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Journal of Clinical Microbiology, March 1999, p. 570-574, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Identification and Typing of
Staphylococcus aureus by PCR-Restriction Fragment Length
Polymorphism Analysis of the aroA Gene
Javier Yugueros
Marcos,1
Alberto Cascón
Soriano,1
María
Sánchez
Salazar,1
Carmen
Hernanz
Moral,1
Susana Suárez
Ramos,1
Mark S.
Smeltzer,2 and
German
Naharro
Carrasco1,*
Departamento de Sanidad Animal,
Microbiología e Inmunología, Facultad de Veterinaria,
Universidad de León, 24071 León,
Spain,1 and
Department of Microbiology
and Immunology, University of Arkansas for Medical Sciences, Little
Rock, Arkansas 722052
Received 6 July 1998/Returned for modification 13 October
1998/Accepted 19 November 1998
 |
ABSTRACT |
The Staphylococcus aureus aroA gene, which encodes
5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for
the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected
by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans
involved in four confirmed outbreaks). Hybridization with an internal
536-bp DNA fragment probe was positive for all PCR-positive samples. No
PCR products were amplified when other Staphylococcus spp.
or genera were analyzed by using the same pair of primers. The
detection limit for S. aureus cells was 20 CFU when the
cells were suspended in saline; however, the sensitivity of the PCR was
lower (5 × 102 CFU) when S. aureus cells
were suspended in sterilized whole milk. TaqI digestion of
the PCR-generated products rendered two different restriction fragment
length polymorphism patterns with the cow and sheep strains tested, and
these patterns corresponded to the two different patterns obtained by
antibiotic susceptibility tests. Analysis of the 59 human isolates by
our easy and rapid protocol rendered results similar to those of other assays.
| |
INTRODUCTION |
Staphylococcus aureus is
the causative agent of many opportunistic infections in humans and
animals (8). As a human pathogen, S. aureus
causes superficial, deep-skin, and soft-tissue infections, endocarditis, and bacteremia, as well as a variety of toxin-mediated diseases including gastroenteritis, staphylococcal scalded-skin syndrome, and toxic shock syndrome (6, 19). Among animals, from whose milk it is frequently isolated, it is the leading cause of
intramammary infections in cows, with major economic repercussions (1, 24). An outbreak on a farm is often caused by a single strain and may lead to further outbreaks among the same species in the
same region. In such cases, it is of crucial importance to isolate and
identify the offending strain in order for appropriate antibiotic
therapy to be initiated. Several methods of identification of S. aureus have been proposed, including those that detect traditional phenotypic properties, and in recent years these methods have been
available in miniaturized form for automation and convenience (2,
23). Molecular methods such as PCR-based DNA fingerprinting or
hybridization have also successfully been used for S. aureus identification and typing (5, 7, 9, 13, 17, 21, 22).
In general, rapid bacterial identification by either PCR or
hybridization uses species-specific and ubiquitous DNA as a target (3, 13). However, the use of universal pathway genes and universal function genes, whose nucleotide sequences are fairly homologous among bacteria, as target DNAs for PCR amplification is
becoming more and more frequent. Gho et al. (7) recently presented data suggesting that a universal DNA target, the chaperoning 60 gene, may be useful for Staphylococcus species
identification. The aroA gene, which encodes
5-enolpyruvylshikimate-3-phosphate synthase (a key enzyme of aromatic
amino acids and the folate universal biosynthetic pathway) of
Aeromonas hydrophila, was used as a target DNA to identify
most species of Aeromonas genus by restriction fragment
length polymorphism (RFLP) analysis-PCR (4). Also, Mollet et
al. (15) were able to assign 20 selected clinical isolates
to the correct enteric species on the basis of RNA polymerase 
-subunit gene (rpoB) sequence comparison after PCR
amplification. The presence of alternating stable and variable regions
within bacterial rpoB genes allowed them to design primers
within stable regions flanking the sequence encoding a variable
polypeptide region and may be used as a good tool for the
identification of enterobacteria.
In this paper we describe a rapid, sensitive, and specific nucleic
acid-based procedure that permits the identification of S. aureus in cows and sheep with intramammary infections by PCR amplification of the aroA gene. The procedure is further
enhanced when it is combined with RFLP analysis, which allows
discrimination among S. aureus isolates. We have also used
our protocol to characterize 59 S. aureus isolates
previously characterized by others (20, 22). When compared
with other methods, our method had an intermediate degree of
discriminatory power, 100% typeability, and 100% reproducibility.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The S. aureus strains used in this study were isolated from milk from
both cows (38 strains) and ewes (6 strains) with acute clinical
mastitis. All of the animals came from farms in northwest Spain, and
the farms belonged to health protection associations under veterinary
surveillance. Animals from a total of 30 farms were analyzed, with from
30 to 50 animals from each farm being analyzed, but animals on only 10 of the farms were positive for S. aureus. The number of
isolates from each farm ranged from 1 to 10. The 59 S. aureus isolates from humans were obtained from individuals
involved in four confirmed outbreaks, named outbreaks I, II, III, and
IV, and one pseudo-outbreak, and were grouped as in previous studies in
groups of 20 (designated groups SA, SB, and SC) including Internal
controls, which were either duplicates of the same isolate or isolates
obtained from the same patient, were included in each group. S. aureus ATCC 12600 was included in all groups (strains SA4, SB7,
and SC3), while a single strain of S. intermedius (ATCC
49052) was included in the SA group (strain SA16). The 59 S. aureus isolates were made available through F. C. Tenover
(Centers for Disease Control and Prevention, Atlanta, Ga.) (20,
22). The reference Staphylococcus strains used in this
study were selected from the Spanish Type Culture Collection (CECT) and
were as follows: S. aureus CECT 86 (ATCC 12600), S. xylosus CECT 237, S. hominis CECT 234, S. saprophyticum CECT 235, S. epidermidis CECT 232, S. warneri CECT 236, S. capitis CECT 233, S. simulans CECT 4538, S. auricularis CECT 4052, and S. carnosus CECT 4491. Other reference
Staphylococcus strains were S. hyicus ATCC 11249, S. delphini ATCC 49171, and S. lugdenensis ATCC
43809. Strains were grown on Luria agar or Luria broth (LB) and manitol salt agar. Escherichia coli C600, Aeromonas
hydrophila ATCC 7966, Pseudomonas putida,
Actinobacillus pleuropneumoniae, Sarcina lutea, Bacillus subtilis, and Streptomyces griseus were
used as negative controls in the PCR assays, and all isolates except
A. pleuropneumoniae were grown on LB. A. pleuropneumoniae was grown on brain heart infusion agar
supplemented with 0.1% NAD. All cells except those of S. griseus and A. hydrophila were incubated at 37°C;
S. griseus and A. hydrophila cells were grown at
28°C.
Identification and susceptibility testing.
Isolates were
identified as S. aureus on the basis of colony morphology,
Gram staining result, the presence of catalase-positive cocci in
clumps, and the detection of coagulase production with fresh rabbit
plasma (with a colony obtained from an overnight culture suspended in
0.5 ml of plasma and incubated at 37°C for 2 h) and by using a
commercial identification system (API Staph; Biomerieux). All isolates
were tested for their susceptibilities to different antibiotics by the
disk agar method as standardized by the National Committee for Clinical
Laboratory Standards (16). The following antimicrobial disks
were used at the indicated concentrations: penicillin, 10 U;
ampicillin, 10 µg; amoxicillin-clavulanate, 20 and 10 µg,
respectively; oxacillin, 1 µg; cloxacillin, 1 µg; erythromycin, 15 µg; oxytetracycline, 30 µg; and gentamicin, 10 µg (Difco
Laboratories, Detroit, Mich.). S. aureus CECT 435 was used
as a control. The results were recorded after 24 h of incubation at 35°C.
Chromosomal DNA isolation and manipulation.
Chromosomal DNAs
from E. coli, A. hydrophila, P. putida, A. pleuropneumoniae, S. lutea,
B. subtilis, and S. griseus were obtained from
overnight cultures grown in LB agar or brain heart infusion agar
supplemented with NAD as indicated above. Samples to be analyzed (a
colony) were suspended in 100 µl of 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 2 mM MgCl2, 50 mM KCl) and were incubated at
95°C for 15 min. A total of 1 µl of the samples was used for PCR
analysis. Chromosomal DNAs from the Staphylococcus strains
were extracted by the following protocol. A colony of
Staphylococcus or 1 µl containing from 103 to
105 CFU was suspended in 100 µl of lysis buffer (20 mM
Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl, 0.5% [vol/vol] Tween
20, 0.45% [vol/vol] Nonidet P-40, 0.01% [wt/vol] gelatin, and 60 µg of proteinase K per ml), and the mixture was incubated at 55°C
for 1 h. The samples were then incubated at 95°C for 10 min. A
total of 5 µl of each sample was used for PCR analysis. Chromosomal
DNA was also obtained directly from milk, once S. aureus had
been detected in the sample, by adding 100 µl of milk to 100 µl of
2× lysis buffer and testing the sample by the protocol mentioned above.
Blotting and hybridization were performed by standard procedures, and
DNA labeling was carried out by random priming with digoxigenin-dUTP.
Hybrids were detected by enzyme immunoassay according to the
manufacturer's instructions (Boehringer GmbH, Mannheim, Germany). For
digestion of the PCR products, a 5-µl sample was used. Restriction
endonucleases were purchased from Boehringer GmbH.
PCR-RFLP assays.
PCR amplification tests were performed with
a pair of primers selected on the basis of the published nucleotide
sequence of the S. aureus aroA gene (length, 1,283 bp;
GenBank database accession no. L05004). A 24-nucleotide forward primer,
FA1 (5'-AAGGGCGAAATAGAAGTGCCGGGC-3'), corresponding to
positions 40 to 63, and a 19-nucleotide reverse primer, RA2
(5'-CACAAGCAACTGCAAGCAT-3'), corresponding to positions 1192 to 1174, were selected. The primers were synthesized by British Bio-Technological Products (Avingdon, England). PCR amplification was
carried out with a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk,
Conn.) by using a PCR kit (Boehringer GmbH) and by following the
instructions of the manufacturer, with some modifications. Briefly, the
reaction mixture consisted of 5 µl of a sample containing DNA, 1.25 U
of Taq DNA polymerase, 5 µl of 10× PCR amplification buffer, 0.6 µM each primer, 0.5 mM each deoxynucleoside triphosphate, and double-distilled water to a final volume of 50 µl. To minimize evaporation, 50 µl of mineral oil was added to the mixture. DNA was
denatured at 94°C for 2 min. A total of 40 PCR cycles were run under
the following conditions: DNA denaturation at 92°C for 1 min, primer
annealing at 58°C for 1 min, and DNA extension at 72°C for 1.5 min.
After the final cycle, the reactions were terminated by an extra run at
72°C for 10 min. The PCR products were analyzed by agarose gel
electrophoresis (with 3% agarose gels run in Tris-borate-EDTA buffer).
The RFLP procedure was carried out by digesting the PCR-amplified products with either TaqI or RsaI endonucleases,
and the products were analyzed by agarose gel electrophoresis as
described above. For determination of the sensitivity of the PCR,
10-fold serial dilutions (106 to 0 bacteria) in both saline
and sterilized whole milk were tested. One hundred microliters of each
dilution was processed as described above, and 5 µl of each sample
was used for PCR amplification. The numbers of viable cells were
counted by determining the numbers of CFU by triplicate plating of the
samples on Luria agar and counting of the colonies after incubation at
37°C for 24 h. When nucleic acids were used, the sensitivity of
the PCR was determined by amplifying 5 µl of 10-fold serial dilutions
(1 ng to 0.1 pg).
| |
RESULTS |
Phenotypic characteristics of S. aureus isolates.
All isolates were gram-positive cocci and coagulase positive and were
identified as S. aureus as described in Materials and Methods. Antibiotic sensitivity testing (Table
1) showed two patterns. The isolates with
one of the patterns, designated group 1, were represented by eight
S. aureus isolates (two from cows and six from ewes) which
were sensitive to penicillin G, ampicillin, and
amoxicillin-clavulanate. However, the isolates with the second pattern,
designated group 2, represented by 36 isolates, originated from 36 samples collected from cows, and the isolates were resistant to the
same three antibiotics. Very similar patterns of susceptibility to five
other antibiotics were observed for all S. aureus isolates, with sensitivity to oxacillin, erythromycin, and oxytetracycline, resistance to cloxacillin, and an intermediate response to gentamicin. Two isolates from group 2 were resistant to erythromycin and one was
resistant to oxytetracycline. The susceptibility pattern of the
reference strain, S. aureus CECT 86, was identical to those of the group 1 isolates.
PCR-RFLP analysis.
The pair of primers used in this study, FA1
and RA2, from the S. aureus aroA gene, successfully primed
the synthesis of an expected 1,153-bp fragment which represents most of
the aroA gene sequence (Fig. 1, lane
1) of all isolates and strains of
S. aureus tested. No PCR amplification products were
obtained when other Staphylococcus spp. or genera were used
as sources of high-molecular-weight target DNA (Fig.
2). Also, a single 1,153-bp band was
obtained when the PCR products hybridized with the internal 536-bp
TaqI fragment of the aroA gene which was cloned
from the S. aureus CECT 86 PCR-amplified product and
digested with TaqI endonuclease (data not shown). These
results demonstrate that PCR amplification of the aroA gene
could be a useful tool for the rapid identification of S. aureus DNA not only from bacterial cells but also from biological materials, such as milk. The sensitivity of our PCR assay was 20 viable
S. aureus cells or 40 pg of extracted DNA; however, when
S. aureus was serially diluted in sterilized whole milk, the
lower detection limit was about 500 CFU.
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FIG. 1.
Agarose gel electrophoresis of PCR amplification
products from high-molecular-weight chromosomal DNA from S. aureus and fragments produced by TaqI endonuclease
digestion. Lanes: 1, 1,153-bp PCR amplification product; 2, RFLP
patterns 1 and A; 3, RFLP patterns 2 and B; 4, RFLP pattern C; 5, RFLP
pattern D; M, DNA molecular mass marker (100-bp ladder; bottom band,
100 bp).
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FIG. 2.
Agarose gel electrophoresis of PCR amplification
products from S. aureus chromosomal DNA and other
Staphylococcus spp. and genera used as negative controls.
Lanes: 1, 1,153-bp PCR amplification product from S. aureus
CECT 86; 2, E. coli; 3, S. intermedius; 4, S. epidermidis; 5, S. xylosus; 6, S. hominis; 7, S. delphini; 8, A. hydrophila;
9, B. subtilis; 10, P. putida; 11, S. lutea; 12, A. pleuropneumoniae; M, DNA molecular mass
marker (100-bp ladder; bottom band, 100 bp).
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PCR products of all
S. aureus isolates were digested with
TaqI, and the resulting fragments were separated by agarose
gel
electrophoresis. Two distinct RFLP patterns were observed (Fig.
1)
among the 44 isolates. One of them, RFLP pattern 1, represented
by
reference strain CECT 86, two isolates from cows, and six isolates
from
sheep, rendered five bands of 536, 254, 244, 87, and 32 bp
after
TaqI digestion (Fig.
1, lane 2). The other one, RFLP pattern
2, was represented by the 36 group 2 isolates from cows. As shown
in
Fig.
1 (lane 3), four fragments of 536, 341, 244, and 32 bp
were
generated after digestion with
TaqI endonuclease. A good
correlation between the results of testing of susceptibility to
antibiotics and the results of genotyping were found. All isolates
with
RFLP pattern 1 were sensitive to penicillin G, ampicillin,
and
amoxicillin-clavulanate, although the isolates with RFLP pattern
2 were
resistant to these three
antibiotics.
Characterization of 59 S. aureus isolates by PCR-RFLP
analysis.
The 59 S. aureus isolates from humans and
S. intermedius ATCC 49052 (strain SA16) were analyzed by our
PCR protocol described above. A 1,153-bp fragment, which represents
most of the S. aureus aroA sequence, was amplified from all
59 isolates, as expected (Fig. 1, lane 1). The pair of primers used,
FA1 and RA2, did not prime the synthesis of the expected PCR product
when S. intermedius ATCC 49052 cells were used as the source
of target DNA (Fig. 2, lane 3). When the 1,153-bp PCR product was
digested with TaqI endonuclease, four RFLP patterns, RFLP
patterns A, B, C, and D, were obtained.
RFLP pattern A (Fig.
1, lane 2; Table
2),
which was identical to RFLP pattern 1 (see above), included 23 isolates
comprising
the complete SC group of strains and strains SA4, SB7
(
S. aureus ATCC 12600), and SB8, an isolate with no clear
epidemiological
link to the other isolates in the SB group. Our
fingerprinting
protocol was not very discriminatory because the SC
group contained
isolates from two relatively well-defined outbreaks, as
well as
an unrelated control strain (strain SC8) and
S. aureus ATCC 12600
(strain SC3). However, the 23 isolates with RFLP
pattern A were
split into two RFLP patterns, RFLP pattern A1 and RFLP
pattern
A2, after digestion of the 1,153-bp PCR product with
RsaI (Fig.
3), which clearly
separated the isolates included in each of the
well-defined outbreaks
III and IV.
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TABLE 2.
RFLP patterns of SA, SB, and SC groups of S. aureus isolates from humans after digestion of PCR products
with TaqI or RsaI
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FIG. 3.
Agarose gel electrophoresis of fragments produced by
RsaI digestion of the 1,153-bp PCR amplification products
from S. aureus SC group. Lanes 1 to 20, strains SC1 to SC20,
respectively; lane M, molecular mass marker (from top to bottom, 12,216 to 75 bp).
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|
RFLP pattern B, which was identical to RFLP pattern 2 (see above), was
generated by
TaqI digestion of the 1,153-bp fragment
PCR
product for eight isolates (Fig.
1, lane 3; Table
2), including
all
isolates from outbreak II (isolates SB2, SB4, SB6, and SB11),
three
epidemiologically unrelated isolates within set B (isolates
SB9, SB13,
and SB17), and isolate
SA14.
RFLP pattern C was generated by
TaqI digestion of the
1,152-bp fragment PCR product for isolates SA8 and SA11 (Fig.
1, lane
4; Table
2), which rendered four fragments of 536, 499, 87, and
32 bp.
These two isolates and isolate SC8 had identical fingerprints
when they
were analyzed previously (
20).
RFLP pattern D (Fig.
1, lane 5; Table
2) had five bands of 341, 300, 244, 220, and 50 bp after
TaqI digestion of the previously
mentioned 1,153-bp fragment for 26 isolates included in sets SA
and SB;
all of these isolates have identical fingerprints according
to Smeltzer
et al. (
20). In general, our fingerprinting protocol
was
easily able to group the 59
S. aureus isolates from humans
and was able to do so with a reasonable discriminatory
power.
| |
DISCUSSION |
The use of nucleic acid amplification by PCR has applications in
many fields, especially for the rapid identification of bacteria. In
this study we were able to identify S. aureus strains
isolated from cows and sheep with clinical mastitis by PCR
amplification of the aroA gene. The pair of primers used in
this study did not recognize the other bacteria tested. The
aroA gene has previously been used with success as a tool
for examination of the taxonomy of Aeromonas genus, a
gram-negative bacterium (4). The sensitivity of PCR analysis
for S. aureus accords with that described for other
bacteria, that is, between 1 and 20 CFU or between 1 and 100 pg for DNA
extracted from S. aureus (3, 4, 11, 18).
On the basis of the published nucleotide sequence of the
aroA gene from S. aureus (GenBank accession no.
L05004), we analyzed this gene by means of a computer program (RESTRI,
PC-GENE) to carry out RFLP analysis. Good results were obtained when
PCR-amplified DNAs from all S. aureus isolates were digested
with TaqI endonuclease. For all S. aureus
isolates from cows and sheep, a good relationship between antibiotic
susceptibility type and RFLP pattern after TaqI endonuclease
digestion was found. Most isolates from cows (36 of 38) had identical
antibiotic susceptibility types and RFLP patterns; however, 2 isolates
from cows, 6 isolates from sheep, and the reference strain, S. aureus CECT 86, had quite different RFLP patterns and antibiotic
susceptibilities. The two patterns found for isolates of S. aureus from animals were in contrast to those found for isolates
from humans, for which a high degree of diversity has been found
(20). The results of this analysis may be limited for an
epidemiological study, although our data support those found by others
with isolates from cows, in which only a small number of S. aureus genotypes were isolated from cows with clinical mastitis
(14, 10, 12). Three possible explanations were given. The
first and most likely explanation was that a small number of different
genotypes of S. aureus in cows with mastitis may be due to
contagion of the pathogen among the animals on a farm. Second, perhaps
only a small number of strains have enough virulence to cause mastitis.
The third explanation was that not all different S. aureus
strains can be adequately differentiated by currently available methods.
Our PCR-RFLP protocol was also used to characterize 59 S. aureus isolates which had previously been analyzed by other
fingerprinting protocols (20, 22), including reference
strains, isolates from four well-defined outbreaks, and unrelated
strains. Our results indicate that the PCR-RFLP protocol used is
relatively accurate with regard to the identification of
epidemiologically related isolates, but it tends to include unrelated
strains falsely within epidemiologically related groups. As suggested
by Tenover et al. (22), the correct epidemiological typing
of S. aureus might require a combination of methods.
Initially, the analysis could be performed by a simple method that is
able to identify all potentially related strains, after which a second
method capable of discriminating among individual isolates could be
applied. Our PCR protocol is one of the best alternatives for the
former, because we have clearly demonstrated that PCR amplification of
the aroA gene is specific for S. aureus
identification and is further enhanced when it is combined with RFLP
analysis, which allows a reasonable discrimination among strains and
isolates, while it can be performed quickly and easily.
| |
ACKNOWLEDGMENTS |
We thank Laboratorio Pecuario Regional (León, Spain) for
providing us with valuable material for this study and Fred C. Tenover for providing us strains of S. aureus.
This work was supported by a grant from the Spanish Ministry of
Education and Science (grant DGICYT PB94-0136). J.Y.M. is the holder of
a fellowship from the University of León.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Sanidad Animal, Microbiología e Inmunología, Facultad
de Veterinaria, Universidad de León, 24071 León, Spain.
Phone: 34 987 291294. Fax: 34 987 291304. E-mail:
dsagnc{at}unileon.es.
| |
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Journal of Clinical Microbiology, March 1999, p. 570-574, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
