Enumeration of Legionella CFU by Colony Hybridization Using Specific DNA Probes

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Oligonucleotide probes and colony hybridization (CH) were appliedto enumerate organisms of the genus Legionella in cooling towerwater. The CH counts indicated almost the same results as CFUcounts in cultivated samples derived from the water. It wasconcluded that it is possible to substitute the CH procedurefor the conventional one.

INTRODUCTION

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Introduction

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Legionella is the etiologic agent of Legionnaires' disease.The combination of isolation on selective medium and identificationby DNA probe is useful in attempting to detect proliferatingLegionella organisms (1, 2). Here we report two kinds of oligonucleotideprobes, one targeting 16S rRNA and the other targeting a newtarget region, namely, an antigen common to the genus Legionella.Subsequently, we applied these probes to enumerate viable legionellaein various cooling tower water samples by using colony hybridization(CH). As a result of this investigation, we successfully identifiedand detected pinpoint Legionella colonies by using each of theprobes.

Design and labeling of specific oligonucleotide probes for Legionella.

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Design and Labeling of...

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The probes LEG225 (6), CA/G L253, CA/G L301 (3, 4, 8), and L5S-1(5), together with their sequences and target positions, arelisted in Table 1. The probe L5S-1 was used as a positive control.

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TABLE 1. Probe sequences and target positions

The synthesized oligonucleotides were labeled with a digoxigeninoligonucleotide tailing kit (Boehringer Mannheim, Mannheim,Germany), in accordance with the manual.

Bacterial strains.

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Bacterial strains.

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The bacterial strains used in this study, along with their sources,are listed in Tables 2 and 3. Legionella strains were obtainedfrom the stock strains of the Department of Microbiology, GifuUniversity School of Medicine. The reference strains were suppliedby the Department of Bacteriology, Juntendo University.

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TABLE 2. Legionella strains used for probe specificity examinations and dot blot hybridization results

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TABLE 3. Reference strains used for probe specificity examinations and dot blot hybridization results

Cell lysis for DNA extraction and purification.

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Cell lysis for dna...

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Legionella strains were cultivated at 37°C for 3 to 5 dayson a 0.1% (wt/vol) ${alpha}$ -ketoglutarate (BCYE ${alpha}$ ) agar plate (Eiken ChemicalCo., Tokyo, Japan). About 10⁸ to 10⁹ cells were supplied in533 µl of 10 mM Tris-HCl-1 mM EDTA buffer (pH 8.0). Sixtyµl of 10% sodium dodecyl sulfate (SDS) (final concentration,1%) and 6 µl of 10-mg/ml proteinase K solution (finalconcentration, 100 µg/ml) were added. After the solutionswere mixed, they were incubated for 1 h at 37°C.

Reference microorganisms except Staphylococcus aureus were culturedin Luria-Bertani broth overnight and harvested. About 10⁸ to10⁹ cells were suspended in 2 ml of 10 mM Tris-HCl-1 mM EDTAbuffer (pH 8.0). One hundred µl of 10% SDS (final concentration,0.5%) and 20 µl of 10-mg/ml proteinase K solution (finalconcentration, 100 µg/ml) were added. After the solutionswere mixed, the mixture was incubated for 1 h at 37°C. Then353 µl of 5 M NaCl and 282 µl of cetyltrimethylammoniumbromide-NaCl were added, and the solution was incubated for10 min at 65°C. Staphylococcus aureus was cultured overnightin Luria-Bertani broth and harvested. The cells were washedtwice in 2 ml of 10 mM Tris-HCl-10 mM EDTA buffer (pH 8.0).The pellet was suspended in 2 ml of 10 mM Tris-HCl-10 mM EDTAbuffer (pH 8.0) and then mixed sufficiently with 40 µlof achromopeptidase (50,000 U/ml in 10 mM NaCl). The solutionwas incubated at 55°C until the cells were lysed.

The subsequent DNA extraction and purification procedures wereperformed as described previously (7).

Detection of legionella colonies.

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Detection of legionella...

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The cultivation and identification of legionella colonies fromthe cooling tower water were performed as described previously(1). The replicated agar plates were reincubated for another2 to 3 days at 37°C until the colonies grew to a visiblesize.

Testing of oligonucleotide probe specificity.

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Testing of oligonucleotide probe...

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One hundred ng of nucleic acid was blotted onto a 0.22-µm-pore-sizeBiodyne A nylon membrane (Poll Europe Ltd.). Subsequent dotblot hybridization was performed under the standard 5x SSC procedure(1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) in a digoxigeninsystem (Boehringer Mannheim).

CH.

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The concentrated cooling tower water was cultured on a WYO ${alpha}$ agarplate at 37°C for 2 to 5 days. Colonies were transferredfrom plates to 0.22-µm-pore-size (82-mm-diameter) BiodyneA nylon membranes (Poll Europe) by using procedures describedpreviously (7). After replication, the membranes were placedcolony-side-up on the paper (3MM; Whatman) and were saturatedwith 5% Triton X-100 in 10 mM Tris-HCl-1 mM EDTA (pH 8.0) for1 h at 60°C. The membranes were transferred onto the paper(3MM); supplemented with 0.1 mg of proteinase K per ml in 10mM Tris-HCl (pH 8.0) containing 10 mM NaCl, 1 mM sodium citrate,and 1.5% SDS; and incubated for 1 h at 60°C to lyse thecells. Subsequently, denaturing and neutralization of the DNAwere performed as described previously (7). In order to removethe protein, the DNA fixed in the membranes was incubated overnightat 37°C with gentle agitation in a solution including 0.1mg of proteinase K per ml, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl,and 0.5% SDS. The membranes were washed twice for 5 min at roomtemperature with about 50 ml of 6x SSC per 100 cm².

Hybridization was carried out by using the same procedure describedfor dot blot hybridization, except that 10% blocking reagentwas used.

Specificity of the oligonucleotide probes.

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Specificity of the...

Probe LEG225 hybridized to 32 of the tested strains of Legionellaspp. (91%) (Table 2). No false-positive hybridization occurredwith the reference strain (Table 3). Probes CA/G L253 and CA/GL301 could hybridize 26 strains (74%) and 18 strains (54%) ofthe Legionella species tested, respectively. A mixed probe consistingof probes CA/G L253 and CA/G L301 together could hybridize 100%of the Legionella species tested (Table 2), but false-positivehybridization occurred with Klebsiella pneumoniae (Table 3).

Other bacteria and fungi which grew on the WYO ${alpha}$ agar plates frequentlydisturbed the growth of legionellae and of the screened legionellacolonies. However, as shown in Fig. 1, the signals from Legionellacolonies clearly discriminated among these other microorganisms.A Legionella colony formed within a fungus colony was also detected(Fig. 2).

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FIG. 1. Cultivated colonies of Legionella spp. from cooling tower water samples and CH results. The sample water was concentrated and mixed with 0.2 M KCl-HCl buffer (pH 2.2) for 4 min at 25°C. Then, 0.1 ml of mixture was streaked on WYO ${alpha}$ agar. The plate was incubated at 37°C. (A) The plate after 4 days of incubation, just before the membrane was laid on the culture. The white arrows indicate Legionella colonies. (B) Signals of X-ray film developed after the plate shown in panel A was exposed to a membrane which was hybridized with a mixed probe consisting of probes CA/G L253 and CA/G L301.

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FIG. 2. Cultivated colonies of Legionella spp. from cooling tower water samples and CH results. The sample water was concentrated and mixed with 0.2 M KCl-HCl buffer (pH 2.2) for 4 min at 25°C. Then, 0.1 ml of mixture was streaked on WYO ${alpha}$ agar. The plate was incubated at 37°C. (A) The plate after 4 days of incubation, just before the membrane was laid on the culture. The white arrows indicate Legionella colonies. (B) Signals of X-ray film developed after the plate shown in panel A was exposed to a membrane which was hybridized with a mixed probe consisting of probes CA/G L253 and CA/G L301.

The results of CH counts of organisms from cooling tower waterby using probe LEG225 and the results of CFU counts by cultivationrevealed a close relationship, with a regression coefficientof 0.96 (Fig. 3).

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FIG. 3. Regression analysis of results of Legionella colony counts from samples of cooling tower water by CH with probe LEG225 against results of CFU counts by cultivation on WYO ${alpha}$ agar plates. Regression coefficient, 0.96.

In the case of the mixed probe consisting of probes CA/G L253and CA/G L301 together, the results of CH counts were generallyin agreement with those of CFU counts by plate culture; theregression coefficient was 0.98 (Fig. 4). The results of CHcounts were slightly lower than those yielded by CFU countsof WYO ${alpha}$ agar plate cultures. This tendency became remarkablein the samples that contained more than 100 CFU per 100 ml.This phenomenon is considered to result from the creation oflarge spots on the membrane by individual adjacent coloniesbecause the colonies were aggregated after the process of colonytransfer and lysing. But this phenomenon did not significantlyaffect the result of Legionella enumeration. The CH method hasexcellent features, including the ability to simultaneouslyperform enumeration of colonies and identification of everycolony on a single plate.

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FIG. 4. Regression analysis of results of Legionella colony counts from samples of cooling tower water by CH with a mixed probe consisting of probes CA/G L253 and CA/G L301 against results of CFU counts by cultivation on WYO ${alpha}$ agar plates. Regression coefficient, 0.98.

Grimont et al. (2) reported that pinpoint Legionella pneumophilacolonies were detectable by the use of a DNA probe labeled witha radioactive isotope after 4 days of incubation on BCYE ${alpha}$ agar.In our study, we demonstrated the ability to enumerate Legionellacolonies by DNA probe without radioactive-isotope labeling afterthe samples were cultured for 3 days.

ACKNOWLEDGMENTS

We thank Hirohisa Tasiro and Takao Numakura for stimulatingdiscussions. We are grateful to Eiko Yabuuchi and KatsunoriFuruhata for their invaluable suggestions.

FOOTNOTES

* Corresponding author. Mailing address: Technology and Development Laboratory, Kurita Water Industries Ltd., 7-1, Wakamiya Morinosato, Atsugi-city, Kanagawa 243-0124, Japan. Phone: 81 046 270 2103. Fax: 81 046 270 2149. E-mail: wakako.Satoh{at}kurita.co.jp.

REFERENCES

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