valster ea asm legion ella 2006

8/3/2019 Valster Ea ASM Legion Ella 2006

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DETECTION A ND IDENTIFICATIONOF FREE-LIVING PROTOZOA PRESENT

IN DRINKING WATER

Rinske W lster,Bavt W ullings, Stga n L%ost, Geo Bakker,Hauke Smidt, and Dick vand e r Koog

Free-living protozoa, e.g., Acanthamoeba, Nae-gleria, Saccamoeba, Hartmannella, and Vxillijra,serve as hosts for Legionella to proliferate innatura1 and man-made freshwater environ-ments. These protozoa multiply on biofilms,and their grazing behavior is influenced bythe composition and density of the biofilm(3). Protozoa not only provide nutrients forthe intracellular growth of L. pneumophila, butalso form a shelter when environmental con-ditions become unfavorable. Defining effec-tive measures to prevent proliferation of L.pneumophila in tap water installations requires asolid understanding of biotic as well as abioticfactors affecting the persistence and multipli-cation of this organism, including the presenceand behavior of host protozoa. However, in-formation about presence and identi ty of bac-terial free-living protozoa in such installationsis still scarce.The objective of this study was tooptimize and apply cultivation-independentmethods to obtain information about the pres-ence and identity of free-living protozoa in

river water (RW, reated water ( T m , and tapwater (Tap) in The Netherlands. To ourknowledge, this is the first description of pro-tozoal diversity in freshwater by using molecu-lar techniques-

WATER TYPES AND DNA ISOLATIONThe examined water samples consist of twoRW samples (temperature [q = 13OC), fiveT W samples (T = 10 to 15OC;TW 1 andTW 3were prepared h-om aerobic groundwater,TW 2 andTW 5 were prepared fiom anaerobicgroundwater, and TVCT 4 was prepared h m ur-f x e water), and three Tap samples (T = 16to 21C;Tap 1 was taken afier 15 min flush-ing,and Tap 2 and Tap 3 were taken from stagnantwater). One to two liters ofTW andTap and 20mi of RW were filtered through a 1.2-ym-pore-size, 55-mm diameter RTTP IsoporeMernbrane (Millipore, The Netherlands) in avacuum not: exceeding 0.3 bar. DNA was ex-tracted h-om the fdters using the FastDNA SPINKit for Soil (Q-Bio-gene, France).

PCR AN D T-RFLP ANALYSISRinske kilster, Bart Wullings,Stefan Voost, and Dick van der KoogKiwa N.V. Water Research, 3430 BB Nieuwegein, The PCR was performed with a GeneAmp PCRNetherlands. Hauke Smidt Laboratory of ~i cro b~ ol og y, System 9700 (Applied Biosystems) in a reac-Wageningen University, Hessehnk van Suchtelenweg 4,6703 tion (50 containing 10 tem-CT Wageningen, The Netherlands Ge o Bakker VitensWatertechnology, Oude Veerweg 1 , 8019 BE Zwolle, The plate DNA, each primer at a

Netherlands 0.5 FM, a 0.2 mM concentration of each

Legionella State ofthe Art 30Years 4 t e r Itr RecognitionEdited by Nicholas P Cianciotto ei al02006 ASM Press,Washington, D C

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deoxynucleoside triphosphate, 1.5 rnM MgCl,,2.5 U of %q DNA polymerase (Invitrogen,TheNetherlands). 18s rRNA gene fkagrnents wereamplified with the universal eukaryotic primers(3'FAM-labeled) Eukla-f (6) and Euk516-r (1).Arnplification conditions were as follows: pre-heating at 94OC for 130 s, 35 cycles of denatura-tion at 94OC for 30 s, annealing at 56OC for 45s, and extension at 72OC for 130 s; and a termi-nal extension at 72OC for 7 min. Fluorescentlylabeled P CR products (45 p1) were purified byusing the DNA Clean & Concentrator-5 Kit(Zyrno Research, Calif.) and redissolved in 20p1 of DNA-hee water. The digestion reactionmixture (20 pl) contained 5U of HhaI, 2 p1 ofbuffer C, 12.5 p1 of water and 5 p1 of the PCRproduct and was incubated at 37OC for 6 h.Themixture was cleaned and redissolved in 15 p1 of

DNA-h-ee water.The restriction digest product (5 pl) was

mixed with 15 p1 of loading buffer (15 p1 ofHi-Di formamide and 1 p1 of GS-500 ROX[Applied Biosystems] as the internal standard)Th e injection time was 5 s for analysis of andthe run time was 35 min.The fluorescently la-beled T-RFs were analyzed by electrophoresison an AB 1 PRHSM 310 genetic analyzer (Ap-plied Biosysten~s) n Genescan mode. T-RFswere entered int0 a genomic fingerprint analy-sis program, Bionumerics v. 3.5 (Applied Maths,Belgium), and hagrnent sizes were calculated.Banding patteriis were compared using a den-sitometric curve-based method that evaluatesthe position of the bands to generate pairwisesimilarity scores (Pearson coefficient) that weresubsequently used for cluster analysis.

CONSTRUCTION AN D ANALYSISOF CLONE L,IBRARIES18s rRNA genes were amplified by theabove- described PC R conditions, except thatnonlabeled Eukla primer was used.The PCRproducts were purified and cloned int0 Es-

cherichia coli JM109 by using the PromegapGEM-T easy Vectors system (Promega,United Kingdom). PCR was performed oncel1 lysates of ampicillin-resistant transfor-mants by using pGEM-T specific primers T7

and Sp6. Sequence analysis was done by Base-Clear Lab services (Leiden,The Netherlands).Th e primers used for sequencing the 18 srRNA gene fragment were Eukla andEuk516, and sequences were compared to se-quences in the National Center for Biotech-nology Information (NCB1)-database byBLAST searches.

PCR and terminal restriction fragmentlength polymorphism (T-RFLP) analysis ofclones were performed as described above, ex-cept that the digestion products were 10-folddiluted and the injection time was 1 s at the T-RFLP-assays.

DIVERSITY AN D IDEN TITYOF EUKARYOTIC ORGANISMSThe T-RFLP analysis showed a high eukary-

otic diversity in all water samples (Fig. 1). Thehighest diversity was observed inTW 4,T W 5,and Tap 1, while the lowest diversity was ob-served in RW 2,T W 1, andTap 3. Clones mostclosely related to identical genera showed T-RFs with identical lengths (data not shown).Occasionally, clones affiliated with differentgenera also produced T-RFs with identicallengths (data not shown). Free-living protozoarepresented a major part of the total eukary-otic comrnunity (Fig. 2). Th e clone librariesshow that the eukaryotic community in sur-face water, treated water, and tap water con-

sisted, respectively, of 44, 62 and 20% of free-living protozoa. Nematodes (metazoa) wereobserved in treated water and tap water, butnot in river water samples.

Blast analysis of the clones confirmed thepresence of a highly diverse group of free-liv-ing protozoa in all three water types. InT W 3,prepared from aerobic ground water, 13 generaof free-living protozoa could be distinguished.This protozoa community consisted for 84%of seven protozoa genera (each protozoa genushad a minima1 contribution of 7 % to the totalprotozoa community).The dominant protozoawere uncultured Cercozoan clone (25.4%),Cercomonas (1 4.3%), Acantlzamoeba (12.7%),Bodo (7.9%), Neobodo (7.9%), Rhynchomonas(7.9%), and Exill$era (7.9%). Many clones had


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101. FREE-LIVING PR OTOZOA IN DRIN KING WATER 429

FIGURE 1 Eukaryotic diversity in river water (RW), treated water ( T T , and tap water (Tap) as deter-mined by T-RFLP analysis.

similarities lower than 95% to sequencesdeposited in accessible public databases. Twoclone types had the highest similarity to proto-zoa described as hosts for Legionel la ,viz. A c a n -thamoebawith >95% similarity and E x - i l - f e r awith a similarity of >94% to thesequence in the database (7).

CONCLUDING REMARKSThis study shows that T-RFLP assays are apowerful technique to obtain a fast indication

of the diversity of the eukaryotic community.Further optimization of T-RFLP assays isneeded to achieve better resolution, distributingthe T-RFs of dominant fiee-living protozoaover the T-RFLP pattern fiom 50 to 500 bp.Clone libraries are a powerful t001 to determinethe diversity and identity of eukaryotic com-munities, but this technique is time-consuming.Despite their ecological irnportance, few studieshave dealt with fiee-living fieshwater protozoa,and thus few 18s rRNA gene sequences are

FIGURE 2 Classification of eukaryotic clone libraries made from river water (RW), reated water (Tm, nd tapwater (Tap) coliected in The Netherlands.


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430 VALSTER E S AL .

available. Consequently, many of the cloneshad less than 95% similarity with sequences inthe NCBI-database. We als0 found that the eu-karyotic diversity can be underestimated withT-RFLP assays, as T-RFs of clones most closelyrelated to different genera can have identicallengths. In most eukaryotes 18s rRNA genesare organized in tandem repeat units, but organ-isms can signifcantly differ in the genomniccopy number of ribosomal RN A genes, and notall eukaryotic organisms are multicellular (5) .Hence, clone libraries generated hom 18srR NA genes can give incorrect proportions be-tween different genera in the total eukaryoticcomrnunity. Still, the clone libraries revealedthat free-living protozoa are ubiquitous in riverwater, treated water, and tap water, and some ofthese organisms probably serve as hosts for L.

pneumophila.The application of real-time P C Rassays for selected protozoa genera and cultiva-tion methods will further extend our knowl-edge of free-living protozoa serving as hosts forL. pneumophila in tap water installations.

ACKNOWLEDGMENT

This study was cond ucted within the framework ofthe Joint Research Program of the water supply com-

panies in The Netherlands and was cofinanced byDelft Cluster.

REFERENCES1. Arnann, R. I., B. J. Binder, R. J. Olson, S.W.

Chisholm, R. Devereux, and D. A. Stahl.1990. Combination of 16s rRNA-targetedoligonucleotide probes with flow cytometry foranalyzing mixed microbial populations. Appl.Environ. Microbiol.56:1919-1925.

2. Fields, B. S., R. F. Benson, and R. E. Besser.2002. Legionella and Legionnaires' disease: 25 yearsof investigation. Clin. Microbiol. Ra! 15:506-526.

3. Hahn, M. W., and M . G. Htle. 2001. Grazingof protozoa and its effect on populrttions ofaquatic bacteria. FEMS Microb. Ecol.35:113-121.

4. Kuiper, M. W., B. A. Wullings, A. D. L. Akker-mans, R. R. Beumer, and D. van der Kooij.2004. Intracellular proliferation of Legionella pneu-mophila in Hartmannella vermijurmis in aquaticbiofilms grown on plasticized PVC. Appl. Environ.Microbiol. 70:68264833.

5. Long, E. O., and I. B. Dawid. 1980. Repeatedgenes in eukaryotes. Annu. Reu. Biochem.49:727-764.

6. Sogin, M. L., and J. H. Gunderson. 1987.Structural diversity of eukaryotic smali subunit ri-bosomal RNAs . Ann . NYAcad. Sci.503:125-139.

7. Steinert, M., U, Hentschel, and J. Hacker.2001. Legionella pneumophila: an aquatic microbegoes astray. FE MS Microbiol. Reu 26:149-162.

valster ea asm legion ella 2006

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