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Journal of Clinical Microbiology, December 2000, p. 4351-4355, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding
Gene as a Useful Taxonomic Tool for Staphylococcus
spp.
Javier
Yugueros,1
Alejandro
Temprano,1
Beatriz
Berzal,1
María
Sánchez,1
Carmen
Hernanz,1
José María
Luengo,2 and
Germán
Naharro1,*
Departamento de Sanidad Animal,
Microbiología e Inmunología1 and
Departamento de Bioquímica y Biología
Molecular,2 Universidad de León, 24071 León, Spain
Received 22 June 2000/Returned for modification 4 September
2000/Accepted 24 September 2000
 |
ABSTRACT |
The gap gene of Staphylococcus aureus,
encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target
to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and
25 nucleotides in length. PCR products, detected by agarose gel
electrophoresis, were also amplified from 12 Staphylococcus
spp. analyzed previously. Hybridization with an internal 279-bp DNA
fragment probe was positive in all PCR-positive samples. No PCR
products were amplified when other gram-positive and gram-negative
bacterial genera were analyzed using the same pair of primers.
AluI digestion of PCR-generated products gave 12 different
restriction fragment length polymorphism (RFLP) patterns, one for each
species analyzed. However, we could detect two intraspecies RFLP
patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus
simulans which were different from the other species. An
identical RFLP pattern was observed for 112 S. aureus
isolates from humans, cows, and sheep. The sensitivity of the PCR
assays was very high, with a detection limit for S. aureus
cells of 20 CFU when cells were suspended in saline. PCR amplification
of the gap gene has the potential for rapid identification
of at least 12 species belonging to the genus
Staphylococcus, as it is highly specific.
| |
INTRODUCTION |
The staphylococci are the causative
agents of many opportunistic human and animal infections and are
considered among the most important pathogens isolated in the clinical
microbiology laboratory (8, 14, 29). Staphylococcus
aureus, which is frequently isolated from milk, is the leading
cause of intramammary infections in cows, with major economic
repercussions (1, 35). However, several coagulase-negative
staphylococci are increasingly recognized as etiologic agents of
infections in humans and animals (5, 13, 16, 28, 33). For
this reason it is of crucial importance to isolate and identify the
offending species in order to initiate appropriate antibiotic therapy.
Several methods of identifying Staphylococcus spp. have been
proposed, including those that detect traditional phenotypic
properties, which are available in miniaturized form for automation and
convenience (2, 10, 21, 34), and gas-liquid chromatography
analysis of cellular fatty acids (31). However, many
isolates are still poorly identified by these methods, and
supplementary test are often required for a complete identification.
Molecular methods such as PCR-based DNA fingerprinting and
hybridization have also been used successfully for staphylococcus identification at the species level (6, 9, 15, 20, 36). In
general, rapid bacterial identification by either PCR or hybridization uses species-specific and ubiquitous DNA as a target (3, 4). However, the use of universal pathway genes and universal function genes, whose nucleotide sequences are fairly homologous in bacteria, is
becoming more and more frequent as target DNAs for PCR amplification (9, 19, 26).
It has been described that S. aureus as well as a number of
coagulase-negative staphylococci, including S. epidermidis,
S. capitis, S. haemolyticus, and S. hominis, have a 42-kDa transferrin-binding protein (Tpn) in common
located within the cell wall, which is a member of the newly emerging
family of multifunctional cell wall-associated
glycerahdehyde-3-phosphate dehydrogenases, which catalyze the
conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate and
incorporate binding sites for both transferrin and the serine protease
plasmin (22, 23, 24, 25). However, this protein is absent
from the cell wall of S. saprophyticus and S. warneri, which are unable to bind human transferrin
(24).
In this paper we describe a rapid, sensitive, and specific nucleic
acid-based procedure allowing the identification of 12 Staphylococcus species analyzed previously when PCR and
restriction fragment length polymorphism (RFLP), using AluI
restriction endonuclease, were combined. We selected the gap
gene as a target for PCR amplification. The gap gene is a
part of the glycolytic operon in S. aureus, whose product,
Tpn, is widely distributed and very well conserved in bacteria. We also
show evidence for the presence of gap genetic information in
S. saprophyticus and S. warneri in spite of their failure to bind human transferrin.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The bacterial
strains used in this study are detailed in Table
1. Forty-four S. aureus
strains were isolated from milk from both cows and ewes (36)
with acute clinical mastitis, all of which came from farms in the
northwest of Spain. The 59 S. aureus human isolates were
made available through F. Tenover (Centers for Disease Control and
Prevention, Atlanta, Ga.) (30, 32). Clinical isolates from
humans and animals, CI-1 to CI-6, were tested in our laboratory by
standard procedures. Reference strains used in this study were selected
from the Spanish Type Culture Collection (CECT; other reference is
given when known): 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 strains were
S. intermedius ATCC 49052, and S. haemolyticus.
Strains were grown on Luria agar or Luria broth (LB) and mannitol salt
agar. Other gram-positive and gram-negative bacteria, used as negative controls in PCR assays, were grown on LB and are listed in Table 1.
Cells were incubated at 37°C except for Aeromonas
hydrophila and Yersinia ruckeri, which were grown at
28°C. Human and animal staphylococcus isolates were obtained from
clinical samples and identified using a commercial identification
system (Api-Staph; Biomerieux).
Chromosomal DNA isolation and manipulation.
Chromosomal DNA
from bacteria other than Staphylococcus spp. was obtained
from overnight cultures grown in LB-agar. Samples to be analyzed (a
colony) were suspended in 100 µl of 1 × PCR buffer (10 mM
Tris-HCl [pH 8.3], 2 mM MgCl, 50 mM KCl) and incubated at 95°C for
15 min. A 1-µl amount of the samples was used for PCR analysis.
Chromosomal DNA from Staphylococcus strains was extracted
following the procedure detailed elsewhere (36), and 5 µl
of each sample was used for PCR analysis.
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
following the
manufacturer's instructions (Boehringer GmbH, Mannheim,
Germany). For
digestion of PCR products, a 5-µl sample was used.
Restriction
endonucleases were purchased from Boehringer
GmbH.
PCR-RFLP assays.
PCR amplification tests were performed
using a pair of primers selected on the basis of the gap
gene nucleotide sequence of S. aureus (933-bp long, from the
GenBank database under accession number AJ133520). A 26-nucleotide
forward primer, GF-1 (5'-ATGGTTTTGGTAGAATTGGTCGTTTA-3'), corresponding to positions 22 to 47 of the gap gene,
and a 25-nucleotide reverse primer, GR-2
(5'-GACATTTCGTTATCATACCAAGCTG-3'), corresponding to
positions 956 to 932 of the previously mentioned gene, were selected.
For comparative studies, another pair of primers were used: G1
(5'-GAAGTCGTAACAAGG-3') and L1 (5'-CAAGGCATCCACCGT-3'), which were selected as described (20) so as to analyze
the staphylococcal 16S-23S ribosomal DNA (rDNA) intergenic spacer
region. 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 following the instructions of the manufacturer with some modifications. Briefly, the reaction mixture consisted of 5 µl of DNA-containing sample, 1.25 U of Taq DNA polymerase, 5 µl of 10× PCR amplification buffer, 0.8 µM each primer, 0.4 mM
deoxynucleoside triphosphate, and double-distilled water to a final
volume of 50 µl. In order to prevent 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 94°C for 20 s, primer annealing at 55°C for
30 s, and DNA extension at 72°C for 40 s. After the final
cycle, reactions were terminated by an extra run at 72°C for 5 min.
PCR products were analyzed by agarose gel electrophoresis (3% agarose
gels were prepared with MetaPhor agarose for fine separation and
resolution of small nucleic acids; FMC Bioproducts, Rockland, Maine) in
Tris-borate-EDTA (TBE) buffer.
The RFLP procedure was carried out by digesting PCR-amplified products
with
AluI endonuclease and analyzing them by agarose
gel
electrophoresis as previously mentioned. The procedure mentioned
above
(DNA extraction, PCR amplification, and RFLP) was done in
triplicate,
obtaining identical results. To determine the sensitivity
of the PCR
assays for staphylococci, 10-fold serial dilutions
(from
10
6 to 0 bacteria) in saline were tested. One hundred
microliters
of each dilution was processed as previously mentioned, and
5
µl of each sample was used for PCR amplification. Viable cells
were
counted as CFU by triplicate plating of samples on Luria
agar, and
colony counts after incubation at 37°C for 24 h were
determined.
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 |
PCR-RFLP analysis.
The pair of primers used in this study,
GF-1 and GR-2, selected from the S. aureus gap gene sequence
successfully primed the synthesis of an expected 933-bp fragment, which
represents most of the gap gene sequence (Fig.
1A), when DNAs from 12 species of
Staphylococcus tested in this study were used as a target, although a limited number of strains from each species were tested. However, no PCR amplification products were obtained when other genera
were used as sources of high-molecular-weight target DNA (Fig. 1B). A
single 933-bp hybridization band was also obtained when PCR products
hybridized with the AluI 279-bp internal fragment of the
gap gene, which was cloned from the S. aureus
CECT 86 PCR-amplified product and digested with AluI
endonuclease (data not shown). These results demonstrate that PCR
amplification of the gap gene could be a useful tool for the
rapid identification of staphylococci not only from bacterial cells but
also from biological materials, such as milk. The sensitivity of our
PCR assay was 25 viable cells or 40 pg of extracted DNA when S. aureus was used as the source of DNA and serial dilutions were
conducted in saline. However, when S. aureus was serially
diluted in sterilized whole milk, the lower detection limit was about
500 CFU. Similar results were obtained with the other 11 Staphylococcus species.
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|
FIG. 1.
Agarose gel electrophoresis of the 933-bp PCR
amplification products from chromosomal DNA from staphylococcal species
reference strains using primers GF-1 and GR-2 (A), from other genera
used as negative control (B), and fragments produced by
AluI endonuclease digestion of PCR amplification products
(C). Lanes: 1, S. aureus CECT 86; 2, S. epidermidis CECT 232; 3, S. capitis CECT 233; 4, S. hominis CECT 234; 5, S. saprophyticus CECT
235; 6, S. warneri CECT 236; 7, S. xylosus
CECT 237; 8, S. auricularis CECT 4052; 9, S. carnosus CECT 4491; 10, S. simulans CECT 4538; 11, S. intermedius ATCC 49052; 12, S. haemolyticus
human isolate; 13, Streptococcus sp. mastitis isolate; 14, S. agalactiae CECT 183; 15, S. dysagalactiae CECT
758; 16, S. suis CECT 958; 17, B. cereus CECT
193; 18, E. faecalis CECT 795; 19, M. luteus CECT
241; 20, A. hydrophila CECT 839; 21, E. coli CECT
434; 22, S. cholerasuis CECT 556; 23, Y. ruckeri
CECT 4319; 24, reaction mixture with no DNA. Lanes M, standard DNA size
markers ( X174 digested with HaeIII), (A and B, from top
to bottom: 1,353, 1,078, 872, and 603 bp) and 50-bp ladder (C); bottom
band, 50 bp.
|
|
The 933-bp PCR products of the 12
Staphylococcus spp. used
in this study were
AluI digested, and the resulting
fragments were
separated by MetaPhor agarose gel electrophoresis. A
distinctive
RFLP pattern was obtained for every species analyzed (Fig.
1C).
A total of 103
S. aureus strains (59 human isolates and
44 animal
isolates) were analyzed in this study, all of them showing an
identical RFLP pattern (Fig.
1C, lane 1), indicating intraspecific
uniformity. All clinical strains of
S. epidermidis (6 strains
isolated from humans and 27 strains isolated from animals)
presented
the same RFLP pattern as the reference strain CECT 232 (Fig.
1C,
lane 2) except for two human isolates, termed CI-1, which presented
a very similar RFLP pattern (Fig.
2A,
lane 2), also different
from the other staphylococcus species RFLP
patterns.
S. epidermidis phenotypic identification by the
API ID32 Staph system was made
with reasonable accuracy. These two
strains were confirmed as
S. epidermidis by 16S-23S
intergenic spacer PCR (ITS-PCR) identification
following the method
described elsewhere (
20).
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|
FIG. 2.
Agarose gel electrophoresis of fragments produced by
AluI digestion of the 933-bp PCR amplification products (A)
and PCR-amplified 16S-23S rDNA spacer regions (B) from
Staphylococcus spp. (A) Lanes: 1, S. epidermidis
CECT 232; 2, S. epidermidis CI-1; 3, S. hominis
CECT 234; 4, S. hominis CI-2; 5, S. warneri CECT
236; 6, Staphylococcus sp. CI-3; 7, S. simulans
CECT 4538; 8, S. simulans CI-4; 9, S. xylosus
CECT 237; 10, Staphylococcus sp. CI-5. (B) Lanes: 1, S. aureus CECT 86; 2, S. epidermidis CECT 232; 3, S. capitis CECT 233; 4, S. hominis CECT 234; 5, S. saprophyticus CECT 235; 6, S. warneri CECT
236; 7, S. xylosus CECT 237; 8, S. auricularis
CECT 4052; 9, S. carnosus CECT 4491; 10, S. simulans CECT 4538; 11, S. intermedius ATCC 49052; 12, S. haemolyticus human isolate; 13 to 17, CI-1 to CI-5,
respectively. Lanes M, DNA molecular mass markers, 50-bp ladder (A);
bottom band, 50 bp; and 100-bp ladder (B); bottom band, 200 bp.
|
|
The CI-2 isolate showed an RFLP pattern very similar to that of
S. hominis CECT 234 (Fig.
2A, lanes 3 and 4), and both
strains
presented identical ITS-PCR patterns when compared. However,
phenotypic
methods did not allow a correct CI-2 identification. It is
known
that
S. hominis, which is one of the most common
species after
S. epidermidis and
S. haemolyticus,
is usually identified with
a low accuracy by the API ID32 system
(
10,
11,
27).
Isolate CI-3 was phenotypically identified as
S. warneri
with a low accuracy. However, the RFLP pattern was quite different
from
that of the reference strain
S. warneri CECT 236 (Fig.
2A,
lanes 5 and 6). CI-3 also presented a different ITS-PCR pattern
when
compared with the reference strain. Probably, this isolate
could be
considered a
Staphylococcus spp. which is highly related
to
S. capitis CECT 233 by both RFLP-PCR and ITS-PCR (Fig.
1C
and
2A). Similar results were obtained by others (
18).
CI-4 isolate was identified as
S. simulans with a low
accuracy by phenotypic identification. The RFLP-PCR pattern was very
similar to that of the reference strain
S. simulans CECT
4538
(Fig.
2A, lanes 7 and 8). CA-4 had an ITS-PCR pattern identical
to
that of the reference strain mentioned (Fig.
2B, lanes 10 and
17).
Isolate CI-5 was identified as
S. xylosus by biochemical
properties with high accuracy. However, biochemical identification
did
not agree with our RFLP-PCR assay. Very different RFLP patterns
were
obtained when CA-5 and the reference strain
S. xylosus CECT
237 were compared (Fig.
2A, lanes 9 and 10), although identical
ITS-PCR
patterns were obtained when these two strains were compared
(Fig.
2B,
lanes 7 and 17). Our results would agree with what is
easily observed,
such as growth and pigment production. Reference
strains grew faster
than isolate CA-5 and presented slightly yellowish
colonies while
isolate CA-5 presented almost white colonies when
both strains were
grown on LB agar
medium.
| |
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 differentiate among 12 reference species of
Staphylococcus as well as to discriminate between strains belonging to the same species by a combination of gap gene
PCR amplification and RFLP with AluI. The pair of primers
used in this study did not recognize the other bacterial genera tested. The sensitivity of PCR analysis accords with that described for other
bacteria, that is, between 1 and 20 CFU, or between 1 and 100 pg of DNA
extracted from Staphylococcus spp. (3, 4, 17,
36).
The gap gene, encoding glyceraldehyde-3-phosphate
dehydrogenase, has proved to be a very well conserved gene that may be
a useful tool in an RFLP-PCR assay for differentiating staphylococcal species. Genetic uniformity was found in S. aureus strains
analyzed by our procedure, although they could be grouped by RFLP-PCR
amplification of the aroA gene (36) and by using
other protocols (7, 12, 15). RFLP-PCR of the gap
gene allowed detection of intraspecies polymorphism among S. epidermidis, S. hominis, and S. simulans strains. However, two isolates, CI-3 and CI-5, presumably identified as
S. warnieri and S. xylosus, respectively,
presented RFLP-PCR patterns quite different from those of their
reference strains. Although CI-5 could be assigned to S. xylosus by ITS-PCR, colony morphology and pigments are very
different from those of the reference strain S. xylosus CECT 237.
The gap gene product, glyceraldehyde-3-phosphate
dehydrogenase, has been discovered to be located within the cell wall
of S. aureus and other coagulase-negative staphylococcal
species (24). Although this enzyme was absent from the
S. warneri and S. saprophyticus cell wall
(24), genetic information coding for it does exist, since a
PCR product with high nucleotide sequence homology (data not shown) was
amplified from these two species. These results suggest that
glyceraldehyde-3-phosphate dehydrogenase may be located in the
cytoplasm of these two species.
In conclusion, our results indicate that the RFLP-PCR protocol used in
this study is relatively accurate in the identification of at least 12 species of Staphylococcus and may be useful for differentiating clinical isolates of staphylococci, especially for
those which often do not allow correct phenotypic identification.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Spanish Ministerio de
Educación y Cultura (DGICYT AGF98-0187). J.Y. is a fellowship holder of the University of León.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Sanidad Animal Microbiología e Immunología, Universidad
de León, 24071 León, Spain. Phone: 34-87-291294. Fax:
34-87-291304. E-mail: dsagnc{at}unileon.es.
| |
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Journal of Clinical Microbiology, December 2000, p. 4351-4355, Vol. 38, No. 12
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
