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Journal of Clinical Microbiology, September 2001, p. 3099-3103, Vol. 39, No. 9
Molecular Genetics Laboratory, Instituto de
Tecnologia Química e Biológica da Universidade Nova de
Lisboa, Oeiras,1 and Centro de Recursos
Microbiológicos2 and Biotechnology
Unit,3 Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Monte da Caparica, Portugal, and
Laboratory of Microbiology, The Rockefeller University, New
York, New York4
Received 21 March 2001/Returned for modification 17 May
2001/Accepted 18 June 2001
The emergence of coagulase-negative staphylococci not only as human
pathogens but also as reservoirs of antibiotic resistance determinants
requires the deployment and development of methods for their rapid and
reliable identification. Internal transcribed spacer-PCR (ITS-PCR) was
used to identify a collection of 617 clinical staphylococcal isolates.
The amplicons were resolved in high-resolution agarose gels and
visually compared with the patterns obtained for the control strains of
29 staphylococcal species. Of the 617 isolates studied, 592 (95.95%)
were identified by ITS-PCR and included 11 species: 302 isolates of
Staphylococcus epidermidis, 157 of S. haemolyticus, 79 of S. aureus, 21 of S. hominis, 14 of S. saprophyticus, 8 of S. warneri, 6 of S. simulans, 2 of S. lugdunensis, and 1 each of S. caprae, S. carnosus, and S. cohnii. All species analyzed had
unique ITS-PCR patterns, although some were very similar, namely, the
group S. saprophyticus, S. cohnii, S. gallinarum, S. xylosus, S. lentus,
S. equorum, and S. chromogenes, the pair S. schleiferi and S. vitulus, and the pair S. piscifermentans and S. carnosus. Four species,
S. aureus, S. caprae, S. haemolyticus, and S. lugdunensis, showed
polymorphisms on their ITS-PCR patterns. ITS-PCR proved to be a
valuable alternative for the identification of staphylococci, offering,
within the same response time and at lower cost, higher reliability
than the currently available commercial systems.
Staphylococci are nosocomial
pathogens associated with multiple antimicrobial resistance mechanisms.
For many years Staphylococcus aureus was the only species
recognized as an important human pathogen, whereas the
coagulase-negative staphylococci (CNS) were viewed mostly as clinically
nonrelevant contaminants. Only recently, the importance of CNS strains
as a major cause of nosocomial infections, mainly associated with the
use of prosthetic and indwelling devices and immunocompromised
patients, began to be ascertained (15). Although
Staphylococcus epidermidis accounts for the majority of
infections caused by CNS, many other species have been identified in
association with human infections (12, 15, 17).
The emergence of CNS as human pathogens and reservoirs of antimicrobial
resistance determinants requires their rapid and reliable identification in order to have an early prediction of the potential pathogenicity or antibiotic susceptibility of each clinical isolate (12, 15, 17) and to clarify the clinical significance of each species.
In recent years, several commercial systems for the rapid
identification of staphylococci have been developed as an alternative to the classical identification protocols (13, 14), which are too laborious and time-consuming to be used in most clinical laboratories. The commercial systems, based on miniaturized biochemical or immunologic reactions, are widely used today for both clinical and
research purposes (12). However, these diagnostic systems present problems, such as cost and response time, but more importantly, they often provide unreliable results. Several of the problems associated with these systems result from the variable expression of
phenotypic characters that are used as diagnostic parameters. Additionally, many of these kits are based on colorimetric results, and
subjectivity in their interpretation may lead to ambiguity.
For these reasons, significant efforts have been made in order to
develop alternative identification methods combining speed, reliability, and low cost. These criteria are met by methods based on
molecular rather than phenotypic characters. One of these methods is
ribosome spacer PCR or internal transcribed spacer-PCR (ITS-PCR), the
rationale for which is described briefly below.
In prokaryotes, the rRNA genetic loci contain the genes for 16S, 23S,
and 5S rRNAs. These genes are separated by spacer regions which show a
high degree of variability in both sequence and size at the genus and
species level (2, 8). The diversity of the intergenic
spacer regions is due in part to variations in the number and type of
tRNA sequences found among these spacers. In staphylococci, there are
several copies of the rrn operon. Gürtler and Barrie
(7) characterized the spacer sequences of S. aureus strains, including methicillin-resistant S. aureus (MRSA) isolates, and identified nine rrn operons
whose 16S-23S spacer region varied from 303 to 551 bp. According to
these authors, three of these spacers contain the tRNAIle
gene and two contain both the tRNAIle and the
tRNAAla genes, while the remaining four 16S-23S spacers
have no tRNA gene (7, 8). Forsman et al. (5)
sequenced the 16S-23S spacer of five staphylococcal species (S. aureus, S. epidermidis, S. hyicus, S. simulans, and S. xylosus) and found that in addition to
S. aureus, S. hyicus and S. simulans
also had a tRNAIle gene in some of their rrn operons.
The highly polymorphic nature of the 16S-23S spacer sequences may be
analyzed by PCR, using as primers conserved sequences from the adjacent
16S and 23S genes. This method, originally described by Barry et al.
(2), is known as ITS-PCR. Identification of staphylococci
by ITS-PCR was first studied by Jensen et al. (10), who
successfully applied this technique to differentiate strains of four
staphylococcal species, S. aureus, S. epidermidis, S. saprophyticus, and S. warneri. Some authors tested ITS-PCR for the identification of
staphylococci from diverse origins, using different protocols (3,
18), whereas others designed PCR primers based on
species-specific sequences of 16S-23S spacers for the detection of
particular staphylococcal species (5, 20).
In this work, ITS-PCR was applied to the identification of over 600 staphylococcal samples received from different hospitals, using as
controls reference strains of 29 of the 32 species that are presently
recognized in the genus Staphylococcus (15).
The experimental conditions used were those described by Jensen et al.
(10), introducing a high-resolution agarose that allowed rapid resolution of the amplification products.
Part of this work was presented at the 40th Interscience Conference on
Antimicrobial Agents and Chemotherapy (I. Couto, S. Pereira, M. Miragaia, I. Sanches, and H. de Lencastre, Abstr. 40th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. 900, 2000).
Bacterial strains. (i) Control strains.
Thirty-three control
strains were used, representing 29 staphylococcal species and 7 subspecies. These controls included the type strains of
Staphylococcus arlettae (ATCC43957T), S. auricularis (ATCC33753T), S. capitis (ATCC27840T), S. caprae
(ATCC35538T), S. carnosus
(ATCC51365T), S. chromogenes
(ATCC43764T), S. cohnii subsp.
cohnii (ATCC29974T), S. cohnii subsp. urealyticum
(ATCC49330T), S. delphini
(ATCC49171T), S. epidermidis
(ATCC14990T), S. equorum
(ATCC43958T), S. felis
(ATCC49168T), S. gallinarum
(ATCC35539T), S. haemolyticus
(ATCC29970T), S. hominis
(ATCC27844T), S. hyicus subsp.
hyicus (ATCC11249T), S. intermedius (ATCC29663T), S. kloosii (ATCC43959T), S. lentus
(ATCC29070T), S. lugdunensis
(ATCC43809T), S. pasteuri
(ATCC51129T), S. piscifermentans
(ATCC51136T), S. saprophyticus
(ATCC15305T), S. schleiferi subsp.
coagulans (ATCC49545T), S. schleiferi subsp. schleiferi
(ATCC43808T), S. sciuri subsp.
carnaticus (ATCC700058T), S. sciuri subsp. rodentium (ATCC700061T),
S. sciuri subsp. sciuri
(ATCC29062T), S. simulans
(ATCC27848T), S. vitulus
(ATCC51145T), S. xylosus
(ATCC29971T), S. warneri
(ATCC27836T), and a well-characterized strain of
S. aureus (NCTC8325) (19).
(ii) Clinical strains.
A total of 617 clinical
staphylococcal samples of human origin from the Molecular Genetics
Laboratory of the Instituto de Tecnologia Química e
Biológica da Universidade Nova de Lisboa, Oeiras, Portugal,
culture collection were studied. The majority of the samples were
isolated during 1997 and 1998 from colonization and infection sites of
hospitalized patients.
Strain purification procedure.
CNS samples received from
clinical laboratories often contained mixed cultures of staphylococci.
These mixtures were more easily detected after a prolonged incubation
for 48 h at 37°C followed by an additional 48 h at room
temperature. This procedure, previously suggested by other authors
(11), was followed for all isolates prior to their
characterization. All strains were grown in tryptic soy agar (Difco
Laboratories, Detroit, Mich.).
Identification criteria. (i) Preliminary identification.
Upon reception and purification, all isolates were tested in our
laboratory for catalase and oxidase assays, mannitol fermentation, and
coagulase production.
(ii) Final identification.
The ITS-PCR amplification
patterns of the clinical isolates were visually compared with those of
the reference strains. Samples showing similar patterns distantly
positioned in the working gels were run side by side in new gels in
order to confirm their similarity. A final identification was assigned
to those isolates with ITS-PCR patterns that matched any of the control
strains, considering the results on the mannitol and coagulase tests.
When additional characterization of isolates was needed, in order to
complete or confirm ITS-PCR results, two commercial identification
systems were used, API STAPH and ID32 STAPH (BioMérieux, Marcy
l'Etoile, France) according to the manufacturer's instructions.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3099-3103.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Identification of Clinical Staphylococcal Isolates
from Humans by Internal Transcribed Spacer PCR
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
ITS-PCR. DNA was isolated by the guanidine isothiocyanate extraction method as described before (1). The ITS-PCR was performed as previously described (10), using primers G1 (5'-GAAGTCGTAACAAGG) and L1 (5'-CAAGGCATCCACCGT) (Gibco-BRL, Life Technology Ltd., Paisley, Scotland). Amplification reaction was performed on a Perkin Elmer Gene Amp PCR system 9600 apparatus (Perkin Elmer Applied Biosystems, Cheshire, England). The program consisted of an initial denaturation step at 94°C for 4 min and 25 amplification cycles, each with 1 min at 94°C, 2-min ramp to 55°C, 7 min at 55°C, 2-min ramp to 72°C, and 2 min at 72°C, followed by an additional extension step of 7 min at 72°C (10).
Amplification products were resolved in high-resolution gels (3% Metaphor agarose [FMC BioProducts, Rockland, Maine]) in 1× TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA [pH 8]) supplemented with 0.25 µg of ethidium bromide per ml for 6 h, using 100-bp ladder molecular size markers (USB, Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) as standards. After photography by standard procedures, DNA patterns were visually analyzed.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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ITS-PCR patterns of control strains.
The usefulness of ITS-PCR
for the identification of staphylococcal species was first tested by
analyzing 33 reference strains, representing 29 staphylococcal species
and 7 subspecies (Fig. 1A and 1B). The ITS-PCR patterns obtained for
the 29 species tested consisted of four to nine bands, ranging from 320 to 750 bp; each species produced a stronger band, usually the smallest
one (Fig. 1).
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Testing of different ITS-PCR conditions. In an attempt to improve the differentiation of the staphylococcal control strains, different ITS-PCR conditions were tested. For this purpose, other ITS-PCR primers were used, namely, the ones proposed by Gürtler and Stanish (8). The primers tested corresponded to region 2 of the 16S ribosomal DNA (rDNA) (5'-TTGTACACACCGCCCGTC), combined with primers for region 7 (5'-GGTACTTAGATGTTT) or 10 (5'-CCTTTCCCTCACGGTACTG) of the 23S rDNA (8). These pairs of primers did not improve the discrimination among the species analyzed; primers 2 and 10 originated compact ITS-PCR patterns, with high-molecular-weight bands of difficult resolution, while primers 2 and 7 produced patterns similar to the ones obtained with G1 and L1, although with larger ITS fragments that needed longer electrophoresis runs in order to be resolved and visually differentiated (results not shown).
We also tested another ITS-PCR protocol described earlier by Mendoza and colleagues (18). These authors used a lower annealing temperature and no ramping in the amplification reaction, as well as a different protocol for DNA preparation. The ITS-PCR patterns obtained following this alternative protocol had lower resolution (data not shown) due to poorer quality of the template DNA and the lower annealing temperature.Identification of clinical staphylococcal isolates.
The
ITS-PCR amplification patterns of the clinical staphylococcal isolates
analyzed were visually compared with those obtained for the reference
strains (Fig. 2).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this work we tested different ITS-PCR protocols described in the literature in order to apply this method to the identification of staphylococci of human clinical origin and found that of the several conditions assayed, the combination of primers G1 and L1 with the amplification procedure described by Jensen et al. (10) provided the best discrimination among the several species tested. In these conditions, ITS-PCR proved to be a reliable method for the identification of staphylococci. Each of the 29 control strains tested showed distinct amplification patterns, which subsequently allowed the identification of nearly 600 clinical isolates, corresponding to 95.95% of the samples tested. Only 25 isolates could not be identified either by ITS-PCR or by the commercial systems used.
The collection of isolates that were analyzed included the three staphylococcal species recognized as clinically more relevant, S. aureus, S. epidermidis, and S. haemolyticus. It also included other species commonly associated with human infections, such as S. saprophyticus, S. hominis, S. lugdunensis, and S. warneri, as well as species less frequently isolated, namely, S. simulans, S. cohnii, and S. caprae. Finally, we identified an isolate of S. carnosus, a species rarely isolated from clinical sources (15, 17).
The ITS-PCR method was simple and met our needs for rapid identification of large numbers of clinical isolates with high reliability. Following strain culture in plates, it was possible to obtain an identification after 18 h, most of this time being spent on the PCR (6 h) and electrophoretic resolution of the amplification products (6 h). Although a shorter time response is desirable, it should not compromise the quality of results. Particularly, we verified that the purity of the DNA samples, the annealing temperature, and the ramping steps in the PCR were essential to obtain reliable and consistent results. Variations in these experimental conditions may explain differences in results obtained by other authors (18), even using the same primers. Moreover, the amplification reaction and programs used in this study were designed to prevent the formation of unspecific products, such as single-stranded or heteroduplex structures (9, 10). All these factors were essential to obtain well-defined ITS patterns and to ensure the stability and reproducibility of the ITS-PCR patterns, which were verified by testing different DNA preparations of the same strains in several independent gel runs performed by different operators. The use of high-resolution agarose avoided the utilization of polyacrylamide gels used in previous works (10), with consequent simplification of the protocol.
In our experience, the restriction of the amplicons with restriction enzymes as proposed by other authors (10, 18) is not necessary for the identification of staphylococci of human origin, since the direct analysis of the amplification products provides enough resolution for the identification of the clinically more frequent species as well as those potentially relevant but not frequently associated with disease. In fact, the majority of these species were easily identified by direct comparison with the control strain profile, and therefore this additional resolution step may not be necessary.
Four staphylococcal species presented more than one ITS-PCR pattern. Of these, S. aureus was the species showing the highest number of different polymorphisms (Fig. 3). Nevertheless, the S. aureus isolates characterized in this work showed an ITS-PCR pattern which was identical or very similar to that of the control strain, which simplified their identification. When working with larger or more heterogeneous collections of isolates, S. aureus identification by ITS-PCR should be based on recognition of the characteristic nuclei of central bands (see Fig. 3 and also reference 10) and confirmed by additional testing, such as production of coagulase.
Because of the highly polymorphic nature of S. aureus ITS regions, several authors have already described the use of ITS-PCR for typing both methicillin-susceptible (MSSA) and -resistant strains (4, 7, 16, 20, 21). However, our own results using ITS-PCR to type strains representative of well-characterized MRSA clones showed that the discriminatory power of ITS-PCR for MRSA typing is limited (I. Couto et al., unpublished data).
In addition to the polymorphisms described in this work, intraspecific variations of the ITS profiles have also been described for S. saprophyticus and S. epidermidis (10, 18). In our work, and despite the number of S. epidermidis and S. saprophyticus isolates studied (302 and 14, respectively), we found no polymorphisms for the ITS-PCR patterns of these two species.
A significant error percentage was found between the identification results provided by the clinical laboratories and those obtained with ITS-PCR. The most often misidentified isolates, also the most significant from the clinical point of view, were S. epidermidis and S. haemolyticus isolates, which were misidentified as S. aureus. The implications of these errors are clearly illustrated by a collection of eight methicillin-resistant staphylococcal isolates identified by the API STAPH system as S. aureus in two independent assays performed in our laboratory. However, they did not produce coagulase and their ITS-PCR profiles were identical to that of the S. haemolyticus type strain. The misidentification of these isolates as S. aureus resulted from an intermediate result in the test for the utilization of D-mannose. This example documents the need for reliable methods to identify clinical staphylococci.
We also detected several S. aureus isolates that failed to react either in the coagulase test tube or in mannitol fermentation. These atypical isolates may represent another important issue in clinical staphylococcus identification. Coagulase-negative variants of S. aureus have been described by other authors (6). In a preliminary study, Mlynarczyk et al. (A. Mlynarczyk, G. Mlynarczyk, M. Luczak, and J. Jeljaszewicz. Abstr. 9th International Symposium on Staphylococci and Staphylococcal Infections, abstr. 21, 2000) described S. aureus strains with a negative reaction for coagulase in the conventional tube test, which accounted for about 20% of all MSSA and MRSA strains tested. The coagulase-negative S. aureus strains found in our study were readily identified by their ITS-PCR patterns, identical to those of typical S. aureus strains.
In conclusion, ITS-PCR proved to be a rapid and reliable identification method for staphylococci of human clinical origin, providing high reliability and reproducibility at low cost. Furthermore, the results obtained are easy to interpret, with no subjectivity in their analysis. The method is simple to implement and to perform, being also versatile, since the DNA prepared can be used for further characterization of the strains, such as searching for antimicrobial resistance genes.
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ACKNOWLEDGMENTS |
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This work was partially supported by contracts FCT 34872/99 and FCT 34842/99, Fundação para a Ciência e a Tecnologia (FCT), Portugal, and a grant from Project FCG/ITQB/HFA from Fundação Calouste Gulbenkian, Portugal awarded to H. de Lencastre. I. Couto and S. Pereira were supported by grants BPD/20187/99 and 023/BIC/2000 from FCT (Portugal), respectively. M. Miragaia received a research grant from Fundação Calouste Gulbenkian.
The help of Mónica Vaz in the preliminary characterization of some of the isolates is acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Microbiology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-8278. Fax: (212) 327-8688. E-mail: lencash{at}mail.rockefeller.edu.
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Journal of Clinical Microbiology, September 2001, p. 3099-3103, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3099-3103.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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