APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p. 3739-37450099-2240/94/$04.00+0Copyright (D 1994, American Society for Microbiology

Detection of Pseudomonas aeruginosa from Clinical andEnvironmental Samples by Amplification of

the Exotoxin A Gene Using PCRASHRAF A. KHAN AND CARL E. CERNIGLIA*

National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas 72079

Received 30 March 1994/Accepted 24 June 1994

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of theexotoxin A (ETA) structural gene sequence. The identity of the amplified 396-bp fragment was confirmed bydigesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments.Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species ofPseudomonas and GC-richbacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%,respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay candetect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5,ul) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected byhybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCRmethod, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. Thismethod is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosastrains. The method described can be used to detect a low level ofP. aeruginosa from environmental and clinicalsamples without the use of selective media or additional biochemical tests.

Pseudomonas aeruginosa is an opportunistic pathogen capa-ble of infecting both humans and animals. P. aeruginosa is animportant cause of bacteremia in patients receiving organtransplants and is responsible for about 28% of most bactere-mia episodes (4). Pulmonary colonization with mucoid P.aeruginosa is also a major cause of morbidity and mortality inpatients with cystic fibrosis (7). Correa et al. (5) have reportedthat vegetables were the main source of P. aeruginosa inhospitals even though 1% hypochloride solution is used forsanitizing the vegetables. Van der Waaij (32) observed that 10to 100 cells of P. aeruginosa can lead to gut colonization inpatients who are in intensive care units and immunosup-pressed. Ohman et al. (27) and Hazlett et al. (14) havereported that 104 cells of P. aenrginosa per ml can lead toocular infection in mice. P. aeruginosa strains producing rela-tively large amounts of exotoxin A (ETA) and proteases at thelevel of 107 cells per ml in drinking water of mice can cause

endogenous bactermia in few days (11, 16).P. aeruginosa produces two different ADP-ribosyltransferase

toxins: ETA and exoenzyme S (2, 3, 17, 21, 34). Exoenzyme Scauses significant tissue damage in lung, burn, and woundinfections (34). The highly toxic ETA is produced by themajority of P. aeruginosa strains and can inhibit eucaryoticprotein biosynthesis at the level of polypeptide chain elonga-tion factor 2, similarly to diphtheria toxin (34). ETA consists oftwo subunits; fragment A is catalytic, and fragment B isresponsible for interaction with eucaryotic cell receptors. ETAis cytotoxic to numerous mammalian cells (24), stimulates invitro production of interleukin-1 in murine peritoneal macro-phages (25), and induces murine cytotoxic T lymphocytes (35).Gray et al. (12) have cloned and sequenced the ETA structuralgene from a P. aeruginosa strain overexpressing ETA.

* Corresponding author. Mailing address: Division of Microbiology,3900 NCTR Rd., Jefferson, AR 72079. Phone: (501) 543-7341. Fax:(501) 543-7307. Electronic mail address: CCerniglia@NTET.NCTR.FDA.GOV.

Because P. aeruginosa is medically important, various meth-ods have been developed to rapidly and accurately identify P.aeruginosa species. P. aeruginosa identification by using a diskof phenanthroline and 9-chloro-9-[4-(diethylamino)]-9,10-di-hydro-10-phenyl-acridine hydrochloride (PC disk) has beenreported recently and compared with a monoclonal antibodydetection method (10). Recently, Kostman et al. (19) usedPCR to detect polymorphisms in the intergenic spacer regionsof bacterial rRNA genes ofPseudomonas cepacia by amplifyinghighly conserved sequences flanking the spacer region of therRNA. Counts et al. (6) developed an immunofluorescent-antibody test for rapid identification of P. aeruginosa in bloodculture. However, the immunofluorescent-antibody test is un-reliable because it produces a dull olive green or yellow colorwhich is hard to distinguish from autofluorescence (6).

Although conventional microbiological methods for identi-fying P. aeruginosa from environmental samples are reliable,they require several days to complete. PCR has the potentialfor identifying microbial species rapidly by amplification ofgene sequences unique to a particular organism (29), andseveral PCR-based, DNA probe methods have been developedto detect various pathogens from clinical, water, and foodsamples (1, 9, 15). However, the potential application of PCRfor environmental monitoring of pathogenic Pseudomonasstrains has not been reported.

In this paper, we report the development of a PCR proce-dure that can be used to rapidly and specifically detect P.aeruginosa strains in environmental samples by amplifying theETA structural gene.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Ninety-five envi-ronmental isolates of P. aeruginosa were obtained from theNational Center for Toxicological Research (NCTR) (Jeffer-son, Ark.) culture collections. These strains were isolated fromlaboratory animal room water samples during the last 10 years.

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TABLE 1. Reference, clinical, and environmental strains used to test the specificity of the assay

Strain No. Source or designation No. of strains ETA positivefor PCR assay

Pseudomonas strainsP. aeruginosa 35 Patient isolates 34

95 NCTR isolates 911 ATCC 29260 11 WR5 01 ATCC 39324 11 ATCC 53308 11 ATCC 27853 11 ATCC 15692 11 ATCC 15442 1

P. acidovorans 1 ATCC 15668 0P. alcaligenes 1 ATCC 14909 0P. cepacia 1 ATCC 25416 0P. fluorescens 1 ATCC 13525 0P. maltophilia 2 ATCC 13637, ATCC 17673 0P. putida 2 ATCC 12633, ATCC 17485 0

Non-Pseudomonas strainsAcinetobacter calcoaceticus 1 NCTR isolate 0Aeromonas hydrophila 4 ATCC 23211, ATCC 7966, ATCC 7965, ATCC 13444 0A. jandaei 1 ATCC 49568 0A. sorbia 1 ATCC 43979 0A. trota 1 ATCC 49657 0Azotobacter vinelandii 1 ATCC 12837 0Escherichia coli 3 ATCC 33694, ATCC 53323, ATCC 33876 0Ewingella americana 1 ATCC 33852 0Flavimonas oryzihabitans 1 NCTR isolateFlavobacterium sp. 1 ATCC 13533 0Vibrio cholerae 2 ATCC 582, ATCC 25870 0V. fluvialis 1 ATCC 33809 0V parahaemolyticus 1 ATCC 17802 0Xanthomonas hydrophila 1 ATCC 13637 0

In addition, 35 clinical isolates of P. aeruginosa were obtainedfrom the Microbiology Department, University of ArkansasMedical School, Little Rock, and the University of WashingtonSchool of Medicine, Seattle. Other bacterial strains used in thisstudy are listed in Tables 1 and 2. All isolates were stored inLuria-Bertani (LB) broth containing 20% glycerol at -70°C.Organisms were grown overnight at 37°C in LB broth or ontryptic soy agar plates supplemented with 5% blood agar. P.aeruginosa from water samples was isolated by plating oncetrimide agar and incubated at 42°C for 48 h.

Bacterial DNA preparation. P. aeruginosa was grown over-night in LB broth at 37°C. Cells were collected from 1-mlbacterial cultures, washed once with TE (10 mM Tris-HCl, 1mM EDTA) buffer, pH 8.0, and resuspended in 100 p.l ofsterile distilled water. Bacteria were lysed by adding 5 p.l of20% sodium dodecyl sulfate, and the tubes were incubated for15 min at 60°C. The clear cell lysate was purified by using theIsogene kit (Perkin-Elmer Cetus, Norwalk, Conn.) with a slightmodification. Briefly, 200 p.l of sodium iodide was added to thelysate, and the tubes were incubated in ice water for 5 min.Three microliters of DNA binder (instead of the 10 ,ulrecommended by the manufacturer) was added, and the solu-tion was mixed for 10 min. Bound DNA was pelleted bycentrifuging for 5 min and was washed twice with washingbuffer. After complete aspiration of the washing buffer, boundDNA was eluted in a final volume of 60 p.1 (three 20-,ulportions) of distilled water and was directly subjected toamplification.

Isolation and identification of environmental bacteria. Bac-

terial populations in water samples from mouse cages weredetermined by serial dilutions which were plated on LB agarand incubated at 37°C for 48 h. Isolated colonies were identi-fied by using the AUTOMICROBIC SYSTEM (bioMerieuxVitek, Inc., Hazelwood, Mo.). The total count of P. aeruginosawas determined by plating the appropriate dilutions of watersamples on cetrimide agar plates and incubating them at 42°Cfor 48 h (Table 2).

Cell recovery from water samples by filtration. P. aeruginosaPA103 (21) was grown overnight in LB broth at 37°C in ashaker, serially diluted in sterile water, and collected byfiltration through 13-mm-diameter Fluoropore FHLP 0013filters (Millipore Corporation, Bedford, Mass.) under vacuum(1). Water samples (10 ml) collected from animal bottles weresimilarly filtered to recover low numbers of cells. BacterialDNA was released by adding 100 p.l of sterile water to eachtube and lysing cells by 10 cycles of freezing and thawing.Samples were frozen in a dry ice-ethanol bath for 1 min andthen thawed in a 55°C water bath for 1 min. The lysed sampleswere heated at 95°C for 5 min to inactivate proteases andnucleases. This protected the template DNA and the addedenzymes and primers from degradation. The released DNA(10 p.l) was used as a template for the amplification of a 396-bpproduct.Primer selection. The primers and probe used in this study

are given in Table 3. The primers (ETAl and ETA2) specificto P. aeruginosa were chosen from the published sequence (12)of the ETA operon (Fig. 1) and amplify a 396-bp region of thestructural gene. Two other primers, ETA7 and ETA8, were

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TABLE 2. Environmental samples

CFU/mlSample nofa PCRb

P. aeruginosa E. coli A. calcoaceticus F. oryzihabitans

4406 NP 3.3x102 NF 3.4x102 +4407 NF 3.2 x 102 2.8 x 102 3.7 x 1024408 2.8 x 102 4.2 x 102 1.9 X 102 2.2 x 102 +4409 NF 4.2x102 NF 3.4x102 +4410 NF 3.8 x 102 3.4 x 102 2.6 x 102 +4411 NF NDd ND ND4413 NF 2.9x102 3.2x102 NF +4415 NF ND ND ND4420 NF ND ND ND7766 4.7 X 102 3.4 x 102 NF 2.8 x 102 +7768 NF ND ND ND7769 NF ND ND ND7860 5.0 x 103 3.9 x 102 3.8 X 102 2.9 x 102 +7880 3.2 x 102 4.0 x 102 2.9 x 102 2.4 x 102 +7893 4.8 x 102 4.2 X 102 2.6 x 102 1.9 x 102 +8156 NF ND ND ND8157 4.5 x 102 4.3 x 102 2.1 x 102 2.8 x 102 +8158 NF ND ND ND8159 NF ND ND ND +8160 NF ND ND ND8171 NF ND ND ND

a Water sample collected from mouse cage.b +, positive for PCR assay; -, negative for PCR assay.c NF, not found by plating method.d ND, not done.

chosen to screen ETA-negative strains and amplify 339 bp of microliters of the reaction mixture was analyzed by standardDNA upstream of the structural gene (Fig. 1). ETA3 was used submarine gel electrophoresis (1.5% agarose; 5 V/cm), and theas the internal probe of the structural gene. The primers and reaction products were visualized by staining with ethidiumprobes were purchased from Genosys Biotechnologies (The bromide (0.5 ,ug/ml in the running buffer). ETA-negative P.Woodlands, Tex.). aeruginosa strains were assayed for the presence of a 339-bp

Amplification. The amplification reaction was performed by product by using ETA7 and ETA8 primers under conditionsusing a DNA thermal cycler (Perkin-Elmer model 480) and similar to those described above except that the amplificationGeneAmp kit with Taq DNA polymerase (Perkin-Elmer Ce- was done as follows: denaturation at 94°C for 1 min andtus) in 0.5-ml microcentrifuge tubes. The reaction mixture annealing and extension at 72°C for 1 min. Five microliters of(50-pI total volume) consisted of 38.75 ,ul of sterile water, 5 pl amplified DNA was subjected to agarose gel electrophoresisof 10x PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, and visualized by staining with ethidium bromide. A reagent15 mM MgCl2, 0.1% [wt/vol] gelatin), 4 pu1 of deoxyribonucleo- blank contained all components of the reaction mixture exceptside triphosphates (2.5 mM each dATP, dTTP, dGTP, and template DNA, for which sterile distilled water was substi-dCTP), 0.5 pul of each primer (stock concentration, 100 ,uM), 1 tuted. This step was included in every PCR procedure. Theto 10 ,u1 of template, and 0.25 p.1 (0.5 U/Iul) of Taq DNA thermocycler, tips, and pipetters used for preparing the PCRpolymerase. After being overlaid with sterile mineral oil, the reagent template were kept in a location different from wheresamples were subjected to 25 to 35 cycles of amplification. the gels were loaded, stained, and photographed. All reagentsPreincubation was at 95°C for 2 min. Twenty-five to 35 PCR used in one experiment were taken in aliquots from the freezercycles were run under the following conditions: denaturation at and discarded at the end of the day.94°C for 1 min, primer annealing at 68°C for 1 min, and DNA Amplified samples (25 p.l) were purified by using a Magicextension at 72°C for 1 min in each cycle. After the last cycle, PCR column (Promega Corporation, Madison, Wis.) accord-the PCR tubes were incubated for 7 min at 72°C. Five ing to the instructions of the supplier and were digested by

TABLE 3. Sequences of oligonucleotide primers and internal probe

Regiona Sizeb Primerc Probe Sequence T (oC)d

1001-1024 396 ETAl 5'-GACAACGCCCTCAGCATCACCAGC-3' 731373-1396 396 ETA2 5'-CGCTGGCCCATTCGCTCCAGCGCT-3' 751199-1222 ETA3 5'-AGCCACATGTCGCCGATCTACACC-3' 71618-637 339 ETA7 5'-TTCCGCTCCCCGCCAGCCTC-3' 65937-956 339 ETA8 5'-AGTAGTGCAGCACGCCCTGG-3' 61

a Positions of the first and last nucleic acids of the primer in the targeted fragment. The structural gene extends from position 959 to 2662.b Number of nucleotides in the amplified fragment.cETAl-ETA2 and ETA7-ETA8 are primer pairs.d The melting temperature (Tm) was calculated by using the following formula: Tm = [4°C(G+C content of oligonucleotide) + 2°C(A+T content of oligonucleotide)]

- 5C.

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5'--AAT CCC ATA AAA GCC CTC TTC CGC TCC CCG CCA GCC TCC600 ETA7 ------>

.... AT CCC GCA CCC TAG ACG CAG GAG CCA TCG CG ATG C...ORF> 1

CGC ATG AGC GTC GAC CCG GCC ATC GCC GAC ACC GGC CAG GGC154 -

GTG CTG CAC TAC TCC ATG GTC CTG GAG GGC GGC AAC GAC GCGETA8

CTC AAG CTG GCC ATC GAC AAC GCC CTC AGC ATC ACC AGC GAC254 ETAl ------->

GGC CTG ACC ATC ............ CTG AAC GCC GGC AAC CAG CTCPvuI 450

AGC CAC ATG TCG CCG ATC TAC ACC ATC GAG ATG GGC .......ETA3

........ CGG GAA AAG CGC TGG AGC GAA TGG GCC AGC GGC AAG<------ ETA2 649

GTG TTG TGC CTG CTC--3'

FIG. 1. Nucleotide sequence of the ETA gene as published by Grayet al. (12) and positions of the primer sets and the internal probe thatwere used.

using PvuI (Bethesda Research Laboratories, Gaithersburg,Md.) at 37°C for 1 h. The fragments were separated on a 1.5%agarose gel at 5 V/cm for 1 h.

Amplification of a 396-bp fragment from environmentalsamples. Water samples collected from the mouse cage waterbottles were analyzed for the presence of P. aeruginosa. Watersamples (50 ,I) were amended with 0.5 il of 1% Triton X-100and incubated for 5 min in a boiling water bath. The sampleswere chilled on ice for 5 min and used directly as a template forin vitro amplification as described above, except the volume oftemplate was 50 RI and the total volume of the reactionmixture was 100 RI. Forty cycles of amplification were done.The amplified samples (10 ,lI) were analyzed on 1.5% agarosegels. Samples negative for P. aeruginosa by the plating methodwere collected on FHLP membrane (Millipore Corporation)filters and were lysed by the freeze-thaw method describedabove.DNA-DNA hybridization analysis of amplified DNA. For

Southern blots, DNA was transferred by vacuum blotting witha Pharmacia LKB Vacu Gene XL blotter from agarose gels tonylon membrane filters (GeneScreen Plus; DuPont, NENResearch Products, Boston, Mass.) according to the manufac-turer's recommendations (22, 31). Filters were air dried,wrapped in plastic, and exposed to UV (302 nm) for 5 min tofix the DNA to the filter matrix. An ETA-specific oligonucle-otide probe (ETA3 [Table 3]) was nonisotopically labeled byusing the 3' end-labeling digoxigenin-dUTP oligo tailing sys-tem (Boehringer Mannheim Biochemicals, Indianapolis, Ind.)and purified by selective precipitation with lithium chlorideand absolute ethanol (30). Hybridization analysis was per-formed according to the manufacturer's recommendations,and hybridization was detected by using the Genius Kit(Boehringer Mannheim Biochemicals).

Sensitivity of PCR detection of P. aeruginosa. P. aeruginosapurified genomic DNA from PA83 (NCTR collection) wasserially diluted to establish a concentration range of 10 fg to 1,ug. A control without P. aeruginosa DNA was also included.The diluted DNA samples were subjected to PCR amplifica-tion, using ETAl and ETA2 primers. After 35 cycles ofamplification, 5 pL of amplified DNA was analyzed on a 1.5%agarose gel and subjected to Southern hybridization analysisusing a nonradioactive internal oligonucleotide probe (ETA3).

RESULTS AND DISCUSSION

Preliminary experiments to determine the optimum PCRamplification conditions utilized genomic DNA from a P.aeruginosa ETA gene-positive strain as the source of template

123 bp

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FIG. 2. Agarose gel electrophoresis of 396-bp ETA amplified DNAfrom environmental sample. Lane 1, 396-bp amplified product fromenvironmental sample; lane 2, products from amplified DNA (246 and150 bp) digested with PvuI; lane 3, 123-bp ladder. The arrowheadindicates the 396-bp amplified PCR product.

DNA. Primer annealing temperatures close to the theoreticalprimer melting points allowed amplification of a single 396-bpproduct (Fig. 2, lane 1) which was confirmed as the internalETA fragment by PvuI digestion of amplified DNA. Twofragments of 246 and 150 bp (Fig. 2, lane 2) were generated, inagreement with previously published sequence data (12). Anal-ysis with different ETA probes of P. aeruginosa strains harbor-ing the ETA gene has shown that ETA genes are highlyconserved and are not duplicated as are the cholera toxin genes(23, 28).The limit of detection by ethidium bromide staining was 5 to

10 P. aeruginosa cells per 10 ml (Fig. 3A), and that by Southernhybridization using the ETA3 internal oligonucleotide-labeled(nonradioactive) probe was 1 to 5 cells (Fig. 3B). The filtrationof water samples through a Fluoropore filter (FHLP) followedby DNA lysis by freeze-thawing enabled us to amplify thetarget sequence from as few as 1 to 5 Pseudomonas cells in10-ml water samples by using a nonradioactively labeled probe(Fig. 3B). The samples were also filtered through cellulose orpolycarbonate filters, and cells were collected. DNA wasreleased and amplified; however, the detection limit was 100cells in 10 ml of water (data not shown). Cellulose acetate andpolycarbonate filters may have decreased the template avail-ability for PCR as a result of the binding of DNA to thecellulose matrix. Bej et al. (1) have successfully amplified aspecific DNA sequence from as few as 1 to 5 cells of coliformbacteria in 100 ml of water by using freeze-thaw lysis of cellscollected on Fluoropore filters (FGLP and FHLP).The detection limit for ethidium bromide DNA visualization

of amplified DNA from purified DNA was 0.1 pg (Fig. 4A). A10-fold-lower detection limit was achieved by using a DNAprobe internal to the PCR-amplified product (Fig. 4B). Theinternal probe (ETA3) was used to confirm the specificity ofthe PCR for ETA gene-positive strains. We did not find anypositive signal other than for P. aeruginosa ETA gene-positivestrains. In addition, this primer was successfully used in colonyhybridization to identify ETA gene-positive strains amongmixed bacterial populations at the ratio of 1:10 (10 to 20 cells

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FIG. 3. Sensitivity of the PCR protocol in detecting the ETA gene from whole cells of P. aeruginosa. Cells of P. aeruginosa were collected from10 ml of water on a Fluoropore FHLP filter, lysed, and amplified with ETAl and ETA2 primers for the ETA gene. Lane 1, 123-bp DNA ladder(Bethesda Research Laboratories as size standard; lane 2, 109 cells; lane 3, 108 cells; lane 4, 107 cells; lane 5, 106 cells; lane 6, 105 cells; lane 7, 104cells; lane 8, 103 cells; lane 9, 102 cells; lane 10, 5 to 10 cells; lane 11, no cells; lane 12, positive control DNA. (A) Ethidium bromide-stained agarosegel. (B) Southern hybridization of agarose gel using a nonradioactive internal oligonucleotide probe (ETA3). The arrowhead indicates the 396-bpamplified PCR product.

ofETA gene-positive P. aeruginosa) for specific detection of P.aeruginosa (data not shown).To determine the presence of P. aeruginosa in the animal

cage bottle water, ETAl and ETA2 primers were used. The

Abp

amplified products (10 pAl) were analyzed on a 1.5% agarosegel (Fig. 5). These results showed a 396-bp PCR-positive DNAfor the samples in lanes 7, 8, 9, 11, 12, and 14 of Fig. 5, andthose water samples contained 10 to 25 cells per ml. These six

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600-400- .4

1 2 3 4 5 6 7 8 91011

FIG. 4. Sensitivity of the PCR protocol in detecting the ETA gene from purified DNA of P. aeruginosa. Lane 1, 123-bp ladder; lane 2, 1 tLg oftemplate; lane 3, 100 ng of template; lane 4, 10 ng of template; lane 5, 1 ng of template; lane 6, 100 pg of template; lane 7, 10 pg of template; lane8, 1 pg of template; lane 9, 100 fg of template; lane 10, 10 fg of template; lane 11, no template DNA (control). (A) Ethidium bromide-stainedagarose gel. (B) Southern hybridization of agarose gel using a nonradioactive internal oligonucleotide probe (ETA3). The arrowhead indicates the396-bp amplified PCR product.

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600

4000

FIG. 5. Agarose gel (1.5%) electrophoresis of the PCR-amplifiedproduct of animal cage bottle water samples with primer pair ETAl-ETA2. Lane 1, 123-bp ladder; lane 2, positive control (P. aeruginosaPA103); lane 3, negative control without template; lane 4, watersample 4409; lane 5, water sample 4406 spiked with strain PA103; lane6, water sample 4406; lane 7, water sample 7880; lane 8, water sample8157; lane 9, water sample 4408; lane 10, water sample 4413; lane 11,water sample 7766; lane 12, water sample 7893; lane 13, water sample4410; lane 14, water sample 7860. Sample numbers are as in Table 2.

samples were also positive by direct plating (Table 2). Wefound that three samples (Fig. 5, lane 4, 10, and 13) were

positive for the 396-bp DNA, although no P. aeruginosa wasdetected on plates (Table 2). These three samples may havehad either dead or injured cells so that we were unable torecover them by plating. One of the water samples (no. 4406)was negative by both the PCR assay and plating (Fig. 5, lane 6).Several P. aeruginosa-negative (by plating) water samples wereassayed for in vitro amplification of 396-bp DNA by PCR afterthe templates were prepared by filtration and freeze-thawing.We found that one of the samples (no. 8159) was positive forthe PCR assay (Table 2). The detection limit of the PCR assayby the boiling method was 2 to 5 cells per 50 [LI. Othermicroflora did not interfere with the assay. The predominantbacterial contaminants in the water samples were Escherichiacoli, Acinetobacter calcoaceticus, and Flavimonas oryzihabitans(Table 2). These results clearly demonstrate that low levels ofP. aeruginosa in water samples can be rapidly detected.We evaluated the specificity of the PCR assay by testing for

amplification of the 396-bp DNA from 33 different strains ofPseudomonas spp. and other bacteria (Table 1). All strains ofnon-P. aeruginosa species failed to produce the 396-bp PCRproduct (Table 1). Ninety-six percent of the 130 strains of P.aeruginosa were positive for the ETA gene tested by PCR(Table 1). Two primers, ETA7 and ETA8 (Table 3), were

designed on the basis of the nucleotide sequence of the P.aeruginosa PA103 upstream region of the ETA gene sequence,which is deleted in strain WR5 (33). The five P. aeruginosastrains in our collection that did not amplify the 396-bp DNAwith ETAl and ETA2 primers were assayed with ETA7 andETA8 primers. Similarly, we found no amplification of the339-bp fragment. These results indicate that sequences imme-diately upstream of the ETA structural gene are absent, as instrain WR5, or are rearranged from strain to strain.There are several methods for the assay of ETA of P.

aeruginosa, and most of them are time-consuming (18). PCRassay can be routinely used to distinguish between ETA-positive and -negative P. aeruginosa strains without growing thebacteria and can save a lot of time and expensive chemicals. ADNA probe to the upstream region of the ETA gene has been

used by Vasil et al. (33) to identify and type P. aeruginosaspecimens taken from cystic fibrosis patients. Interestingly,they found that sequences upstream of ETA genes are rear-ranged between strains.Our results indicate that PCR amplification of P. aeruginosa

is a sensitive and specific method for the detection of Pseudo-monas strains isolated from environmental and clinical sam-ples. Several researchers (10, 13, 20, 26) have detected P.aeruginosa by raising monoclonal antibodies against outermembrane antigens. In addition, P. aeruginosa has been iden-tified with the PC disk method (10). The sensitivity andspecificity of both methods were more than 95%. The PCRassay was tested with cross-reacting organisms, and ourmethod showed no amplification (Table 1). Festl et al. (8) haveconstructed a specific probe for the identification of Pseudo-monas fluorescens group bacteria by cloning 360 bp of therRNA genes (rDNA) of P. aeruginosa. This probe has beenused to distinguish the P. fluorescens group from a wide varietyof microorganisms. The colony hybridization method is time-consuming and is not as sensitive as PCR. The data reportedhere suggest that the PCR method has advantages over all ofthese presently used identification methods in specificity, sen-sitivity, and cost. This assay allows the detection of an impor-tant toxin gene in environmental and clinical isolates. Thisassay may be useful for the early detection of low levels of P.aeruginosa in cystic fibrosis patients. The main advantage of thePCR method is that detection can be completed in less than 6h with reliable results.

In conclusion, we have presented data that suggest that PCRcan be used to provide rapid and specific detection of smallnumbers of P. aeruginosa in environmental samples against abackground of other bacterial species.

ACKNOWLEDGMENTS

We thank Stephen Lory, Department of Microbiology, School ofMedicine, University of Washington, Seattle; Michael Vasil, Depart-ment of Microbiology and Immunology, University of ColoradoHealth Science Center, Denver; David Wennerstrom, MicrobiologyDepartment, University of Arkansas Medical School, Little Rock; andWirt Franklin and Christine Ward of NCTR for providing P. aerugi-nosa strains and for identification of environmental isolates. Wegratefully acknowledge Kay Shuttleworth for critical reading of themanuscript.

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