Biocontrol Science, 2007, Vol.12, No.4, 155-158 Note Evaluation of DuopathLegionella Kit for the Rapid Identification of Legionella Strains Isolated from Water Samples HIROAKI INOUE1•–, TOMOKO TAKAMA1, YUKIKO AGAWA1, JUNKO ONODERA1, TOMOKI ISHIMA1, KUNIO AGATA1, KEIKO SAITOH2, AND KATSUNORI HURUHATA3 1 Tsukuba Research Laboratories , Aquas Corporation, 4-4 Midorigahara, Tsukuba, lbaraki 300-2646, Research and Investigation Department, Building2 Manegement Education Center, 1-4-28, Mita, Minato, Tokyo 108-0073, School of Environmental Health, Azabu University, 3 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan Received 20 August, 2007/Accepted 18 October, 2007 Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a rapid and simple immunochromatographic assay kit for the identification of Legionella species. We evaluated the precision of the kit in identifying 100 strains of Legionella and 35 strains of non-Legionella bacteria isolated from cooling tower and bath water samples. Consequently, of all the Legionella strains tested, 99 strains were judged to be Legionella, and only one strain (Legionella busanensis) was judged to be non-Legionella. All of the 35 non-Legionella strains were judged to be non-Legionella. We therefore conclude that Duopath Legionella is a useful method for the rapid identification of Legionella. Key words: Identification/Immunochromatography/Legionella. Legionella species are gram-negative bacteria are required to obtain the final results of the test, be- ubiquitously found in various aquatic environments. If cause the growth of Legionella on the selective agar humans were to inhale aerosolized water from a plates is very slow. Therefore, the development of a source contaminated with Legionella, such as cooling rapid detection and identification procedure for tower or bath water, they could contract a severe Legionella by the culture method is desired. form of pneumonia called Legionnaires' disease Duopath Legionella (Merck KGaA, Darmstadt, (Horwitz and Silverstein, 1980). Therefore, the con- Germany) is an immunochromatographic assay for trol and management of Legionella contamination in the simultaneous identification of cultured Legionella water systems is very important, and the effective- pneumophila and Legionella species other than L. ness of these efforts must be monitored by screening pneumophila. This method, which is simple and rapid, for the presence of Legionella. The conventional cul- is able to identify a small colony (approximate colony ture method is the standard method (IS011731, diameter, 1 to 2 mm) of bacteria. Here, we evaluated 1998) for the detection of Legionella in water sys- the performance of Duopath Legionella by using iso- tems. However, approximately ten days (including lated strains from cooling tower and bath water. two to three days for the identification of Legionella) Between August and November 2006, 50 strains of Legionella from cooling tower water samples and 50 * Corresponding author. Tel:+81-29-847-6000, Fax:+81-29- strains of Legionella from bath water samples were 847-6080 collected. In addition, 18 strains of non-Legionella 156 H. INOUE ET AL. bacteria from cooling tower water samples and 17 Legionella species-specific primers (Miyamoto et al, strains of non-Legionella bacteria from bath water 1997). Only one strain (L. busanens/s) was judged to samples were collected. These bacterial strains were be non-Legionella (Table 1). Out of 88 strains of L. collected according to the standard method pneumophila, only one strain (L. pneumophila (IS011731, 1998). That is, collected water samples serogroup 5) was judged to be a Legionella species were concentrated 100-fold by centrifugation or filtra- other than L. pneumophila. Therefore, this strain was tion. The concentrated samples were pretreated with tested again and the same result was obtained . All acid buffer, and inoculated onto GVPC selective agar isolated non-Legionella bacteria (35 strains) were plates (Merck, Tokyo, Japan). The plates were incu- judged to be non-Legionella. bated at 37 •Ž for 6 to 8 days. From the GVPC selec- Currently, the genus Legionella contains 52 spe- tive agar plates, three colonies with characteristics of cies (Fields et al., 2002; Park et al., 2003; Scola et al., Legionella were selected for subculturing on BCYE a 2004; Kuroki et al, 2007); all species of Legionella agar (Edelstein, 1981) and blood agar (nutrient agar are thought to cause pneumonia. Therefore, assays containing 5 % horse blood) medium. Subcultures of for the detection of Legionella need to detect as many each colony on both types of agar media were incu- species of Legionella as possible. Helbig et al. bated at 37 •Ž for 2 days. Those colonies that grew (2006) reported that 38 (90.5 %) out of 42 different on BCYE a but failed to grow on blood agar medium species of Legionella gave positive results by were regarded as Legionella, and were subcultured Duopath Legionella, indicating that the method was again on BCYE a agar plates to obtain single colo- able to identify a wide range of Legionella species. nies. The species of the isolated Legionella strains Koide et al.(2007) reported that Duopath Legionella (total 100 strains) were identified by using the im- was not effective in the case of the direct detection of mune serum aggregation assay (Denka Seiken, Legionella from aquatic samples, because the sensi- Tokyo, Japan) and the DNA-DNA hybridization assay tivity of Duopath Legionella was very low. However, (Kyokuto Pharmaceutical Industrial, Tokyo, Japan). we focused on the specificity of Duopath Legionella Legionella strains that could not be identified by the to identify the cultured colonies on GVPC selective DNA-DNA hybridization assay were tested for homol- ogy of the DNA sequences. In addition, non- Legionella bacterial strains were isolated from GVPC selective agar plates. These strains were collected from separate samples. Duopath Legionella was used according to the manufacturer's instructions. That is, subcultured bac- terial cells on BCYE a agar plates were picked up with a disposable inoculation loop, and suspended in a 0.9 % NaCI solution containing 1 % Tween 20. After the addition of polymyxin B (Merck, Darmstadt , Germany), the suspension was incubated for 5 min at room temperature followed by 5 min in boiling water, and cooled to room temperature. The treated cell sus- pension was applied into the sample port of Duopath Legionella. Results of the tests L.pn (for L. pneumophila), Spec (for Legionella species) and control zones were read after 30 min (Fig. 1). Our results in this study are summarized in Table 1. FIG. 1. Duopath Legionella. Out of 100 isolated strains, 98 strains were identified The treated cell suspension (150 ,ƒÊl) is applied into by using the immune serum aggregation assay and the sample port of Duopath Legionella, and a distinct DNA-DNA hybridization assay; one strain, isolated red line appears in the each zone within 30 min . from cooling tower water, was identified as Legionella Results are positive for Legionella species if two dis- busanensis by using the homology of the 16S rDNA tinct red lines appear in the control zone and Spec 5' end partial sequence (490 bp). However, only one (Legionella species) zone. Results are positive for L. strain was not identified, and the identification of this pneumophila if three distinct red lines appear in the strain is now in progress. We expect that this strain is control zone, Spec zone and L.pn (L. pneumophila) zone. Results are negative for Legionella species if a a Legionella species because specific amplification distinct red line appears only in the control zone was observed in a polymerase chain reaction using . RAPID IDENTIFICATION OF LEGIONELLA 157 TABLE 1. Identification of isolated Legionella strains by Duopath Legionella. a Identified as Legionella species by Duopath Legionella Identified as L. pneumophila by Duopath Legionella This strain showed cross reaction with L. pneumophila serogroups 8 and 10. d Unidentified strain agar plate. In this study, we tested nine different spe- ACKNOWLEDGMENTS cies of Legionella including L. busanensis isolated from water samples, and clarified that of all the spe- This work was supported by the Ministry of Health, cies tested only L. busanensis was not detected as a Labour and Welfare of Japan under grant number (H18- Legionella species by Duopath Legionella. This fact Kenki-Ippan-008). was revealed in the current study because L. REFERENCES busanensis was not present in the evaluation by Helbig et al. In this study, Duopath Legionella was un- Fields, B. S., Benson, R. E., and Besser, R. E.(2002) Legionella and Legionnaires' disease: 25 years of investi- fortunately not able to detect only one strain of L. gation. Clin. Microbiol. Rev., 15, 506-526. pneumophila serogroup 5 as L. pneumophila. We Helbig, J. H., Luck, P, C., Kunz, B., and Bubert, A.(2006) considered that this negative result in the identifica- Evaluation of the Duopath Legionella lateral flow assay tion of L. pneumophila was not a serious problem, be- for identification of Legionella pneumophila and cause the strain was judged to be a Legionella Legionella species culture isolates. Appl. Environ. species. Therefore, this negative result will have little Microbiol., 72, 4489-4491. Horwitz, M. A. and Silverstein, S. C.(1980) Legionnaires' effect on the identification of Legionella species in the disease bacterium (Legionella pneumophila) multiplies conventional culture method. Duopath Legionella intercellularly in human monocytes. J. Clin. Investig., 66, could detect most of the tested strains, and therefore 441-450. we think that Duopath Legionella can be utilized for International Organization for Standardization (ISO). the rapid identification of Legionella. Consequently, (1998) Water quality Detection and enumeration of the use of Duopath Legionella is expected to reduce Legionella. IS011731. the time required for the detection of Legionella by Koide, M., Haranaga, S., Higa, F., Tateyama, M., Yamane, N., and Fujita, J.(2007) Comparative evaluation of approximately two to three days compared to the Duopath Legionella lateral flow assay against the con- conventional culture method.