APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1994, p. 3739-3745
Vol. 60, No. 10
0099-2240/94/$04.00+0 Copyright (D 1994, American Society for Microbiology
Detection of Pseudomonas aeruginosa from Clinical and Environmental Samples by Amplification of the Exotoxin A Gene Using PCR ASHRAF 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 the exotoxin A (ETA) structural gene sequence. The identity of the amplified 396-bp fragment was confirmed by digesting 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-rich bacteria 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 can detect 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 by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests. Pseudomonas aeruginosa is an opportunistic pathogen capable of infecting both humans and animals. P. aeruginosa is an important cause of bacteremia in patients receiving organ transplants and is responsible for about 28% of most bacteremia episodes (4). Pulmonary colonization with mucoid P. aeruginosa is also a major cause of morbidity and mortality in patients with cystic fibrosis (7). Correa et al. (5) have reported that vegetables were the main source of P. aeruginosa in hospitals even though 1% hypochloride solution is used for sanitizing the vegetables. Van der Waaij (32) observed that 10 to 100 cells of P. aeruginosa can lead to gut colonization in patients who are in intensive care units and immunosuppressed. Ohman et al. (27) and Hazlett et al. (14) have reported that 104 cells of P. aenrginosa per ml can lead to ocular infection in mice. P. aeruginosa strains producing relatively large amounts of exotoxin A (ETA) and proteases at the level 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 S causes significant tissue damage in lung, burn, and wound infections (34). The highly toxic ETA is produced by the majority of P. aeruginosa strains and can inhibit eucaryotic protein biosynthesis at the level of polypeptide chain elongation factor 2, similarly to diphtheria toxin (34). ETA consists of two subunits; fragment A is catalytic, and fragment B is responsible for interaction with eucaryotic cell receptors. ETA is cytotoxic to numerous mammalian cells (24), stimulates in vitro production of interleukin-1 in murine peritoneal macrophages (25), and induces murine cytotoxic T lymphocytes (35). Gray et al. (12) have cloned and sequenced the ETA structural gene from a P. aeruginosa strain overexpressing ETA.
Because P. aeruginosa is medically important, various methods have been developed to rapidly and accurately identify P. aeruginosa species. P. aeruginosa identification by using a disk of phenanthroline and 9-chloro-9-[4-(diethylamino)]-9,10-dihydro-10-phenyl-acridine hydrochloride (PC disk) has been reported recently and compared with a monoclonal antibody detection method (10). Recently, Kostman et al. (19) used PCR to detect polymorphisms in the intergenic spacer regions of bacterial rRNA genes of Pseudomonas cepacia by amplifying highly conserved sequences flanking the spacer region of the rRNA. Counts et al. (6) developed an immunofluorescentantibody test for rapid identification of P. aeruginosa in blood culture. However, the immunofluorescent-antibody test is unreliable because it produces a dull olive green or yellow color which is hard to distinguish from autofluorescence (6). Although conventional microbiological methods for identifying P. aeruginosa from environmental samples are reliable, they require several days to complete. PCR has the potential for identifying microbial species rapidly by amplification of gene sequences unique to a particular organism (29), and several PCR-based, DNA probe methods have been developed to detect various pathogens from clinical, water, and food samples (1, 9, 15). However, the potential application of PCR for environmental monitoring of pathogenic Pseudomonas strains has not been reported. In this paper, we report the development of a PCR procedure that can be used to rapidly and specifically detect P. aeruginosa strains in environmental samples by amplifying the ETA structural gene. MATERIALS AND METHODS
Bacterial strains and growth conditions. Ninety-five environmental isolates of P. aeruginosa were obtained from the National Center for Toxicological Research (NCTR) (Jefferson, Ark.) culture collections. These strains were isolated from laboratory animal room water samples during the last 10 years.
Corresponding author. Mailing address: Division of Microbiology, 3900 NCTR Rd., Jefferson, AR 72079. Phone: (501) 543-7341. Fax: (501) 543-7307. Electronic mail address:
[email protected]. FDA.GOV. *
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TABLE 1. Reference, clinical, and environmental strains used to test the specificity of the assay Strain
Pseudomonas strains P. aeruginosa
P. acidovorans P. alcaligenes P. cepacia P. fluorescens P. maltophilia P. putida
Non-Pseudomonas strains Acinetobacter calcoaceticus Aeromonas hydrophila A. jandaei A. sorbia A. trota Azotobacter vinelandii Escherichia coli Ewingella americana Flavimonas oryzihabitans Flavobacterium sp. Vibrio cholerae V. fluvialis V parahaemolyticus Xanthomonas hydrophila
Source or designation
No.
35 95 1 1 1 1 1 1 1
Patient isolates NCTR isolates ATCC 29260 WR5 ATCC 39324 ATCC 53308 ATCC 27853 ATCC 15692 ATCC 15442
34 91 1 0 1 1 1 1 1
1 1 1 1 2 2
ATCC ATCC ATCC ATCC ATCC ATCC
1 4 1 1 1 1 3 1 1 1 2 1 1 1
NCTR isolate ATCC 23211, ATCC 7966, ATCC 7965, ATCC 13444 ATCC 49568 ATCC 43979 ATCC 49657 ATCC 12837 ATCC 33694, ATCC 53323, ATCC 33876 ATCC 33852 NCTR isolate ATCC 13533 ATCC 582, ATCC 25870 ATCC 33809 ATCC 17802 ATCC 13637
15668 14909 25416 13525 13637, ATCC 17673 12633, ATCC 17485
In addition, 35 clinical isolates of P. aeruginosa were obtained from the Microbiology Department, University of Arkansas Medical School, Little Rock, and the University of Washington School of Medicine, Seattle. Other bacterial strains used in this study are listed in Tables 1 and 2. All isolates were stored in Luria-Bertani (LB) broth containing 20% glycerol at -70°C. Organisms were grown overnight at 37°C in LB broth or on tryptic soy agar plates supplemented with 5% blood agar. P. aeruginosa from water samples was isolated by plating on cetrimide agar and incubated at 42°C for 48 h. Bacterial DNA preparation. P. aeruginosa was grown overnight in LB broth at 37°C. Cells were collected from 1-ml bacterial cultures, washed once with TE (10 mM Tris-HCl, 1 mM EDTA) buffer, pH 8.0, and resuspended in 100 p.l of sterile distilled water. Bacteria were lysed by adding 5 p.l of 20% sodium dodecyl sulfate, and the tubes were incubated for 15 min at 60°C. The clear cell lysate was purified by using the Isogene kit (Perkin-Elmer Cetus, Norwalk, Conn.) with a slight modification. Briefly, 200 p.l of sodium iodide was added to the lysate, and the tubes were incubated in ice water for 5 min. Three microliters of DNA binder (instead of the 10 ,ul recommended by the manufacturer) was added, and the solution was mixed for 10 min. Bound DNA was pelleted by centrifuging for 5 min and was washed twice with washing buffer. After complete aspiration of the washing buffer, bound DNA was eluted in a final volume of 60 p.1 (three 20-,ul portions) of distilled water and was directly subjected to
amplification. Isolation and identification of environmental bacteria. Bac-
No. of strains ETA positive for PCR assay
0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
terial populations in water samples from mouse cages were determined by serial dilutions which were plated on LB agar and incubated at 37°C for 48 h. Isolated colonies were identified by using the AUTOMICROBIC SYSTEM (bioMerieux Vitek, Inc., Hazelwood, Mo.). The total count of P. aeruginosa was determined by plating the appropriate dilutions of water samples on cetrimide agar plates and incubating them at 42°C for 48 h (Table 2). Cell recovery from water samples by filtration. P. aeruginosa PA103 (21) was grown overnight in LB broth at 37°C in a shaker, serially diluted in sterile water, and collected by filtration through 13-mm-diameter Fluoropore FHLP 0013 filters (Millipore Corporation, Bedford, Mass.) under vacuum (1). Water samples (10 ml) collected from animal bottles were similarly filtered to recover low numbers of cells. Bacterial DNA was released by adding 100 p.l of sterile water to each tube and lysing cells by 10 cycles of freezing and thawing. Samples were frozen in a dry ice-ethanol bath for 1 min and then thawed in a 55°C water bath for 1 min. The lysed samples were heated at 95°C for 5 min to inactivate proteases and nucleases. This protected the template DNA and the added enzymes and primers from degradation. The released DNA (10 p.l) was used as a template for the amplification of a 396-bp product. Primer selection. The primers and probe used in this study are given in Table 3. The primers (ETAl and ETA2) specific to P. aeruginosa were chosen from the published sequence (12) of the ETA operon (Fig. 1) and amplify a 396-bp region of the structural gene. Two other primers, ETA7 and ETA8, were
PCR FOR DETECTION OF P. AERUGINOSA
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TABLE 2. Environmental samples CFU/ml
Sample nofa P. aeruginosa
E. coli
A. calcoaceticus
F. oryzihabitans
NP NF 2.8 x 102 NF NF NF NF NF NF 4.7 X 102 NF NF 5.0 x 103 3.2 x 102 4.8 x 102 NF 4.5 x 102 NF NF NF NF
3.3x102 3.2 x 102 4.2 x 102 4.2x102 3.8 x 102 NDd 2.9x102 ND ND 3.4 x 102 ND ND 3.9 x 102 4.0 x 102 4.2 X 102 ND 4.3 x 102 ND ND ND ND
NF 2.8 x 102 1.9 X 102 NF 3.4 x 102 ND 3.2x102 ND ND NF ND ND 3.8 X 102 2.9 x 102 2.6 x 102 ND 2.1 x 102 ND ND ND ND
3.4x102 3.7 x 102 2.2 x 102 3.4x102 2.6 x 102 ND NF ND ND 2.8 x 102 ND ND 2.9 x 102 2.4 x 102 1.9 x 102 ND 2.8 x 102
4406 4407 4408 4409 4410 4411 4413 4415 4420 7766 7768 7769 7860 7880 7893 8156 8157 8158 8159 8160 8171
PCRb
+
+ + + + + + + + +
ND
ND ND ND
+
Water sample collected from mouse cage. +, positive for PCR assay; -, negative for PCR assay. c NF, not found by plating method. d ND, not done. a
b
chosen to screen ETA-negative strains and amplify 339 bp of DNA upstream of the structural gene (Fig. 1). ETA3 was used as the internal probe of the structural gene. The primers and probes were purchased from Genosys Biotechnologies (The Woodlands, Tex.). Amplification. The amplification reaction was performed by using a DNA thermal cycler (Perkin-Elmer model 480) and GeneAmp kit with Taq DNA polymerase (Perkin-Elmer Cetus) in 0.5-ml microcentrifuge tubes. The reaction mixture (50-pI total volume) consisted of 38.75 ,ul of sterile water, 5 pl of 10x PCR buffer (100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2, 0.1% [wt/vol] gelatin), 4 pu1 of deoxyribonucleoside triphosphates (2.5 mM each dATP, dTTP, dGTP, and dCTP), 0.5 pul of each primer (stock concentration, 100 ,uM), 1 to 10 ,u1 of template, and 0.25 p.1 (0.5 U/Iul) of Taq DNA polymerase. After being overlaid with sterile mineral oil, the samples were subjected to 25 to 35 cycles of amplification. Preincubation was at 95°C for 2 min. Twenty-five to 35 PCR cycles were run under the following conditions: denaturation at 94°C for 1 min, primer annealing at 68°C for 1 min, and DNA extension at 72°C for 1 min in each cycle. After the last cycle, the PCR tubes were incubated for 7 min at 72°C. Five
microliters of the reaction mixture was analyzed by standard submarine gel electrophoresis (1.5% agarose; 5 V/cm), and the reaction products were visualized by staining with ethidium bromide (0.5 ,ug/ml in the running buffer). ETA-negative P. aeruginosa strains were assayed for the presence of a 339-bp product by using ETA7 and ETA8 primers under conditions similar to those described above except that the amplification was done as follows: denaturation at 94°C for 1 min and annealing and extension at 72°C for 1 min. Five microliters of amplified DNA was subjected to agarose gel electrophoresis and visualized by staining with ethidium bromide. A reagent blank contained all components of the reaction mixture except template DNA, for which sterile distilled water was substituted. This step was included in every PCR procedure. The thermocycler, tips, and pipetters used for preparing the PCR reagent template were kept in a location different from where the gels were loaded, stained, and photographed. All reagents used in one experiment were taken in aliquots from the freezer and discarded at the end of the day. Amplified samples (25 p.l) were purified by using a Magic PCR column (Promega Corporation, Madison, Wis.) according to the instructions of the supplier and were digested by
TABLE 3. Sequences of oligonucleotide primers and internal probe Regiona
Sizeb
Primerc
1001-1024 1373-1396 1199-1222 618-637 937-956
396 396
ETA2
339 339
ETA7 ETA8
Probe
Sequence
T (oC)d
ETA3
5'-GACAACGCCCTCAGCATCACCAGC-3' 5'-CGCTGGCCCATTCGCTCCAGCGCT-3' 5'-AGCCACATGTCGCCGATCTACACC-3' 5'-TTCCGCTCCCCGCCAGCCTC-3' 5'-AGTAGTGCAGCACGCCCTGG-3'
73 75 71 65 61
ETAl
Positions of the first and last nucleic acids of the primer in the targeted fragment. The structural gene extends from position 959 to 2662. 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)] a
b Number of nucleotides in the
-
5C.
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123 bp
5'--AAT CCC ATA AAA GCC CTC TTC CGC TCC CCG CCA GCC TCC ------> ETA7 600 .... 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 GGC 154 GTG CTG CAC TAC TCC ATG GTC CTG GAG GGC GGC AAC GAC GCG ETA8 CTC AAG CTG GCC ATC GAC AAC GCC CTC AGC ATC ACC AGC GAC -------> ETAl 254 GGC CTG ACC ATC ............ CTG AAC GCC GGC AAC CAG CTC 450 PvuI 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 649
