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Reliable probe-based microbial typing systems are not yet commonplace in microbiological practice (1, 4, 7, 8, 12). In the past we identified domains that are differentially present within the staphylococcal genome on the basis of randomly amplified polymorphic DNA analysis. These probes were used to develop a DNA probe-based typing approach. The strain-specific DNA probes provide a simple binary output and have been presented before (13-15). We describe here the development of a new format for the binary typing technique. In the newly described procedure DNA is extracted from overnight Staphylococcus aureus cultures, labeled with the digoxigenin (DIG)-universal linkage system (ULS) (9), and reversibly hybridized to strips containing the immobilized probes. Signal is generated by chromogenic staining, and binary types can be read visually. This novel binary typing system will be introduced, and its versatility will be discussed.

Well-characterized strains of S. aureus (n = 20) were obtained from a reference collection (10). Isolates were cultured onto blood agar plates at least once, and single colonies were used for further testing.

DNA was isolated (i) by the protocol described by Boom et al. (3), (ii) with a miniprep of bacterial genomic DNA with a cetyltrimethylammonium bromide-NaCl solution (2), (iii) by extraction with phenol-chloroform (5), and (iv) by proteinase K-sodium dodecyl sulfate (SDS)-treatment and boiling. Another method (method v) consisted of simple DNA isolation by 10 min of boiling only. An alkaline method (method vi) for DNA extraction was used as well. Finally, a method (method vii) used the Wizard genomic DNA purification kit. The extraction of staphylococcal DNA was performed according to the manufacturer's instructions. The DNA size distribution and the concentrations of all preparations were estimated by gel electrophoresis and were compared to those of a reference series of bacteriophage  DNA (5). DNA was stored at 20°C for labeling and hybridization experiments.

For the optimization of DNA labeling, purified S. aureus DNA was labeled by using three different ratios (micrograms of DNA versus units of ULS), namely, 1:1, 1:2, and 1:4 (digoxigenin-ULYSIS nucleotide labeling kit; KREATECH Diagnostics, Amsterdam, The Netherlands). A serial dilution of labeled DNA from all strains (1 µl) was spotted onto a membrane to check the labeling efficiency. The procedures for the generation, validation, and application of the 12 strain-specific DNA probes have been described in detail elsewhere (13-15). Purified DNA probes (n = 12) were spotted (1 µl) onto a membrane strip (5 by 80 mm; Hybond N⁺; Amersham Life Science, Little Chalfont, United Kingdom). For hybridization quality control, 10 ng of petunia DNA (LMC 1322) and 0.25 ng of the S. aureus amplified nuc gene (6) were spotted as negative and positive hybridization controls, respectively. The DNA probes were denatured by 0.5 M NaOH-1.5 M NaCl treatment for 15 min at room temperature. Subsequently, the strips were neutralized with 0.5 M Tris-HCl-1.5 M NaCl (pH 7.4) for 15 min at room temperature (RT). The strips were washed with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate (5), and the denatured DNA was cross-linked on the strip with UV light (280 nm). Each strip was transferred into a Greiner tube (15 ml), and 1 ml of DIG Easy Hyb buffer (Roche Molecular Biochemicals, Almere, The Netherlands) was added. The labeled DNA was denatured for 5 min at 100°C and quenched on ice. The sample was centrifuged to collect condensate, and 1 ml of DIG Easy Hyb buffer was added. The labeled DNA was then added to the hybridization mixture, and the mixture was incubated overnight at 42°C in a rotation oven. After hybridization, the strips were washed twice in 2× SSC-0.1% SDS for 5 min each time at RT and twice in 0.5× SSC-0.1% SDS for 15 min each time at 68°C. The strips were processed by the protocol accompanying the Roche Molecular Biochemicals hybridization kit. After posthybridization wash steps, the strips were equilibrated in 1× maleic buffer for 1 min. The strips were blocked for 30 to 60 min at RT with 1× blocking buffer under slight agitation. The strips were incubated with conjugate (anti-DIG alkaline phosphatase conjugate, 150 mU/ml) for 10 min at RT. The strips were washed twice for 15 min each time in 1× wash buffer at RT and were equilibrated twice for 5 min each time in detection buffer. Finally, the strips were incubated in freshly prepared color substrate (nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate ready-to-use tablets; Roche Molecular Biochemicals) for a maximum of 3 h at RT in the dark. Hybridization of labeled genomic DNA with each of the 12 different DNA probes (probes AW-1 through AW-12) was scored with a 1 or a 0 according to the presence or absence of the hybridization signal, respectively. The combination of hybridization results forms the binary type.

The current conditions for the binary typing procedure are summarized below and are outlined in Fig. 1. Genomic staphylococcal DNA was purified with the Wizard genomic DNA purification kit and labeled with DIG-ULS (target DNA versus label at a ratio of 1:1). A concentration of 200 ng of labeled DNA per ml of hybridization buffer was used.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Schematic diagram showing the novel binary typing protocol. hyb., hybridization.

The hybridization results for the S. aureus strain collection, determined by the currently developed reversed hybridization procedure, are depicted in Fig. 2 and are specified in Table 1. Strains 1 to 5 (Fig. 2, lanes 1 to 5, respectively) showed identical binary codes. Strains 6 to 10 (Fig. 2, lanes 6 to 10, respectively), representing genetically related strains, displayed binary patterns that varied for no more than 2 of the 12 probes. Of the 10 unique strains (Fig. 2, lanes 11 to 20, respectively), all except strains 18 and 19 exhibited unique binary codes.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   Binary typing results for the methicillin-resistant S. aureus strain collection obtained after hybridization on the strip-immobilized DNA probe panel (probes AW-1 through AW-15).

Here we present a new format of binary typing as an example of a single-species typing test developed for the characterization of S. aureus strains. In previous studies, we defined the resolution, reproducibility, and stability of the epidemiological markers of the binary typing technique (11, 13-15). Technically speaking, the initial method, which involves repeated hybridization of the probe to digested staphylococcal DNA, was complex and time-consuming. We therefore developed a simple and fast format for the characterization of S. aureus strains based on reversed hybridization with 12 strip-immobilized DNA probes. The major advantage of ULS labeling for this application is the direct labeling of culture-amplified total genomic S. aureus DNA (target labeling). The additional value of binary typing is its simplicity, speed, reproducibility, and lack of need for expensive peripheral equipment. The efficiency of the labeling reaction with DIG-ULS (9) depends on the time, the temperature, the label-to-DNA ratio, and the purity of the DNA. A simple and standardized procedure resulted in optimal labeling and hybridization results. Hybridization conditions and probe and target concentrations were optimized.

The data obtained by the novel binary typing protocol are identical to those that were obtained by the coventional binary typing method. Epidemiologically linked strains were again identified as clusters, whereas unique isolates were well differentiated. The reversed hybridization data are corroborated by those obtained by other typing techniques such as pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis (Table 1).

The new binary typing protocol described here provides a simple and fast probe-based molecular typing strategy for the characterization of S. aureus strains and generates easily interpretable results, which can be compiled in a database. This culture-amplified nucleic acid probe technology can be developed for the characterization of other species and can be easily expanded with probes that directly detect genes associated with virulence factors and resistance determinants. It may be concluded that this technique comprises a compact, user-friendly S. aureus typing system suitable for use in the development of an international database for both methicillin-susceptible and methicillin-resistant S. aureus strains. In addition, the binary typing system has the clear potential to be useful in peripheral laboratories as well. The interlaboratory reproducibility of the assay is currently the subject of a multicenter study.