Clinical isolates and reference strains for cross-hybridization studies
A total of 102 clinical isolates and reference strains of various bacteria from American Type Culture Collection (ATCC, VA), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Germany), or Helsinki University Central Hospital Laboratory (HUSLAB, Finland) were used for the cross-hybridization comparisons. Bacteria were grown in cystine lactose-electrolyte-deficient (CLED), blood, or chocolate agar plates. Culturing was performed under aerobic or anaerobic conditions depending on the bacterial species. All strains were incubated at 37°C for at least for 24 hours.
Clinical isolates and reference strains of Staphyloccusspecies
The Staphylococcus samples comprised ATCC strains and clinical isolates that were characterized by conventional methods. A total of 18 CNS samples including S. capitis (ATCC27840), S. cohnii (ATCC29972), S. haemolyticus (one clinical isolate), S. hominis (ATCC25615, ATCC27844), S. lugdunensis (two clinical isolates), S. saprophyticus (two clinical isolates), S. warnerii (one clinical isolate, ATCC25614), S. xylosus (ATCC29971, ATCC35033), S. schleiferi (DSMZ4809), and S. epidermidis (two clinical isolates, ATCC14990, ATCC49134) were obtained for testing. Coagulase-positive staphylococcus S. intermedius (ATCC29663), S. aureus (four clinical isolates, ATCC29213), and MRSA were also included (three clinical isolates).
Clinical isolates and reference strains of Staphylococcus species were grown using the standard methodologies. Briefly, lyophilized bacterial strains were diluted by Luria-Bertani (LB) or tryptic soy broth. After dilution, nearly all bacterial species were grown on blood agar plates. The three exceptions were S. epidermidis ATCC14990 and S. capitis ATCC27840 that were both grown on tryptic soy agar plates, and S. epidermidis ATCC49134 that was grown on a nutrient agar plate. Culturing was performed under aerobic conditions with the exception of S. saprophyticus, which was grown under anaerobic conditions. All strains were incubated at 37°C for least 24 hours.
Blood samples were drawn into aerobic and anaerobic blood culture bottles (BacT/Alert®, bioMérieux, France) and were incubated in the blood culturing equipment BacT/ALERT 3 D (bioMérieux) for up to 5 or 6 days, at which time they were reported as negative when no sign of micro-organism growth was detected. If during the cultivation period possible growth was observed by the blood culturing instrument, it was identified and reported according to CLSI guidelines http://www.clsi.org in the Department of Bacteriology, HUSLAB (Finland). The cultivation took 1–3 days, with a further 1–2 days culture needed for the identification of pathogen from a positive blood culture. In total, 186 blood cultures were collected between May 2007 and June 2007. These were used as references to evaluate the performance and feasibility of the assay with that of standard routine diagnostic testing. Of these, 146 were blood culture positive and 40 were blood culture negative.
The susceptibility to oxacillin of the staphylococcal species was determined by disc diffusion according to CLSI guidelines, using Mueller-Hinton II agar base (cat no 212257, Becton, Dickinson and Company, USA) and antibiotic discs (Oxoid, UK), incubated at +35°C. Minimal inhibitory concentrations (MIC) values for oxacillin were determined by E-tests (Biodisk, Sweden) on Mueller-Hinton agar supplemented with 4 percent NaCl, and incubated at +30°C.
The extraction of DNA from clinical isolates and reference strains was carried out as follows: one bacterial colony was picked from the plate and suspended in 100 μl of PBS. After centrifugation (at 3000 rpm, for 3 minutes), the supernatant was discarded and the pellet was suspended in 100 μl of TE. Two heating steps of 95°C for five minutes were performed sequentially with a 2 minutes cooling step between them. Finally, the solution was centrifuged (at 13 000 rpm, for 10 minutes) and the supernatant containing DNA was collected. In the case of the blood culture samples, 100 μl of the samples were collected for DNA extraction. The DNA was extracted using an automated nucleic acid extraction instrument Nuclisens®easyMAG™ (bioMérieux, France) according to the manufacturer's protocol (Generic 1.0.6). The eluation volume was 55 μl. A negative control, i.e., sterile water was included in each test series.
Dna Amplification and Labelling
The broad-range PCR primers gBF (5'-CGICCIGGKATGTAYATHGG-3') and gBR (5'-RMICCWACICCRTGYAGICCICC-3') were modified from primers introduced by Roth and colleagues (2004) . We reduced the number of degenerated regions in primers by using inosines. The primers amplified a ~300 bp region of the bacterial gyrB and parE genes. In addition, specific primers for mecA gene, mecAR (5'-TTACTCATGCCATACATAAATGGATAGACG-3') and mecAF (5'-AATACAATCGCACATACATTAATA-3'), were designed. To enhance S. aureus amplification SaurF (5'-AGACCTGGTATGTATATTGG-3') and SaurR (5'-CCAACACCATGTAAACCACC-3') primers were further designed. All the reverse primers were biotinylated at their respective 5'-end.
The PCR reaction mixture contained 1 μM of gBF primer mixture (Metabion, Germany), 1 μM of biotin-labeled gBR primer mixture (Metabion, Germany), 0.165 μM of SaurF primer (Metabion, Germany), 0.165 μM of biotin-labeled SaurR primer (Metabion, Germany), 0.25 μM of mecAF primer (Metabion, Germany), 0.25 μM biotin-labeled mecAR primer (Metabion, Germany), 1× Hot Start Taq® PCR buffer (Qiagen, Germany), in which the final concentration MgCl2was 2.0 mM, 300 μM of each of dNTP (Finnzymes, Finland), 1.5 g/l BSA (EuroClone, Italy), 0.125 U/μl Hot Start Taq® DNA polymerase (Qiagen, Germany), 1.5 μl of isolated DNA, and water to bring the total volume to 15 μl. In the blood culture dataset, 1.5 μl of PCR control template was added in the reaction and the equivalent amount of water was reduced. A negative control, i.e., sterile water was included in each test series.
The PCR was performed using a Mastercycler® epgradient S thermal cycler (Eppendorf, Germany). The following PCR program was used: a denaturation step at 95°C for 15 minutes, 36 cycles of 10 seconds at 96°C, 35 seconds at 52°C, 10 seconds at 72°C, 5 cycles of 5 seconds at 96°C, 30 seconds at 65°C, 5 cycles of 5 seconds at 96°C and finally 30 seconds at 68°C. After the PCR, the success of the amplification of double-stranded DNA and single-stranded DNA was ascertained by gel electrophoresis using a 2% agarose gel containing SYBR® Green II (Invitrogen, USA) or using Agilent BioAnalyzer (Agilent Technologies, USA).
The microarray platform was an ArrayTube™ (Clondiag, Germany). The design of specific oligonucleotide probes were carried out according to the principles and methods described previously . One to three different species-specific oligonucleotide probes were selected for each target species. In total, 22 species-specific probes for 12 bacteria, 2 CNS-specific, and 4 mecA resistance marker specific probes (Metabion, Germany) were chosen for spotting on the microarray (Table 1). All oligonucleotide probes were spotted as duplicates on the array. Two different oligonucleotides per spot were used for the mecA probes. Position control oligonucleotides containing a biotin label were attached to the array for verifying the correct function of the hybridization reagents.
Hybridization and Scanning
The hybridization on microarray was performed as described previously  with only slight modifications. All incubation steps except that of the last precipitation reaction were performed under continuous agitation of 550 rpm at 25°C. Briefly, a first a prewash with 500 μl of water from 30 to 55°C for 5 to 10 minutes was done. Hybridization at 55°C for 10 minutes, of 1 μl of the biotinylated target and 99 μl hybridization buffer (250 mM Na2HPO4, 4.5% SDS, 1 mM EDTA, 1×SSC) took place on a microarray. When hybridization control oligonucleotides were included, they were added to the hybridization buffer. After hybridization, the microarray was washed in 500 μl of 0.2×SCC at 20°C for 5 minutes. Incubation with 100 μl of blocking buffer (2% milk powder, 6×SSPE, 0.005% Triton-X100) was performed for 5 minutes at 30°C. Then 100 μl of 1:5000 dilution of streptavidin-conjugated horseradish peroxidase in PBS was applied for 10 minutes at 30°C followed by a similar washing step as described above. Finally, 100 μl of 3, 3', 5, 5'-tetramethylbenzidine (TMB) analog (Seramun Grün; Seramun Diagnostica, Germany) was added for the precipitation reaction at 25°C for 10 minutes. Microarray images were generated by ATR-01 Reader (Clondiag).
The array images were analyzed with the Prove-it™ Advisor software (Mobidiag, Finland, http://www.mobidiag.com). The software performed image analyses and result reporting, including the identification of the bacterial targets and the evaluation of the control probes. This took place automatically without user involvement in adjusting any of the parameters. The target identifications were made by software using multiple parameters such as signals from the probe oligonucleotides on the array. These were interpreted using built-in rules and parameters specific for each assay type. All the probes for a specific bacterial target were required to be positive for that target to be classified as positively identified, except for the CNS probes of which only 2 of 4 specific oligonucleotides were required to be positive. If both CNS and S. epidermidis probes in the analyses were positive, only S. epidermidis was reported due to its identification by species-specific probes. The original array layout contained spots, which were not included in the final probe panel. Microarray data files have been deposited in NCBI's Gene Expression Omnibus database and are accessible through GEO Series accession number GSE17221.
Sequencing of CNS Samples
For sequencing of the CNS samples 16S_rRNA_F (5'-AGAGTTTGATCYTGGYTYAG-3')  and 16S_rRNA_R (5'CTTTACGCCCARTRAWTCCG-3')  were used as reported earlier. The primers amplified a ~550 bp region of the bacterial 16S rRNA genes. The PCR reaction mixture contained F and R primer mixture at a final concentration of 0.4 μM (Sigma, USA), 1× Hot Start Taq® PCR buffer (Qiagen, Germany), in which the final concentration of MgCl2 was 2.0 mM, 200 μM of each of dNTP (Finnzymes, Finland), 0.8 g/l BSA (EuroClone, Italy), 0.05 U/μl Hot Start Taq® DNA polymerase (Qiagen, Germany), 2.5 μl of isolated DNA, and water to bring total volume to 25 μl. The PCR was performed using a Mastercycler® epgradient S thermal cycler (Eppendorf, Germany). The PCR program was initialized by a 15 minute denaturation step at 95°C followed 36 cycles of 30 seconds at 95°C, 30 seconds at 54°C, and 30 seconds at 72°C. The PCR program ended with 10 minute step at 72°C. After the PCR, the success of the amplification of dsDNA was verified by gel electrophoresis using 2% agarose gel containing ethidiumbromide (Sigma, USA). The amplified PCR product was purified using the QIAquick® PCR purification Kit (250) (Qiagen, Germany) and a minimum of 50 ng of product was mixed with either the forward or reverse primer (0.42 μM). Water was added to bring the total volume up to 12 μl. Sequencing was performed using cycle sequencing with Big Dye Terminator kit (version 3.1) supplied by Applied Biosystems (ABI, CA, USA) and the reactions were run on ABI 3130xl capillary sequencer according to the manufacturer's instructions.
Sequences were edited and analyzed with the Vector NTI Advance™ (Invitrogen, USA) and BioEdit http://www.mbio.ncsu.edu/BioEdit/bioedit.html programs using the ClustalW alignment algorithm version 1.4 . We used the BLAST algorithm  to search for homologous sequences in the European Bioinformatics database and the National Center for Biotechnology Information database http://www.ebi.ac.uk/Tools blast.ncbi.nlm.nih.gov/Blast.cgi).
We compared the results and calculated the sensitivity, specificity, and confidence interval (CI) values according to CLSI guidelines (EP12-A2, User protocol for evaluation of qualitative test performance, http://www.clsi.org. Briefly, these analyses were performed using the following definitions: true-positive (TP), true-negative (TN), false-negative (FN), and false-positive (FP). The sensitivity was calculated as follows: TP/(TP+FN), and the specificity was calculated as TN/(TN+FP).