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1 Department of Microbiology and Infectious Diseases, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438540, Japan
2 Clinical Laboratory Department, Toho University Omori Hospital, Japan
Correspondence
Yoshikazu Ishii
yoishii{at}med.toho-u.ac.jp
Received 6 July 2005
Accepted 23 November 2005
Abbreviations: MRSA, meticillin-resistant Staphylococcus aureus.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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A total of 271 S. aureus isolates were isolated by the Clinical Laboratory Department in Toho University Omori Hospital, Japan, from January to April 2002. These isolates, including 34 biochemically atypical isolates, were analysed using Vitek® 2 and its optional tests. Thirty-four (12·5 %) were found to be biochemically atypical, because they did not produce acid from mannitol salt agar or did not agglutinate in Staphaurex testing but were categorized as MRSA by PCR analysis and antibiotic susceptibility. Because of an increased frequency of isolation of physiologically atypical S. aureus and the need to identify them accurately, this collection of isolates was used to evaluate three automatic identification systems, AutoScan-4®, BD PhoenixTM and Vitek® 2.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. All atypical isolates were isolated at Toho University Omori Hospital between February and April 2002. Data from pulsed-field gel electrophoresis provided by the hospital infection control team indicated that the 34 S. aureus isolates used did not have a common origin (data not shown).
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Confirmation of S. aureus by species-specific PCR and detection of the mecA gene. DNA amplification of the 34 isolates was performed with colony direct PCR (Tsuchizaki et al., 2000). A small portion of a colony was picked up by a toothpick, transferred directly to 50 µl of PCR mixture containing 50 pmol of each oligonucleotide primer from the Sa442 set (Martineau et al., 1998), 25 µl SYBR® Green Master Mix (Applied Biosystems) and autoclaved MilliQ water. S. aureus FDA 209P and Staphylococcus epidermidis ATCC 14990 were used as positive and negative controls, respectively, for the PCR. The thermal cycling protocol was as follows: 5 min at 95 °C for hot start of DNA polymerase and initial denaturation followed by 40 cycles of two steps consisting of 1 s at 95 °C for denaturation and 55 °C for the annealing and extension steps. Real-time detection of the PCR product was performed on an ABI PRISM 7000 Sequencing Detection System (Applied Biosystems) by measuring the fluorescence signal. Specificity of the fluorescence signal was estimated by a denaturation protocol to compare with a theoretical Tm value of the PCR product after 40 cycles.
The mecA gene was used as the gold standard for detection of meticillin resistance by PCR assay. The specific primer set for mecA reported by Reischl et al. (2000) was used. PCR conditions used were as previously described (Martineau et al., 1998). S. aureus N315 (Hiramatsu et al., 1992) and S. aureus FDA 209P were used as positive and negative controls for the mecA gene, respectively.
Biochemical identification. Inocula for the following studies were prepared using a nephelometric device to adjust the turbidity to McFarland standard 0·5. S. aureus FDA 209P and S. epidermidis 14990 were used as positive and negative controls, respectively. Analysis of the results was based on the computerized reports from each identification system.
Identification with AutoScan-4® system. Preparation of the AutoScan Pos ID panel (Dade Behring), inoculum preparation, panel rehydration and inoculation, biochemical overlays (Pos ID only), incubation, reading of the panels and quality control were performed according to the manufacturer's instructions. Pos ID panels were read visually after 24 h incubation at 35 °C. The test reactions were read by the AutoScan-4® (Dade Behring) and the results were converted to compare with the AutoScan updated database.
Identification with Vitek® 2. The test panels (ID-GPC; bioMérieux) were automatically filled by a vacuum device, sealed and inserted into the Vitek® 2 reader-incubator module (bioMérieux) and subjected to a kinetic fluorescence measurement every 15 min. The results were interpreted by the ID-GPC database and final results were obtained automatically.
Identification with the PhoenixTM system. The PhoenixTM system (Becton Dickinson) was used according to the manufacturer's instructions with PMIC/ID14 panels (Becton Dickinson) for strain identification and oxacillin-susceptibility testing. Test suspensions were prepared from pure bacterial cultures grown on trypticase soy agar II with 5 % defibrinated sheep blood (Becton Dickinson). The ID suspension was inoculated within 30 min into the panel, which was then loaded into the instrument for incubation at 35 °C and continuous reading. The results were interpreted by the ID-GPC database and final results were obtained automatically.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Once an isolate has been positively identified, cases of disease can be treated appropriately, and other procedures such as patient isolation, decontamination of exposed areas and increased hygiene measures can take place. Thus, identification is crucial.
All 34 isolates in this study were confirmed as S. aureus by PCR using the Sa442 primer set. Free coagulase production was confirmed for all isolates by the tube method with rabbit plasma. Nine of 34 isolates did not aggregate in the Staphaurex test, 25 of 34 isolates did not produce acid from mannitol, 26 of 34 had haemolysin activity and only 30 of these clinical isolates produced yellow pigment (Table 1). In addition, these 34 strains were confirmed as MRSA (mecA gene positive) by PCR analysis. Antibiotic susceptibility testing also confirmed all isolates as MRSA by the PhoenixTM system.
Out of 34 isolates tested, a concordant identification to the species level was obtained by the PhoenixTM system, AutoScan-4® (Table 2) and genetic determination by PCR for all the isolates tested. On the other hand, only 16 (47·1 %) isolates were identified as S. aureus by Vitek® 2, with good or better confidence levels, without the use of supplementary tests such as Staphaurex and/or haemolysin activity and pigment of colony on sheep blood agar (Table 2). One strain was identified incorrectly as Staphylococcus chromogenes by the Vitek® 2 instrument (Table 1).
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Toho University Omori Hospital uses the Vitek® 2 system for identification of clinical isolates. This system had previously proved to provide accurate and acceptable identification and antibiotic susceptibility for Gram-positive cocci (Ligozzi et al., 2002). Recently, the frequency of isolation of S. aureus with atypical physiological characteristics has increased to approximately 12·5 % (data not shown) in Toho University Omori Hospital, and it is very difficult to identify atypical S. aureus by the Vitek® 2 system unless extra tests are used. Furthermore, there have been several reports on the limitations of this identification system in distinguishing staphylococcal species (Becker et al., 2004; Ben-Ami et al., 2005).
Several genes have been targeted for PCR analysis of S. aureus but among these genes, tst genes, eta and etb and staphylococcal enterotoxin genes are not always detected in S. aureus (Becker et al., 2003; Pinto et al., 2005). The nuc gene has been widely used for species-specific detection, although it has also been reported in Staphylococcus intermedius strains (Becker et al., 2005). Thus, the Sa442 DNA fragment was used to confirm S. aureus by PCR in the present study. Recently, Klaassen et al. (2003) reported that the Sa442 primer set did not work against the clinical isolate S. aureus 550226. They concluded that a number of S. aureus strains may have been misidentified in the past or the presence of S. aureus in clinical isolates may have been overlooked when identification was based solely on the Sa442 PCR assay. However, in our study, all 34 atypical S. aureus isolates were identified by PCR using this primer set.
Extra tests were required to confirm the identity of the strains when using the Vitek® 2 system: Staphaurex for membrane-bound coagulase and protein A, haemolysin activity and production of pigment on sheep blood agar plate. Staphaurex is a method commonly used for S. aureus identification, even though it only detects clumping factor and protein A. Staphaurex has also been reported as too insensitive for reliable detection of MRSA (Rappaport et al., 1988). We obtained negative results with Staphaurex for several isolates. Fortunately, isolates negative for clumping factor and/or protein A produced acid from mannitol, and all 34 isolates produced free coagulase, were identified by PCR and as oxacillin resistant by antibiotic susceptibility testing.
The semi-automatic identification system of AutoScan-4® conforms to the requirements of the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) and requires a longer incubation period than Vitek® 2 or PhoenixTM. However, it permits technologists to check biochemical reactions visually by observing the panels. We believe that this point is very important for clinical technologists, because some like to reconfirm the results of biochemical reactions by eye. The AutoScan-4® correctly identified the 34 S. aureus strains in this study without any extra tests.
The PhoenixTM system also correctly identified all S. aureus strains without extra testing within 6 h. This was faster than the 18 h needed by the AutoScan-4® system and the more than 24 h needed by the Vitek® 2 system when additional tests were necessary. The confidence levels of identification were above 90 % for AutoScan-4® and PhoenixTM. On the other hand, Vitek® 2 had low discrimination levels for 17 strains (Table 2).
It should be noted that molecular biological techniques, such as DNA sequencing (Becker et al., 2004), hybridization (Sogaard et al., 2005; Trindade et al., 2003) or the use of DNA microarray technology (Charbonnier et al., 2005), could provide a more accurate identification and classification tool, but such techniques are difficult to apply in a routine clinical laboratory. A rapid, conventional and automated identification method, based on phenotypic characters, is a more practical approach for daily clinical laboratory procedures.
In conclusion, the PhoenixTM system and AutoScan-4® could provide accurate information for the identification of S. aureus with atypical physiological characteristics without any extra tests. The merit of AutoScan-4® is that technologists can check biochemical reactions by observing the panels. This study shows that biochemically atypical S. aureus strains were not identified as S. aureus by Vitek® 2 unless extra tests were used. The PhoenixTM system identified all strains correctly within 6 h. Accordingly, this report suggests that PhoenixTM, a fully automatic system, can be used for rapid identification in the clinical laboratory.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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