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Nosocomial Staphylococcus aureus infections require rapid identification and source recognition so that further spread, particularly of multiply resistant or unusually pathogenic strains, can be controlled. A number of methods to identify and distinguish bacterial strains have been developed. More-traditional phenotypic characterization has been gradually replaced by DNA sequence-based technology. PCR amplification has been particularly useful for strain typing in a clinical setting because it is rapid, relatively easy to automate, and less labor-intensive (14) than methods such as pulsed-field gel electrophoresis or Southern blotting (12). As DNA sequencing technology becomes increasingly more available in a clinical setting, nucleotide-level analysis is likely to be used more often, perhaps in combination with PCR fingerprinting, for strain identification.

A strategy currently used for strain typing of S. aureus relies on the amplification of defined tandemly repeated DNA sequences, often within surface-associated proteins, that are present in a variable number of copies in different strain isolates (for reviews, see references 7 and 13). This technique usually requires the analysis of several marker loci to differentiate strains that may by chance alone be identical at one locus.

Here, we describe a DNA sequence that is highly variable among S. aureus strains and that is found in many copies throughout the genome. An analysis of PCR products whose sizes may differ by only a few nucleotides and/or nucleotide sequence analysis at several loci would allow the rapid identification and detailed differentiation of clinical isolates.

Methods. The primers listed in Fig. 1A lie within the uvrA and hprK coding regions and were used to amplify the uvrA-hprK intergenic sequence for sizing as well as subcloning and sequencing. The primers shown in Fig. 1B lie just downstream of the icaC and geh coding sequences and were used for fragment sizing. The following two primers within the coding region of each gene were used to amplify a slightly larger fragment from each strain for subcloning and sequencing: SA18, 5'-GTATAGGTGTCGGCATGATATTGCGTG; SA19, 5'-T GAAAACTGAAAAGCTGACTGATACTAAGC. Primers were obtained from MWG-Biotech (Ebersberg, Germany). Fragments were amplified using Taq (AGS, Heidelberg, Germany) or Vent (New England Biolabs GmbH, Schwalbach, Germany) polymerase and annealing temperatures of 62 (uvrA-hprK) or 52°C (icaC-geh). PCR products were blunt-end ligated into vector pUC18 using the SureClone ligation kit (Amersham Pharmacia Biotech, Braunschweig, Germany) and sequenced on both strands by MWG-Biotech, with the following exceptions. The uvrA-hprK intergenic sequence from strain 601055 (Zeneca Pharmaceuticals strain collection) was obtained using Pathoseq S. aureus contig sequence information available from Incyte Pharmaceuticals (Palo Alto, Calif.) and contained on contig Sau1c0627. The icaC-geh intergenic sequence from strain ATCC 35556 was previously published by our group and was not resequenced for this work (2). Computer sequence analysis was performed using Multalin, version 3.5.5 (1), with manual adjustments. Preliminary S. aureus sequence data were obtained from The Institute for Genomic Research at http://www.tigr.org and the S. aureus Genome Sequencing Project (University of Oklahoma) at http://www.genome.ou.edu. Southern blots were hybridized and washed (0.1× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate]-0.1% sodium dodecyl sulfate) at 55°C and detected using the DIG DNA labeling and detection kit from Boehringer (Mannheim, Germany).

View larger version (53K): [in this window] [in a new window]   FIG. 1.   Intraspecies diversity in STAR element size. The intergenic sequences between uvrA-hprK (A) and icaC-geh (B) in 12 S. aureus strains, listed below each agarose gel, were amplified by PCR. The fragments were grouped into size classes (I to VI and I to V, respectively), and a representative from each class was sequenced. A schematic representation of the chromosomal organization in each region is shown beneath each agarose gel. The primers used for PCR amplification are listed at the bottom.

Identification of the STAR element. A comparison of the hprK sequence from Staphylococcus xylosus (3a) with the S. aureus sequence available from a commercial provider (Incyte Pharmaceuticals) identified a noncoding intervening sequence in the region between uvrA and the hprK promoter that was not present in S. xylosus. The sequence contained two large and several smaller direct repeats that were unusually GC rich, including several copies of the sequence GGGGCCCC. The presence of multiple copies of this sequence, which contains the recognition site for restriction enzyme ApaI (GGGCC^C), in close proximity is highly improbable in these AT-rich organisms.

Size variability. Initial sequence comparisons revealed that the intergenic regions between icaC and geh in strain ATCC 35556 and strain U500 in the database were not identical, the latter being smaller by a 58-bp directly repeated sequence. This led to a size analysis of the icaC-geh intergenic sequences by PCR amplification of a number of different S. aureus strains, which revealed that the sequence showed significant size variability. A similar PCR analysis, which also showed size variability among strains, was carried out at the uvrA-hprK locus. Figure 1A shows six different size classes ranging from 573 to 1,127 bp among representative strains in which the intergenic region between uvrA and hprK was examined. Five size classes (380 to 577 bp) were discernible among the same strains when the intergenic region between icaC and geh was investigated (Fig. 1B). In addition, a sixth size class (520 bp) is represented by the icaC-geh intergenic sequence of strain U500 (9), not shown here.

Sequence comparison. The sizes of each sequenced fragment and the corresponding STAR element are listed in Table 1, along with the database nomenclature. A sequence comparison of the STAR elements, including part of each intergenic region, is shown in Fig. 2. The alignment is structured around the largest STAR element, star01, from the uvrA-hprK intergenic region of strain 601055. Figure 3 graphically depicts the structure of the star01 sequence, including the sequences flanking the STAR element.

View larger version (73K): [in this window] [in a new window]   FIG. 2.   Comparison of sequenced STAR elements. Presented is an alignment of STAR sequences, showing the core element sequence (capital letters) and a short flanking sequence (lowercase letters) for each sequence, with the exception of the hprK region of strain 12601, which contains no detectable STAR element. The numbering refers to star01 (601055) and corresponds to bases 345 to 838 of the entire sequenced region included in the database entry. The beginning (start) and end of the element are indicated. Five (A to E) 14-bp signature sequence direct repeats are found in star01, and subsets are found in the other sequences (boldface underlining). A possible 8-bp recombination site that could account for the deletion of the "beginning" of the element in star08 and star09 is indicated for the strains in which it is present (lightface underlining).

View larger version (4K): [in this window] [in a new window]   FIG. 3.   Graphic representation of the star01 sequence structure. The end and beginning of the uvrA and hprK coding regions are indicated with an arrow and an arrowhead, respectively. The portion of the sequence containing the STAR element is marked. Grey boxes, directly repeated 14-bp signature sequence; black box, extended signature/end sequence; white rectangles, two copies of a 22-bp sequence, the second of which is the beginning of the element(s). The Tn557 attachment site (att) is marked. Black bars, inverted repeats, including the uvrA transcription terminator (t).

Abundance of the STAR element in S. aureus. Due to the presence of two STAR elements in different and apparently unrelated chromosomal locations, it seemed reasonable to assume that there were more STAR elements elsewhere in the S. aureus genome. Chromosomal DNA from the strains shown in Fig. 1 was digested with restriction enzyme HindIII, blotted, and hybridized with the uvrA-hprK intergenic region from strain 601005, the largest STAR sequence from the two loci we investigated in detail. We expected that a single hybridizing band would be specific for the fragment containing the uvrA-hprK region used as the probe and that any other bands would represent other STAR elements. The Southern blot shown in Fig. 4 reveals 13 to 21 cross-hybridizing bands, depending on the strain. The number of cross-hybridizing bands may be an underestimate, however, because each band could contain more than one STAR element, but it is clear that the STAR element is present in tens but not thousands of copies in the S. aureus genome.

View larger version (81K): [in this window] [in a new window]   FIG. 4.   Abundance of STAR element sequences in S. aureus. Chromosomal DNA from 12 S. aureus strains was digested with restriction enzyme HindIII and hybridized with a labeled probe corresponding to the sequence between the uvrA and hprK primers listed in Fig. 1A in strain 601055. Lanes 1 to 12 correspond, respectively, to the strains listed in Fig. 1B. Size marker (M), lambda/HindIII DNA.

Presence and abundance of the STAR element in other staphylococci. Cross-species staphylococcal DNA hybridization was also performed, using the same S. aureus uvrA-hprK STAR element as a probe. No signal was detected with DNA from S. carnosus, S. hyicus, S. saprophyticus, S. simulans, or S. xylosus; however, cross-hybridization with the STAR probe was observed with DNA from S. epidermidis (6 bands), S. haemolyticus (16 bands), S. hominis (1 band), S. intermedius (1 band), S. lentus (18 bands), S. sciuri (10 bands), and S. warneri (15 bands) (data not shown).

Concluding remarks. With the ever-expanding number of microbial genome sequences available, it is likely that more small multicopy sequences will be identified. The Bacillus subtilis genome project has produced one such 190-bp sequence, present in 10 nonidentical copies in the strain sequenced (8). The genome of Mycobacterium leprae also contains 28 copies of a sequence that shows strain-to-strain variability (3, 15).

Nucleotide sequence accession numbers. The sequences determined in this study have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence data libraries under the accession numbers listed in Table 1.