However, isolate 6, a dairy cattle isolate, showed RAPD patterns (type
III) distinctly different from those of the other animal and human
isolates. As determined on the basis of GelCompar analysis, the data
showing the genetic relatedness of this isolate to the other isolates
revealed only 65% similarity (Fig. 2). Three MRSA isolates from
chicken specimens were isolated during this study. AP-PCR showed that
all three chicken isolates had identical RAPD patterns (all type I)
and detected genetic homogeneity at 97% similarity. These isolates
were as much as 83% genetically related to the other animal and human
isolates (Fig. 2).

 FIG. 1.
RAPD patterns of MRSA isolates generated by AP-PCR with primers M13
(upper panel) and H12 (lower panel). Isolate numbers (in accordance
with the data presented in Table 2) are indicated above the lanes, and
molecular size markers (M) are indicated to (more ...) 

 FIG. 2.
Dendrogram showing the levels of similarity between RAPD patterns of
19 isolates of mecA-positive MRSA isolates as determined by AP-PCR
with primers M13 and H12 and subsequent GelCompar analysis of
digitized photographs. The scale indicates levels of (more ...) 

Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
REFERENCES  DISCUSSIONThis study was designed to determine the
prevalence of MRSA isolates among S. aureus isolates from major food
animal-related specimens and to determine the antimicrobial
susceptibilities of these isolates and their genetic relatedness to
human MRSA isolates. This was done to evaluate whether animal MRSA
isolates are a possible source of human infections.

Among the specimens from cattle, pigs, and chickens examined in this
study, 421 contained S. aureus. Of the isolates from these specimens,
15 (3.6%) were resistant to oxacillin and were positive for the mecA
gene. The level of oxacillin resistance of these 15 isolates ranged
between 4 and greater than 128 µg/ml (Table 2). The other
antimicrobial susceptibility tests revealed that the isolates had the
characteristics of general multidrug resistance. All MRSA isolates
were resistant to penicillin and ampicillin and were less susceptible
to erythromycin, gentamicin, and kanamycin. Such profiles of
antibiotic resistance occur rather frequently in many of the MRSA
isolates from other countries (1, 10, 15, 26, 27, 32). The isolates
from this study, however, were more susceptible to fluoroquinolones,
such as ciprofloxacin, ofloxacin, and norfloxacin, and all of the
isolates were susceptible to vancomycin, amikacin, and
trimethoprim-sulfamethoxazole. The majority (12 of 15) of the MRSA
isolates were identified in milk specimens of dairy cattle. Among 12
MRSA isolates from milk specimens, 9 showed signs of mastitis, as
indicated by the results of somatic cell count assays. In Korean dairy
farms with livestock with mastitis problems, antibiotics (including
members of the penicillin family such as ampicillin and penicillin)
are largely used as a dry-cow treatment, although oxacillin and
methicillin are rarely used in this veterinary field. This practice
may contribute to the increasing incidence of MRSA strains in cows
associated with mastitis.

RAPD analysis has been extensively applied for epidemiological typing
and taxonomic study of microbial isolates (9, 25, 29, 31). RAPD
patterns were generated to determine the genetic relatedness between
the animal and human MRSA isolates in a community. Eight visually
different RAPD patterns (types I to VIII) from the 15 animal MRSA
isolates were generated (on the basis of combinations of primers M13
and H12) (10) by AP-PCR. Techniques using antibiotypes are also useful
tools for the epidemiological study of bacterial isolates (9, 10, 15,
22, 27). The results of determinations of the susceptibility profiles
of the 15 isolates revealed nine different antibiotypes. RAPD patterns
assigned to isolates were compared with antibiotypes of isolates
(Table 2). General agreement was found between the two types of data
but with some discrepancies. Isolates with the same RAPD types were
within the same antibiotype categories, although isolates with RAPD
type II patterns revealed three different antibiotypes (Table 2). This
finding indicates that categorization of the MRSA isolates by RAPD
pattern was concordant with antibiotype categorization. The most
frequent RAPD pattern was type II, which was characteristic of five
isolates from different milk specimens of dairy cattle. One of the
human isolates generated a type II RAPD pattern identical to that of
these isolates. RAPD type II was also the most prevalent pattern among
the 38 human MRSA isolates in this study. These results indicated that
the MRSA strain generating RAPD pattern type II is most likely the
dominant MRSA strain in the community environment. Of the dairy cattle
isolates, one (isolate 7 [type IV]) also showed a pattern identical to
that of a human isolate (Fig. 1 and 2). There are several study
reports that suggest that the transfer of S. aureus between humans and
cattle is possible (6, 24, 35), although successful transfer of these
bacteria between humans and cattle is not a frequent event. The
present data also indicated that infection of humans by transmission
through food products contaminated with animal MRSA is very plausible.
The animal MRSA isolates may have originally come from humans,
considering that the rate of methicillin resistance among human S.
aureus isolates in Korea is over 50% (13) and that the incidence of
MRSA in animals (3.6% in this study) is relatively low. Once
interspecies transfer has occurred, these isolates can become
widespread in the veterinary environment of Korea, where antibiotics
such as vancomycin, amikacin, and fluoroquinolones (which are more
effective against this organism) are rarely used for animals. This can
lead to an increasing prevalence of MRSA strains in humans.

In the GelCompar analysis, the six isolates generating the type II
RAPD pattern in this study were also grouped together at 95% genetic
similarity (Fig. 2), suggesting a shared genomic background. Isolates
9 and 13, which generated the type VI RAPD pattern, also shared 91%
similarity with the isolates generating the type II RAPD pattern.
Three MRSA isolates from chicken specimens were isolated during this
study. To our knowledge, this is the first report on isolation of MRSA
from chicken. All these three isolates showed the same RAPD pattern
(type I) and had a genetic homogeneity of 97% similarity among the
isolates. These isolates were 83% genetically related to the animal
and human isolates that generated RAPD pattern types II and VI. All of
this may indicate that these isolates are a closely related and
coherent group that may have diverged from a common ancestor.
 
  Acknowledgments
This study was supported by the Technology Development Program for
Agriculture and Forestry, Ministry of Agriculture and Forestry, and by
the Brain Korea 21 Project in 2003, Seoul, Republic of Korea.

I gratefully acknowledge the staff of Veterinary Public Health in
Chonbuk National University for collection efforts with the animal
specimens and the staff of human hospital facilities for providing the
clinical MRSA isolates.

 
Top 
Abstract 
MATERIALS AND METHODS 
RESULTS 
DISCUSSION 
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