Received 27 January 1999/Returned for modification 5 June
1999/Accepted 11 July 1999
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INTRODUCTION |
Staphylococcus aureus is
the leading cause of nosocomial infection in the United States
(6). In New York City (NYC) methicillin-resistant S. aureus (MRSA) accounts for approximately 29% of these infections and 50% of associated deaths (20). Increasingly, S. aureus typing has become an important tool in the study of strain
origin, clonal relatedness, and the epidemiology of outbreaks. Typing
also plays an important role in hospital investigations (1),
as MRSA is now endemic or epidemic in many institutions
(17). Although several different phenotypic and, more
recently, molecular techniques are available for differentiating
S. aureus, no method is clearly superior under all
conditions. Currently, macrorestriction analysis by pulsed-field gel
electrophoresis (PFGE) is the standard at the Centers for Disease
Control and Prevention (CDC) for S. aureus strain typing and
has been used successfully to study strain dissemination, especially in
the identification of nosocomial outbreaks (3, 18, 19, 28).
However, while PFGE has excellent discriminatory power, it is
labor-intensive and difficult to standardize among different
laboratories (33). As with other gel-based typing systems,
the interpretation of PFGE results is often subjective (27).
These problems make the exchange of strain typing information difficult
and complicate the creation of an S. aureus and MRSA typing database.
DNA sequencing is a powerful approach to strain typing with advantages
in speed, unambiguous data interpretation, and simplicity of
large-scale database creation. These advantages have been described by
Maiden et al. (13), who developed the multilocus sequence typing (MLST) approach. This technique combines sequence information from several housekeeping genes to compare strains in a manner similar
to multilocus enzyme electrophoresis (MLEE) (22). Recently, DNA sequencing of the polymorphic X, or short sequence repeat (SSR),
region (32) of the protein A gene (spa) has been
proposed as an alternative to current techniques for the typing of
S. aureus (8). The polymorphic X region (Fig.
1) consists of a variable number of 24-bp
repeats and is located immediately upstream of the region encoding the
C-terminal cell wall attachment sequence (9, 21, 29). The
diversity of the SSR region seems to arise from deletion and
duplication of the repetitive units and also by point mutation
(4). While the biological function is not known, the protein
A domain encoded by the X region may serve to extend the N-terminal
immunoglobulin G binding portion of the protein through the cell wall
(34). The existence of well-conserved regions flanking the X
region coding sequence in spa allows the use of primers for
PCR amplification and direct sequence typing (Fig. 1). The sequencing
of the spa SSR region combines many of the advantages of a
sequencing-based system such as MLST but may be more rapid and
convenient for outbreak investigation in the hospital setting since
spa typing involves a single locus. Inasmuch as the protein
A X region has a high degree of polymorphism, it may have a variation
rate (or clock speed) that provides suitable discrimination for
outbreak investigation.
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FIG. 1.
Protein A gene map. Boxes indicate segments of the gene
coding for the signal sequence (S), the immunoglobulin G-binding
regions (A-D), a region homologous to A-D (E), and the COOH terminus
(X), which includes the SSRs (Xr) and the cell wall
attachment sequence (Xc). Primers are numbered from the 5'
end of the primer on the forward strand of S. aureus
(GenBank accession no. J01786 [29]).
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Despite the potential of spa typing, the clinical and
epidemiological validity of protein A polymorphism analysis has not been clearly established (31, 32). The present study was
undertaken to evaluate spa typing based on typeability,
discriminatory power, reproducibility, ease of interpretation, and ease
of use (14, 27). Fifty-nine isolates from the CDC were used
to compare spa typing to a broad range of techniques in
terms of their abilities to correctly group outbreak-related strains of
S. aureus. In addition, we compared the utility of
spa typing with that of two current molecular techniques for
the identification of major MRSA clusters and specifically the clone
I:A:A (type determined by restriction fragment length polymorphism
(RFLP) typing with mecA and Tn554 combined with
PFGE [mecA/Tn554/PFGE type]) in a larger study
of MRSA in NYC hospitals (18). spa typing
displayed a high degree of reproducibility and typeability and adequate
resolving power and was simple to use and interpret.
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MATERIALS AND METHODS |
Bacterial strains.
The three hundred and twenty MRSA and
methicillin-susceptible S. aureus isolates used in this
study for the validation of spa typing were obtained from
two previously characterized collections (26).
(i) CDC collection.
Fifty-nine strains were from a CDC
collection previously analyzed by several typing techniques (24,
27, 30). Typing results and key features of these isolates are
presented in Table 1 (adapted from
Tenover et al. [27]). This collection included 29 isolates from four well-documented outbreaks (I-IV), 30 epidemiologically unlinked isolates, and one Staphylococcus
intermedius isolate (Table 1). Isolates are divided into three
sets, SA, SB, and SC (26).
S. aureus ATCC 12600 (SA-4, SB-7, SC-3) was included as a
control in all groups. Group SA contains nine isolates from two nursing
homes (labeled NH1 and NH2) that did not have a clear epidemiological
link but were characterized as belonging to a pseudo-outbreak
(26). Seven additional isolates from seven states and
S. intermedius isolate ATCC 49052 (SA-16) were included in the SA group and are labeled NO. Among the SA isolates, SA-1 and SA-9
are duplicates as are SA-2 and SA-15. Strains SA-12, SA-18, and SA-20
all have the same bacteriophage type but are epidemiologically unlinked.
SB contains eight unrelated isolates labeled NO and isolates obtained
during two outbreaks, identified as I and II. Strains SB-3, -5, -10, -12, -15, -19, and -20 from outbreak I were obtained from the Iowa
Veterans Affairs Medical Center. Outbreak II strains (SB-2, -4, -6, and
-11) were isolated from a contaminated anesthetic.
SC contains isolates from outbreaks III and IV, strain SC-3 (ATCC
12600), and an unrelated control (SC-8) labeled NO. Outbreak III
strains were from the Sepulveda Veterans Affairs Medical Center, Sepulveda, Calif. Outbreak IV strains were from an anesthetic contamination unrelated to outbreak II. Strains SC-17 and -20 were
duplicates included as internal controls.
(ii) NYC collection.
Two hundred and sixty-one isolates were
from a consecutive single-patient MRSA study and were collected over a
6-month period from 12 NYC hospitals (18). These isolates
had been typed by our laboratory previously via Southern blot
hybridization with the two gene probes mecA and
Tn554 following ClaI digestion (18). Isolates were also analyzed by macrorestriction analysis using PFGE of
SmaI-digested chromosomal DNA. Both RFLP and PFGE were performed as described previously (5, 12). Five major clones were identified and assigned the codes I:A:A, I:D:C, V:NH:E, IV:M:B and
I:E:F (mecA/Tn554/PFGE type).
spa sequencing.
Amplification and sequencing of
the SSR region of the spa gene were performed with
chromosomal DNA purified from each isolate as a template
(18); several rapid techniques proved sufficient (26). Primer sites for PCR amplification were designed
according to published sequence data (29) (Fig. 1).
PCR amplification of the SSR region of the spa gene was
accomplished by adding 1 µl of a 1:200 dilution of genomic DNA and 24 µl of water to 25 µl of PCR master mixture in 0.2-ml PCR tubes (Perkin-Elmer Applied Biosystems Division [PE-ABI]). Master mixture buffer contains 0.5 µM PA 1095F forward PCR primer, 0.5 µM PA 1517R
reverse primer, 2.5 U of AmpliTaq Gold DNA polymerase, 2 mM
MgCl2, 350 µM total dNTPs, and 25 mM KCl. A negative
control (sterile deionized water) and a positive control (from our
laboratory's S. aureus collection) were included. Tubes
were capped and placed in a GeneAmp PCR System 9600 Thermocycler
(PE-ABI). Thermal cycling parameters included an initial 10 min at
95°C; 30 cycles of 30 s at 95°C, 30 s at 60°C, and
45 s at 72°C; and a final extension at 72°C for 10 min. PCR
products were purified and concentrated twofold prior to sequencing in
distilled water with a Microcon-100 (Millipore) microconcentrator.
Completed reaction mixtures were stored at 
20°C.
DNA cycle sequencing reaction mixtures had a total volume of 20 µl,
and reactions were carried out with a GeneAmp PCR System 9600 with the
following reaction conditions: 13 µl of AmpliTaq FS Dye Terminator
ready reaction mixture and sequencing primer (PA 1095F and PA 1517R), 3 µl of purified PCR product, and 4 µl of deionized water. The cycle
sequencing profile consisted of a 96°C denaturation step for 10 s followed by an annealing/extension step starting at 65°C and
decreasing 1°C every six cycles until a touchdown temperature of
55°C was reached, for a total of 66 cycles. Dye terminator cycle
sequencing reaction products were purified with a Sephadex column
(Pharmacia) and a Silent Screen filter plate (Nalge Nunc). The column
filtrate was evaporated in a vacuum centrifuge and resuspended in 2 µl of blue dextran-EDTA-deionized formamide (1:5). Sequences were
determined by electrophoresis with the ABI PRISM 377 DNA sequencer.
Consensus sequences were assembled from both orientations with PE-ABI software.
Identification and classification of spa types.
Since the major source of variation in the X region seems to be
duplication or deletion of the repetitive units, strain lineages cannot
be obtained by comparing the sequences with an algorithm based on
sequence alignment. This precludes the use of a dendrogram to visually
represent typing results because dendrograms rely on sequence
alignments. Therefore, we attempted to establish strain relatedness by
first identifying all possible variations of the repeat units and then
assessing how these repeat units were organized in the X regions of the
different isolates. The program FINDPATTERNS from the Genetics Computer
Group (GCG) Wisconsin Package 9.1 was used to identify repeat units
matching the ambiguous patterns AAAGAAGAXXXXAAXAAX {1,4} CCXXXX
and GAGGAAGAXXXXAAXAAX {1,4} CCXXXX. This strategy allowed the
identification of all SSRs contained in the spa regions
analyzed from all isolates. A customized Perl script (NEWREPEATS;
M. Bergman) was used to identify, from the output of FINDPATTERNS,
unique repeat sequences that were assigned a type based on a random
alphabetical code (Fig. 2A). Another Perl
script (CLEANFP; M. Bergman) was used to assign, to each of the
spa regions analyzed, a spa repeat code based on
the order of SSR codes as defined by the output of NEWREPEATS. The
spa repeat code, therefore, represents the structure of the
SSR region of the S. aureus isolates studied (Tables 2 and
6). All unique spa repeat codes were assigned a random
numerical code, or spa type, for identification.
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FIG. 2.
DNA (A) and amino acid (B) sequences of individual
spa repeats and their letter codes based on variation from a
DNA consensus sequence. Repeats were obtained by a GCG FINDPATTERNS
search with the ambiguous search patterns AAAGAAGAXXXXAAXAAX {1,4}
CCXXXX and GAGGAAGAXXXXAAXAAX {1,4} CCXXXX.
Identical residues are identified by dashes, and gaps are identified by
tildes.
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In this paper, isolates are considered members of the same clone based
on identical spa sequences (spa types). The
relatively large number of isolates (n = 261) from the
NYC collection were also grouped based on similarity of the patterns of
repeat sequences at both the DNA and amino acid levels (Table 6). This
association is based on the assumption that accumulated point mutations
are consistent between strains (4) and that, as such,
similar repeat patterns may be an indication of genetic relatedness and
a more recent common ancestor.
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RESULTS |
spa typing results.
The spa SSR regions
of 320 isolates from two strain collections was sequenced. The analysis
of the sequences resulted in the identification of 24 unique SSR types
that were assigned letter codes (as suggested by Frenay et al.
[8]). In the two collections analyzed in this study,
repeat types were 24 bp long with the exception of one (repeat I1),
which was 27 bp. The analysis of the SSR regions from additional
S. aureus strains from our laboratory's collections
(unpublished data) allowed the identification of 13 new unique SSRs.
The combined list of 37 unique SSRs and their amino acid conversion are
presented in Fig. 2A and B, respectively. Most nucleotide changes in
the repeats result in synonymous mutations, indicating evolutionary
pressure to conserve amino acid sequence. Therefore, positive Darwinian
selection does not appear to be driving sequence diversity, as has been
described for other highly polymorphic loci (25). This may
account for the observation that, while variable enough to provide
adequate strain discrimination, this region has the stability to group
related strains for use as a typing tool (this study).
The organization of the repeats in the spa SSR region from
each of the isolates was represented as a spa type repeat
code that was used as a typing criterion. Thirty-three different
spa (strain) types were defined. The strain types and their
frequencies in both the CDC and NYC collections appear in Tables 2 and
5, respectively. In the 59-strain CDC collection there were 22 unique short repeats that defined 13 different strain types (Table
2). spa types 21 and 52 have
the same repeat organization but have sequence alterations in the
normally conserved adjacent region and are assigned a unique type. In
the 260-isolate NYC collection, there were 20 repeat types and 24 different strain types (Table 6). From all strains studied, SSR regions
composed of 4, 5, 7, 8, 9, 10, and 11 repeats were characterized by PCR
products that ranged from 250 to 637 bp in length. All SSRs regardless
of length were successfully sequenced and assembled. The length of the
variable region was not an accurate indicator of strain type, as many
had the same number of repeats (for example, 11 unique strain types had
eight repeat elements). Type 2, the most common spa type, had 10 repeats and a 556-bp PCR product. Strain types 2, 7, 17, and 21 were present in both collections.
Typeability and reproducibility.
All 320 isolates of S. aureus were typeable by spa sequencing. As indicated in
Table 3, the 100% typeability of
resistant and susceptible isolates of the 59-strain CDC collection by
spa sequencing is an advantage over phage typing, plasmid
typing, insertion sequence mapping, and most of the RFLP methods. The reproducibility of spa typing was tested by including
duplicate isolates twice in set SA (SA-1 and SA-9 and SA-2 and SA-15)
and in set SC (SC-17 and SC-20) of the CDC collection. In addition, strain ATCC 12600 was included in all three sets. There were no variations in the results for the duplicate isolates by spa
sequencing, which is not the case for 5 of the 13 other methods
including PFGE, (Table 1) (18).
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TABLE 3.
spa typing of CDC collection: typed, subtyped,
and nontypeable isolates by set and number of isolates correctly
identified and misclassified by each method from four
outbreaks (n = 29)a
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The in vitro and in vivo stability of spa typing results was
investigated previously (8). We independently confirmed in vitro stability by examining the effects of multiple passages on blood
agar plates. Ten different strains were subcultured on a daily basis
for 6 weeks. All 10 strains had unique spa types that ranged
in size from 7 to 11 repeats with virtually all repeat groups (defined
by organization of repeat sequence code) represented. No changes in
either PFGE pattern or spa type were seen (data not shown).
Previously, in vivo stability was assessed by using three isolates
collected over a 5-year period in a chronic MRSA carrier cystic
fibrosis patient. The spa type remained the same over this
time period (8).
Discriminatory power with the CDC collection.
Many of the
pseudo-outbreak strains of the SA group had an identical spa
type (type 2). It has been suggested by Smeltzer et al. (24)
that this may represent the correct grouping of related strains
(similarly, Smeltzer et al. found a unique type for pseudo-outbreak
strain SA-14, in agreement with spa typing). Three strains,
SA-12, SA-18, and SA-20, that were included in the SA group are known
to be epidemiologically unrelated but have a common phage type.
spa typing was more sensitive than phage typing and was able
to classify SA-18 as different. Immunotyping, MLEE, field inversion gel
electrophoresis (FIGE) and plasmid restriction analysis were able to
distinguish all three. All other typing methods were unable to discern
differences between these strains. This observation is in agreement
with the previous results of Frenay et al. (8) that
spa typing has sufficient stability to group strains in
concordance with phage typing but with greater discrimination.
In the SB group, outbreak II strains SB-2, -4, and -6 had an identical
type (type 52). This did not include outbreak II strain SB-11 (type
21), which was therefore misclassified. It has been noted that strains
SB-2, -4, and -6 came from the same patient and that 8 of 13 other
techniques distinguished SB-11 as different (24). All
outbreak I strains were correctly grouped together as type 2, but four
strains (SB-1, -14, -16, and -18) that were originally judged to be
unrelated to this outbreak based on epidemiological information were
also assigned this type. This result is in agreement with the typing of
Smeltzer et al. (24), who have also shown that strains SB-1
and -16 may be related to each other and outbreak I strains. In the SC
study group, without exception, strains from outbreak III were grouped
together as type 7. Outbreak IV strains were grouped as type 57 with
the exception of strain SC-16 (type 2), which was also incorrectly
classified by immunotyping, HindIII ribotyping, and
MLEE. In light of the epidemiological link between SC-16 and outbreak
IV strains (25), this appears to be the only unambiguous
example of a failure of spa typing to group strains appropriately.
A summary of the total number of types, subtypes, and nontypeable
strains identified by each method and of the number of isolates correctly identified and misclassified by each typing system is given
in Table 3. The information in this table is derived from the 29 strains that were epidemiologically linked to four different outbreaks
(I-IV) from strain sets SB and SC from the CDC collection. SB and SC
also contained isolates that were not outbreak related and that
therefore were omitted from Table 3. Similarly, SA strains were not
included since an outbreak was not clearly established for these isolates.
spa typing was able to group 27 of the 29 outbreak strains
into four clusters corresponding to the outbreaks (outbreaks I-IV corresponded to spa types 2, 52, 7, and 57, respectively).
Four additional strains from sets SB and SC that were unrelated, based on epidemiological information, to these outbreaks had the same spa type and are deemed misclassified. Therefore, the
sensitivity and specificity of this method compare favorably with those
of current techniques (Table 3) (average: 25 correct and 5 misclassified; PFGE: 28 correct and 7 misclassified).
Discriminatory power with the NYC collection.
Table
4 describes the ability of the three
different molecular methods to differentiate the NYC isolates.
spa typing identified 24 unique strain types. The
combination of mecA and Tn554 hybridization patterns with PFGE subtype and spa type produced a total of
107 types for the 261 isolates. There were 39 mecA/Tn554/PFGE types, and there were 53 types
when spa typing was added. Similarly, there were 21 mecA/Tn554 types and 47 mecA/Tn554/spa types. PFGE subtyping provides the
greatest discrimination of the four methods, describing 80 subtypes.
Typing with spa appears to provide discrimination similar to
that of mecA/Tn554 polymorphism. However,
spa typing was able to provide a clonal assessment for the
isolates (Table 5) that was previously
provided only with by mecA/Tn554/PFGE information
(18). All five major clonal groups in the NYC collection were identified by spa typing with greater than 95%
agreement for 196 isolates (Table 5; mecA/Tn554/PFGE) types I:A:A,
I:D:C, I:E:F, IV:M:B, and V:NH:E correspond to spa types 2, 16, 4, 3, and 7, respectively). spa types for both major and
minor clones (all other isolates) of the NYC collection are presented
in Table 6. The minor clones represent 67 strains or 25% of the NYC study total (n = 261). The
minor clones consist of isolates that did not display a prevalent
clonal type (less than 10 isolates with one PFGE/hybridization pattern,
including a few isolates that did not hybridize with the
mecA probe) and that were categorized separately from the
major clones in the NYC study (18). Interestingly, the 20 spa repeat patterns seen among the minor clones are the same
as or similar to those of the five major clonal groups, indicating a
relatedness which would otherwise have been overlooked by PFGE and
hybridization alone.
In addition to clonal assessment, by analyzing the pattern of repeats,
spa typing allowed the grouping of NYC strains that have
similar repeat organizations and therefore may have a relatively recent
common ancestor. This grouping (Table 6; types labeled A-C) is more
subjective than the strict and simple criteria used for clonal
assessment (based on identical spa SSR sequence as indicated
in Materials and Methods). However, by doing so we observed three major
repeat pattern groups for all but 10 NYC strains (additional sequence
information from coagulase repeat regions confirms these groupings
[unpublished data]. Of the six repeat types that do not fall into the
three repeat similarity groups, four of them (types 21, 37, 15, and 22)
are unique among strains that do not hybridize with the mecA
gene, which confers methicillin resistance (MRSA phenotype). The
apparent limited variation of repeat patterns (see A, B, and C groups
in Table 6) for the MRSA strains may be a reflection of their clonal
origin (2, 10, 11, 15).
Ease of use and interpretation.
The typing systems used to
analyze the CDC collection have been previously assessed for ease of
use and interpretation (27). We compared the ease of use and
interpretation of spa typing with that of the molecular
techniques of RFLP and PFGE by using the isolates from the NYC
collection (18). PFGE demanded the use of special
electrophoretic equipment and the preparation of DNA in agarose disks,
which required delicate handling to provide the distinct banding
patterns necessary for analysis. Similarly, a fair amount of expertise
and time for analysis was required for the interpretation of the
complex patterns produced by PFGE and the application of grouping
criteria to relate them along clonal lines. The original analysis of
the NYC strains was a collaborative effort at two institutions (Public
Health Research Institute and Rockefeller University) (18).
We found that it was necessary to standardize virtually all reaction
conditions and apparatuses to permit a unified interpretation of
results. Interpretation of PFGE also required the standardization of
parameters for the computer software (Bioimage Whole Band Analyzer)
used to analyze and compare banding patterns. The complex PFGE
patterns, doublet bands, and partial digests also made the
interpretation of results moderately subjective. RFLP typing with the
gene probes mecA and Tn554 required separate
electrophoretic equipment in addition to a transfer apparatus and
labeling products for the probes. The patterns were more easily
interpreted than PFGE patterns and were compared visually. The amount
of time required to obtain, analyze, and compare typing information for
the original 270 strains by RFLP and PFGE was approximately 1 year.
spa typing required DNA preparation (possible by several
rapid techniques) for PCR amplification and sequencing with a PE-ABD
TC9600 sequencer. The identification of unique sequences required
computer software from PE-ABD (sequence assembly) and GCG (analysis of
repeats). The interpretation of repeats for assessing strain types is
unambiguous because only isolates with the identical repeat region
sequences are considered clonal, allowing rapid identification of
related and unrelated strains. Sequencing and analysis of all strains in this study were accomplished in approximately 3 weeks. Cost analysis
of spa sequencing was not performed and may vary depending on the apparatus and reagents chosen. However, in light of rapid improvements in automated sequencing technology, the cost (less than
$10 per sample) will likely continue to decrease.
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DISCUSSION |
S. aureus is the most frequent cause of
hospital-acquired infection in the United States (6). A
number of different phenotypic and, recently, genotypic techniques are
available to classify strains for epidemiological investigation in the
detection and tracking of nosocomial outbreaks (27, 28). The
goal of this study was to evaluate a new typing system for S. aureus based on the DNA sequencing of the variable region of
protein A as an alternative to current techniques for use in research
and clinical applications. Sequencing this variable region has been
described as a typing tool by Frenay et al. (8) but has not
been rigorously compared to current methods. As previously suggested,
the availability of well-described collections such as those in this
study can be used to establish the value of novel typing tools
(30).
We found that spa typing compared favorably to other
techniques and was able to identify and differentiate 27 of 29 epidemiologically related strains and misclassified only 4 unrelated
strains in the four outbreaks of the CDC collection (27).
Significantly, spa typing exhibited only one unambiguous
discrepancy with epidemiological information. All strains included as
internal controls were accurately identified. spa typing was
able to distinguish the five major clones of a 261-strain NYC hospital
MRSA collection originally described by a combination of PFGE and RFLP
analysis (18). This included the correct clustering (14 of
14 with no misclassifications) of I/E/F isolates recently reported as
the first outbreak of the "Iberian clone" in the United States
(19). The observation that spa typing can group
isolates in congruence with other methods in the two collections
directly addresses concerns over the instability of this region for use
in epidemiological studies (31).
For the 320 isolates in this study, there were 24 repeats, which were
organized to describe a total of 33 different strain types. Unambiguous
spa types were achieved for all isolates. The ability of
spa typing to discriminate strains was similar to that of
Southern blot hybridization with the gene probes mecA and
Tn554 for the NYC study isolates. While spa
sequencing does not have the resolving power of PFGE subtyping, it has
several advantages in terms of speed, ease of use, ease of
interpretation, and database creation. Significantly, spa
typing also provides clonal groupings that RFLP and PFGE techniques
cannot individually identify. This is accomplished without the use of
subtypes, which are difficult to clearly define and which introduce a
high degree of subjectivity that affects reproducibility among
laboratories. The difficulties we encountered in coordinating PFGE
typing of the NYC strains between two laboratories confirm the
conclusions of a recent study of intercenter PFGE typing
reproducibility, which stated that due to variability and bias, true
standardization may never be achieved in this system (33).
The cost of sequencing also compares favorably to that of techniques
such as PFGE.
The main advantage of spa typing over current methods may be
the unambiguity and portability of sequence data. This greatly simplifies the sharing of information between laboratories and facilitates the creation of a large-scale database for the study of
global as well as local epidemiology (the electronic portability of
sequence data allows rapid exchange of strain typing information without having to transfer bacterial strains). This is especially important in light of recent observations that MRSA outbreak strains from intercontinental sources have been documented within and among
hospitals (19). Sequencing can facilitate the creation of an
Internet Web site for the downloading of spa typing
sequences, which could then be analyzed by software available at the
site and added to a database. Such advantages have been ascribed to other systems, such as MLST (13), which was recently used to describe Streptococcus pneumoniae strains (7,
23).
The requirements for sequence typing are the ability to perform PCR and
access to an automated sequencer, both of which are increasingly
available to clinical laboratories and public health facilities
worldwide. An additional advantage of spa typing is that
adequate typing information is obtained from a single locus, as opposed
to MLST, which requires the combination of allelic information from
many genes (7) (seven loci for S. pneumoniae). This is because spa typing utilizes a single hypervariable
SSR locus as opposed to the several housekeeping genes used in MLST and
MLEE. Interpretation of the sequence information from spa sequencing does not require sophisticated algorithms and utilizes readily available sequence analysis software (GCG Wisconsin Package 9.1) that allows the description of strain types by a simple number code and alphabetical repeat designation. Thus, spa typing
lends itself to use in a wide range of laboratories as well as the
clinical environment.
While MLST provides information on strain lineage that is important for
research, this may not be relevant from a clinical point of view, where
the main goal is to rapidly identify if an outbreak is occurring. Even
so, it is possible that groupings based on spa repeat
sequence similarity could reflect chromosomal relationships (Table 6)
and therefore allow the strain lineage to be inferred. To validate this
hypothesis, we will assess whether spa repeat types (either
alone or in combination with other alleles) can be accurately compared
to the described S. aureus population genetic framework
(unpublished data) characterized by MLEE (15, 16). A
temporally and geographically diverse collection of worldwide MRSA
isolates and a comparison group of MRSA and methicillin-susceptible S. aureus isolates that represent a wide breadth of
genetically diverse S. aureus strains previously analyzed by
MLEE will be sequenced. For S. aureus, identification of
lineages may be simplified by the clonal nature of MRSA, which could
limit the diversity of chromosomal backgrounds seen in clinical
isolates (2, 10, 11, 15). In this way, guidelines to define
an S. aureus strain type and assign a clonal grouping for an
isolate with the use of the protein A repeat region alone may be
established (as suggested by Maiden et al. (13), not all
MLST genes may be necessary). Thus, in addition to its use for outbreak
investigation, spa typing may prove useful as a practical
method for describing a natural population of S. aureus
organisms. This may aid in the identification of strains that have
special virulence properties or drug resistance since in many bacteria
these are believed to be nonrandomly distributed along clonal lines
(16).
In summary, we have evaluated spa typing by comparing it to
several currently utilized techniques for the ability to differentiate well-defined collections of S. aureus strains.
Spa typing appears to have significant advantages over many
existing techniques in terms of speed, ease of use, ease of
interpretation, standardization, and data management and dissemination.
As mentioned by Tenover et al. (27), no single typing method
appears to be clearly superior in all cases. However, the current
ability of spa typing to distinguish both molecularly and
epidemiologically linked strains rapidly and easily makes it
particularly well suited for the initial screening that may be used to
identify and direct epidemiological studies.
We thank Mark Bergman (PHRI) for computer programming, Harriet
Marasco (PHRI) for help in preparing the manuscript, and Nicole Ellis
(PE) for her technical support.
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Abstract
Introduction
