Modification of reagents in the EnviroAmpTM kit to increase recovery of Legionella organisms in water

ROBIN K. OSHIRO Utiiversity of' CclljfOi-ilia, School qf Social Ecology, Irvirze, CA 9271 7-5/50, U. S.A.

TERESA PICONE Roche Moleculnr Systeiii.s, Alniiieclcl, CA 94501, U.S.A.

AND

BETTY H. OLSON~ Uilivei.~irv of C~rliforizi~, Srhool rf Socicrl Ecology, 11-vii~e, CA 92717-5150, U.S.A.

Received November 2, 1993

Revision received February 2 1, 1994

Accepted February 2 1, 1994

OSHIRO, R.K., PICONE, T., and OLSON, B.H. 1994. Modification of reagents in the ~ n v i r o ~ m ~ ~ ~ kit to increase recovery of Legioi~elln organisms in water. Can. J. Microbiol. 40: 495-499.

Organisms of the bacterial genus Legioilelln, comnlonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionella pneumonia, caused by Legioizelln pize~ri i~o~~l~i ln) and Pontiac fever (nonpneumonic legionellosis). EnviroAmpTb' Legioilell~r sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-Elmer Corp.) were developed for rapid detection of DNA from organisms of the genus Legioi~el l~~ and the species L. pize~riizo~~hila from environmental water samples. The kits are based on molecular techniques incorporating polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The manufacturer states that the EnviroAmpTh' Legioi7ellrr sample preparation, polymerase chain reaction amplification, and detection kits can detect approxin~ately 100 Legioiiellrr organisms/mL (I0 000 organisms1100 mL) in the original water sample. The sensitivity of the kits was increased to 0. I colony-forming unitslml (10 colony-forming units1100 mL), at least for cl~ltured organisms, by modifying the EnviroAmpr" Le~ioi7ellcr sample preparation kit protocol. Data obtained in this study indicated that sample volume could be increased from 100 to 1000 mL (in the absence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mL to increase the ability of the kit to detect lower numbers of Legioiielln spp. or L. /)ize~tit~ophilci per volume.

Key +vorrls: Legioi~ellcr, environment, water. EnviroAmpTM kit.

O ~ H I R O , R.K., PICONE, T., et OLSON, B.H. 1994. Modification of reagents in the ~ n v i r o ~ m ~ ~ ~ kit to increase recovery of Legioi7elln organisms in water. Can. J. Microbiol. 40 : 495-499.

Des me~nbres du genre bactCrien Legioilelln, communCment retrouvC dans les rCservoirs d'eau. ont CtC associt h la maladie du ltgionnaire ( pneun~onie h legionelle causCe par Legior1elln piie~rnio/)hila) et h la f ittvre de Pontiac ( ICgionellose nonpulmonaire). Des trousses de prCparation de l'tnchantillon pour Legioilella ~ n v i r o ~ ~ n ~ ~ ~ , d'amplification par la PCR et de dCtection ont CtC dCveloppCes par la firme Perkin-Elmer pour dCtecter rapide~nent le DNA de bactCries du genre Legioiiellrr et de I'espece L. pi~e~rii~o/)l~iln B partir d'Cchantillons d'eau de l'environnement. Les trousses utilisent des outils ~nolCculaires qui associent I'amplification par PCR, l'hybridation 5 un genre spici f iq~~e par buvardage en point inversC et des sondes spCcifiques. Le lnanufacturier affir~ne que les trousses EnviroAmpTM pour prkparation de I'Cnchantillon pour Legiorlelln, d'amplification par la PCR et de dktection peuvent dCtecter environ 100 ICgionelleslmL (10 000 bacttries/100 mL) dans un Cchantillon d'eau. La sensibilitC des trousses peut &tre augmentCe j. 0,l unitis formatrices de colonies1mL ( I 0 unitCs formatrices de colonies/lOO mL), du moins pour les bactCries cultivCes, en modifiant le protocole de la trousse ~ n v i r o ~ m ~ ~ ~ pour la preparation de I'Cchantillon. Les rtsultats obtenus dans cette Ctude indiquent que le volume de I'Cchantillon po~lrrait &tre augment6 de 100 h 1000 mL (en absence de substances pouvant interfkrer colnrne I'acide humique) et que le volume de DNA extrait poun-ait &tre rCduit de 2 5 0,5 mL, ce qui augmenterait la sensibilitC de la trousse pour des quantitis plus faibles de Legiorlella ou de L. pnerriilo/~hiln par volume d'eau.

Mots cl6.s : Legioilell~i, environnement, eau, trousse E n v i r o ~ r n p ~ ~ . [Traduit par la Rtdaction]

Members of the Legio~zellc/ceae family of bacteria are known to cause Legionnaires' disease and Pontiac fever (Fang et al. 1989). Since the initial identification of the type species in 1977, 40 specles and 52 serogroups have been characterized (Dennis et al. 1993; Thacker et al. 1992). Of these species, at least 18 have been linked to human respiratory disease (Fang et al. 1989).

Leglonell~ species are aquatic bacteria found in drinking water (Stout et al. 19920), hospital (Hart and Makin 1991) and residential water supplies (Stout et al. 1992b), lakes, and rivers

l ~ ~ ~ t h o r to whom all co~respondence should be addressed.

(Fliermans et al. 1981). In the environment Legiolzella species have been found in concentrations ranging from 28-31 colony- forming units (cfu)/L (Marrao et al. 1993) to lo7 cfu/L (Lee and West 1991). In water distribution systems Legiolzellaplzeu- mophila appears to be almost impossible to isolate. Colbourne and Trew (1986) have had some success using 10-L samples. How Legionella species are transported from untreated surface waters to hot water heaters where they amplify remains unclear. Thus, increasing the sensitivity of tests for waters with a low occurrence of legionellae, such as distribution systems, would greatly improve our understanding of this issue.

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496 CAN. J. MICROBIOL. VOL. 40, 1994

Auloclave , Filter , Add Filler Fillers and 100 mL lo 2 mL DNA 10 m ~ n

Filter Holders Water Extraction Sample Reagenl

Add 20 p L Add 1511L Add 40 )IL DNA Sample Denatural~on

to PCR Reacl~on Solul~on MIX Tube

Put Tubes in GeneAmp PCR

lnslrument Syslem

Place Tray l n z e

1 3 mL Hybr~d~za l~on Solut~on Gn2y$h 55'C. 2 50 pL Denatured Sample 20 mln

Label Slrips Place in Tray

1. Aspirate 2. ~ d d 3 m~ In* A* Add color lncubale [Ey--

Enzyme Con ugale 55-C, Wash Developmenl ~ ~ x m i n lo dach well 12 min Two limes l o Wash [ L n p o - + ''

1 . 550C Each Three Times 2. Room Temp with 10 mL H 2 0

FIG. I. EnviroAmpTM Legioilelltr sample preparation, PCR amplifica- tion, and detection kit protocol. Sample waters were filtered through previously autoclaved filters and filter holders. The filter was removed, vortexed for 30 s, and then boiled for 10 rnin with DNA extraction reagent. A 20-pL sample was PCR amplified with the appropriate reagents and primers, denatured, briefly centrifuged, and then used for the hybridization reaction.

Methods for the detection, isolation, and identification of Legiotlella species from nonclinical environmental samples have included selective media culture (Kusnetsov et al. 1993; Reinthaler et al. 1993; Steele et al. 1990; Meers et al. 1989; Roberts et al. 1987; Dutka and Walsh 1984; Rushkin et al. 1992), immunological techniques including use of monoclonal anti- bodies, antisera, or immunofluorescence (Pelaz et al. 1992; Ott et al. 1991; Makin and Hart 1989; Witherell et al. 1988; Brindle et al. 1987; States et al. 1987; Berubeet al. 1989; Bornstein et al. 1989), and a combination of these methods (Mamolen et al. 1993; M a l ~ a o et al. 1993; Bezanson et al. 1992; Marrie et al. 1992; Stout et al. 1992b; Yamamoto et al. 1992; Alary and Joly 199 1 ; Hedges and Roser 199 1 ; Verissirno et al. 199 1 ; Wilkinson et al. 1990; Payment et al. 1989; Barbaree et al. 1987). Other means of detection and identification for environmental samples have included intraperitoneal pig inoculation (Negron-Alvira et al. 1988; Fliermans et al. 1981) as well as genetic methods such as polymerase chain reaction (PCR) and probes (Koide et al. 1993; Bej et al. 1991; Mahbubani et al. 1990; Starnbach et al. 1989) and pulsed-field gel electrophoresis (Luck et al. 1991).

Recently, the legiolysin (Bender et al. 1991; Wintermeyer et al. 1991) and mip (Engleberg et al. 1989) genes have been successfully used for Legiotzella species differentiation. The nzip gene is conserved and found only in L. pne~~mophila, although tnip-like genes have been found in other Legiotzella species (Cianciotto et al. 1990) and mil;,-like proteins have been found in non-Legiotzella species (Lundemose et al. 1992). This gene, along with a genus-specific 5 s rRNA gene, has been adapted in a commercially available kit for the rapid detection of DNAfrom organisms of the genus Legionella and the species L. ptzeumo- phila, from environinental water samples. The EnviroAmpTM Legionella sample preparation, PCR amplification, and detec-

tion kits (Perkin-Elmer Corp., Norwalk, Conn.) utilize PCR amplification on concentrated water samples and a non- radioactive reverse dot blot hybridization.

When the recommended protocol is used, the detection sensitivity of this kit is 100 cfu/mL (100 000 cfu/L). This study was undertaken to increase the sensitivity of this method by altering various parameters of the kit protocol. In light of aquatic environmental concentrations reported in the literature for water distribution systems, such an approved protocol would greatly aid our understanding of the occurrence of Legionella species and the process by which they enter buildings and homes.

The kit protocol is illustrated in Fig. 1. HVLP filters (25 mm) were placed in Swinnex disc filter cartridge holders and autoclaved at 121°C (15 psi; 1 psi = 6.895 kPa) for 30 min, after which they were placed at the tip of 60-mL sterile syringes and attached to a vacuum manifold. Water samples were poured into the syringes and filtered. The membrane was aseptically placed in a tube containing DNA extraction reagent, which was then capped, vortexed for 30 s, and boiled 10-12 min. A 50-FL sample was removed and placed into a PCR reaction tube containing 65 p L of the kit-provided Legiotzella PCR reaction mix containing uracil N-glycosylase. The PCR amplification protocol was followed as specified in the kit instructions. Forty microlitres of denaturing solution (1.2 N NaOH, 0.11 M EDTA) was added to the PCR product, mixed, and then spun in a inicro- centrifuge for 5 s. Denatured PCR product (50 pL) was added to a well of the hybridization tray containing 3 mLof prewarmed kit-supplied hybridization solution and a Legiot7ella detection strip. The tray was covered (as in all further incubation steps) and incubated at 55 + 1°C for 20 + 1 min at 50-90 rpm. The hybridization solution was replaced with 3 mL of kit-supplied enzyme conjugate and incubated at 55 +- 1°C for 12 ? 1 min at 50 rpm (as in all further incubation steps). The conjugate was replaced with 10 mL of prewarmed kit-supplied wash solution and incubated at 55 +- 1°C for 10 ? 2 min. A 10-mLsecond wash solution at room temperature for 5 min was replaced by 10 mL of kit-supplied citrate buffer and incubated for 5 + 1 min. This was replaced by 5 mLof kit-supplied color development solution and incubated in the dark at room temperature for 30 + 2 min. The color developn~ent solution was replaced with 10 mL of distilled water and incubated at room temperature for 5 - 10 min in the dark. The strips were then removed from the tray, blotted dry, and photographed.

The Legionella test strips were interpreted using the following definitions. The "L" dot indicated Legiotzella (5s rRNA DNA sequence), "p" indicated L. pne~lmol~hila (mil;, gene), "-" was the negative control (a blue dot here indicating incorrect hybrid- ization), and "+" was the internal positive control (ablue dot here indicating proper PCR amplification, hybridization, and color development). The intemal positive control (+ dot) represented a final concentration of 10' Legionella organisms/mL. Compar- ison of the intensity of the Legionella genus (L) dot with the intemal positive control estimated the number of Legionella organisms/mL in the original water sample.

Changes in some of the parameters in the manufacturer's protocol were studied independently for their effect on sensitiv- ity, using the kit-provided L. pneutnophila control DNA or seeded Legionella dutnofl cells.

Legionella d~itnofJi (ATCC 33279, American Type Culture Collection, Rockville, Md.) cells were grown on buffered char- coal yeast extract medium (BBL Cockeysville, Md.) incubated at 35°C in a candle jar under humidified conditions for 3 days.

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NOTES 497

Appropriate inoculum concentrations for spiked experiments were determined by plate count of dilutions of cells obtained from the surface of plates and resuspended in sterile distilled water.

The following four aspects of the test kits were altered independently of any other kit parameter: sample water filtration volume, DNA extraction reagent volume, the number of PCR amplification cycles, and the volumes used for PCR detection in the hybridization step, including the PCR amplified denatured sample volume as well as the hybridization buffer volume.

Increasing the amount of filtered sample volume while leaving all other manufacturer-recommended parameters the same should increase the sensitivity of the kit by increasing the likelihood of achieving the lower kit detection limit (100 Legion- ella organisms/mL). This aspect of the kit was tested by increasing the sample volumes filtered through the HVLP kit filters from 100 mL (kit-recommended volume), to 200,500, and 1000 mL. Autoclaved sterile distilled water, autoclaved sterile tap water, and colored water known to contain high concentra- tion of humic substances were tested. Legionella pne~~mol>hila DNA was used as a positive control and Pseudonzonas j7~rorescens DNA was used as a negative control.

The next modification examined was reduction of the DNA extraction reagent (Chelex 100 in Tris-HC1 EDTA with sodium azide) volume to increase sample concentration. Volumes (100 mL) of a 20 cfu/mL suspension of Legionella organisms were filtered and processed using the kit-recommended 2.0-mL volume of DNA extraction reagent as well as 0.25-, 0 . 5 , 0 .75, and 1.0-mL volumes. All other reagent volumes were as specified by the manufacturer.

A third modification focused on the number of PCR am- plification cycles. The number of cycles was increased from the manufacturer-recommended 30 cycles to 32, 35, and 40.

The amount of amplified product DNA was also manipulated by increasing the volume of denatured PCR amplified product used in the hybridization from 50 pL to 75, 100, 125, 150, and 200 pL. This increases the amount of DNA target. Additionally, the volume of hybridization buffer used to dilute PCR amplified sample was decreased independently from 3.0 mL to 2.5, 2.0, 1.5, and 1.0 mL to increase the kit sensitivity.

The PCR product from each modification was validated by electrophoresis on a 4% agarose gel stained with 0.4 kg ethidium bromide/mL. The gel was run for 2 h at 200 V.

Greater sensitivity was achieved by increasing the sample volume. A 1000-mL volume of clean water was as easily filtered as the recommended 100 mL but with the colored water containing humic substances, the flow quickly slowed, reducing the maximum filterable sample volume to 300 mL (Fig. 2). The colored water samples were taken from a basin aquifer whose deeper levels were laid down on a peat bog (Bradford et al. 1994). In addition, both the tap water and colored water samples inhibited the test kit by eliminating the internal positive control dot (+), which is used as an indicator of proper PCR amplifica- tion, hybridization, and color development steps.

Reducing the amount of DNA extraction reagent had no effect on the kit sensitivity except at 0.25 mL (data not shown). This volume was insufficient to cover the filter surface, reducing bacterial removal. The minimum extraction solution volume needed for transfer to a microcentrifuge tube that allowed the Chelex 100 to settle was 0.5 mL. Tubes having a diameter of 15.9 mm require more than 0.5 mL because the liquid portion cannot be withdrawn without removing the bead portion of the

FIG. 2. Effect of increased sample volume size on EnviroAmpTM kit sensitivity. Strip interpretation: Ldot, Legiotzelln organisms (5s rRNA DNA sequence); p dot, L. pnerurlo~~hila (n~ip gene); -, negative control (a blue dot here being indicative of incorrect hybridization); t dot, internal positive control (a blue dot here being an indicator of proper PCR amplification, hybridization, and color development). The internal positive control represents a final concentration of l o 3 Legioizella organismsImL. Comparison of the intensity of the Legiorlella genus (L) dot with the internal positive control estimates the number of Legionella olganisms1mL in the original watersample. A 1000-mLquantity ofclean water was as easily filtered as the kit-recommended sample volume of 100 mL. Both tap water and colored water samples inhibited the kit, resulting in aloss of the internal positivecontrol indicator. Colored water samples containing humic substances quickly slowed the flow rate, resulting in n reduction of the maximum filterable sample volume to 300-400 mL. Strips: a, sterile distilled water ( a ] , 100 mL; 02, 1000 mL); b, tap water (b l , 100 mL; b2, 1000 mL); c, colored sample water (75 platinum color units; c l , 100 mL; c2, 300 mL); d, colored sample water (37 platinum color units; (11, 100 mL; ~12, 400 mL); e, negative control ( ~ s e ~ t d o n l o r ~ a s j1~1ore.rce11.r DNA);f, positivecontrol (L . pr~e~~rnol~hi la DNA).

Chelex 100. This was not a factor with microcentrifuge tubes or with larger extraction solution volumes. Reduction of the kit- recommended 2.0 mL of DNA extraction reagent to 0.5 rnL did not reduce sensitivity. Thus, using the lesser amount of DNA extraction reagent can increase the sensitivity of the kit because more DNA becomes available for PCR amplification.

PCR amplification modification as a method to increase sensitivity was ineffective. There was no difference in sensitivity when the number of cycles was increased from the kit- recommended 30 to 32. Increasing the number of cycles by 5 or 10 (to 35 or 40, respectively) resulted in increased DNA copy number detection (data not shown). However, this result was

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498 CAN. J. MICROBIOL. VOL. 40. 1994

concentrations as low as 0.1 cfu/mL (10 cfu/100 mL) in the spiked water sample (Fig. 3). Therefore, the EnviroAmpTM Legiorzelln kit could be used to detect Legio~zellu species or L. 111zerr1noplzila at concentrations reported for water distribution systems.

Alary, M., and Joly, J.R. 1991. Risk factors for contamination of domestic hot water systems by legionellae. Appl. Environ. Microbiol. 57: 2360-2367.

Barbaree, J.M., Gorman, G.W., Martin, W.T., Fields, B.S., and Moi-rill, W.E. 1987. Protocol for sampling environmental sites for legionellae. Appl. Environ. Microbiol. 53: 1454-1458.

Bej, A.K., Mahbubani, M.H., and Atlas, R.M. 199 1. Detection of viable Legiot~ella pt~ec~nlopl~ilu in water by polymerase chain reaction and gene probe methods. Appl. Environ. Microbiol. 57: 597-600.

Bender, L., Ott, M., Debes, A., Rdest, U.. Heesemann, J., and Hacker, J. 199 1. Distribution, expression, and long-range mapping of legiolysin gene (11~)-specific DNA sequences in legionellae. Infect. Immun. 59: 3333-3336.

Berube, A,, Trudel, M., and Payment, P. 1989. Rapid detection and identification of Legiortella prlenrriophilo by a membrane irnmuno- assay. Appl. Environ. Microbiol. 55: 1640-1 64 1.

Bezanson, G., Burbridge, S., Haldane, D., Yoell, C., and Marrie, T.

Frc. 3. Detection of 0.1 cfu/mL (10 cfu/100 mL) in L. cl~/rtrofii spiked water sample using EnviroAmpTM kit. Strips: cr, negative control. ster~le distilled water; 11, 0.03 cfu/mL; c, 0.3 cfu/mL; cl, 1.0 cfu/mL; e, I0 cfu/rnL; f, 100 cfu/mL; g, negative control, P.se~rc1orr1orla.s

jT~rorescer1.s; 11, L. prles~rr~opl~iln, lo3 DNA copies.

offset by false-positive negative controls (sterile distilled water containing no Legionella DNA). This limitation might be over- come by additional processing of the PCR master mix.

No change in sensitivity was 5een by either increasing the PCR amplified denatured sample volume or reducing the hybridiza- tion buffer volume (data not shown). The pH of the buffer was unchanged as sample volume was increased.

The Legionelln detection limit was found to be 100 DNA copies for both Legio~zella organisms and L. pnellr?zol2lzilu as indicated by the test strip. Gel electrophoresis clearly showed the 5 s rRNA gene band at the 100 DNA copy level, as well as the nzip gene band (indicative of L. p ~ z e a ~ n o l ~ h i l a ) . These bands became faint at the 10 DNAcopy number and were undetectable at 1 DNA copy number (data not shown). The internal positive control band was clearly visible at all copy numbers. However, using a 32P-labelled Southern blot technique in future studies may increase sensitivity at copy numbers of 1 or 10.

Increasing the sainple volume to 1000 mL and decreasing the DNA extraction reagent to 0.5 inL were found to be the two most effective changes that increased sensitivity. The combination of these two manipulations resulted in an increased volume of sample assayed (1000 vs. 100 mL) and a decreased amount of DNA extraction reagent (from 2.0 to 0.5 mL) so that the final outcome was a greater reduction of sample volume. Five 1000-mL sterile water samples were spiked with vaiying con- centrations of L. dllrnofl to achieve a range of 0.01 to 100 cfu/mL in 10-fold increments. A decreased amount of DNA extraction reagent was used (0.5 mL). Using the EnviroAmpTM kit- recommended protocol, L. d~~rnoffi was detected at concentra- tions as low as 100 cfu/mL in the spiked water sample. When the sainple volume was increased and the DNA extraction reagent volume was decreased, L. dunzofl was detected at

1992. Diverse populations of Legior~ellaprlerrrilo1)11iln present in the water of geographically clustered institutions served by the same water reservoir. J. Clin. Microbiol. 30: 570-576.

Bornstein, N., Marmet, D., Surgot, M., Nowicki, M., Arslan, A,, Esteve, J.. and Fleurette, J. 1989. Exposure to Legior~ellaceae at a hot spring spa: a prospective clinical and serological study. Epidemiol. Infect. 102: 31-36.

Bradford, S.M., Palnler, C.J., and Olson, B.H. 1994. Assimilable organic carbon concentrations in southern California surface and groundwater. Water Res. 28: 426-435.

Brindle, R.J.. Stannett, P.J., and Tobin, J. O'H. 1987. Legiorzella pr~e~~rrzopl~ila: non no clonal antibody typing of clinical and environ- mental samples. Epidem. Infect. 99: 235-239.

Cianciotto, N.P., Bangsborg. J.M.. Eisenstein, B.I., and Engleberg, N.C. 1990. Identification of t~ ip - l ike genes in the genus Legiortelln. Infect. Immun. 58: 29 12-29 18.

Colbourne, J.S., and Trew, R.M. 1986. Presence of Legiot~elln in London's water supplies. Isr. J. Med. Sci. 22: 633-639.

Dennis, P.J., Brenner, D.J., Thacker, W.L., Wait, R., Vesey, G., Steigerwalt, A.G., and Benson, R.F. 1993. Five new Legiorlella species isolated from water. Int. J. Syst. Bacterial. 43: 329-337.

Dutka, B. J., and Walsh, K. 1984. Incidence of Legiorlella organisms in selected Ontario (Canada) cities. Sci. Total Environ. 39: 237-249.

Engleberg, N.C., Carter, C., Weber, D.R., Cianciotto, N.P., and Eisenstein, B.I. 1989. DNA sequence of r~lip, a Legiorlella pt1errrno- pl~iln gene associated with macrophage infectivity. Infect. Immun. 57: 1263-1270.

Fang, G.D., Yu, V.L., and Vickers, R.M. 1989. Disease due to Legior~ellaceae (other than Legior~ella p~eroi~ol~hi la) : historical, microbiological, clinical, and epidemiological review. Medicine (Baltimore), 68: 1 16-1 32.

Fliermans, C.B., Cherry, W.B., Omison, L.H., Smith, S.J., Tison, D.L., and Pope, D.H. 198 1. Ecological distribution of Legiorzella pt~err7nophila. Appl. Environ. Microbiol. 41: 9-1 6.

Hart, C.A., and Makin, T. 1991. Legiot~ella in hospitals: a review. J. Hosp. Infect. A, 18: 48 1-489.

Hedges, L. J., and Roser, D. J. 199 1. Incidence of Legior~ella in the urban environment in Australia. Water Res. 25: 393-399.

Koide, M., Saito, A,, Kusano, N., and Higa, F. 1993. Detection of Legioraella spp. in cooling tower water by the polymerase chain reaction method. Appl. Environ. Microbiol. 59: 1943-1946.

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Kusnetsov, J.M., Martikainen, P. J., Jousimies-Somer, H.R., Vaisanen, M.-L.. Tulkki, A.I., Ahonen, H.E., and Nevalainen, A.I. 1993. Physical. chemical and microbiological water characteristics associated with the occurrence of Legiot~ellcr in cooling tower systems. Water Res. 27: 85-90.

Lee, J.V., and West, A.A. 1991. Survival and growth of Legionella species in the environment. J. Appl. Bacterial. 70(Symp. Suppl.): 121s-129s.

Luck, P.C., Bender, L., Ott, M., Helbig, J.H., and Hacker, J. 1991. Analysis of Legiot~ellaptie~rtizo~~hiln serogroup 6 strains isolated from a hospital warm water supply over n three-year period by using genomic long-range mapping techniques and monoclonal antibodies. Appl. Environ. Microbiol. 57: 3226-323 1.

Lundemose, A.G., Rouch, D.A., Birkelund, S., Christiansen, G., and Pearce, J.H. 1992. Chlc~t~iyclin trnchotlzntis Mip-like protein. Mol. Microbiol. 6: 2539-2548.

Mahbubani, M.H., Bej, A.K., Miller, R., Haff, L., DeCesare, J., and Atlas, R.M. 1990. Detection of Legiot7elln with polymerase chain reaction and gene probe methods. Mol. Cell. Probes, 4: 175-187.

Makin, T., and Hart, C.A. 1989. Detection of Legiotzelln p~7eu~7zop17iln in environmental water samples using a fluorescein conjugated monoclonal antibody. Epidem. Infect. 103: 105-1 12.

Manlolen, M., Breiman, R.F., Barbaree, J.M., Gunn, R.A., Stone, K.M., Spika, J.S., Dennis, D.T., Mao, S.H., and Vogt, R.L. 1993. Use of

., . . . . multiple molecular subtyping techniques to investigate a legionnaires' disease outbreak due to identical strains at two tourist lodges. J. Clin. Microbiol. 31: 2584-2588.

Mal~ao. G., Verissimo, A., Bowker, R.G., and Da Costa, M.S. 1993. Biofilms as major sources of Legiotielln spp. in hydrothermal areas and their dispersion into stream water. FEMS Microbiol. Ecol. 12: 25-33.

Marrie, T.J., Haldane, D., Bezanson, G., and Peppard, R. 1992. Each water outlet is a unique ecological niche for Legiotzelln~~tze~~tizophiln. Epidemiol. Infect. 108: 261-270.

Meers, P.D., Goh, K.T., and Lim, E.W. 1989. Legiotielln species, serogroups and subgroups found in the environment in Singapore. Ann. Acad. Med., Singapore, 18: 375-378.

Negron-Alvira, A., Ferez-Suarez, I., and Hazen, T.C. 1988. Legiotlella spp. in Puerto Rico cooling towers. Appl. Environ. Microbiol. 54: 2331-2334.

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