JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/97/$04.0010

June 1997, p. 1295–1299 Vol. 35, No. 6

Copyright © 1997, American Society for Microbiology

Direct Detection and Identification of Pseudomonas aeruginosain Clinical Samples Such as Skin Biopsy Specimens andExpectorations by Multiplex PCR Based on Two Outer

Membrane Lipoprotein Genes, oprI and oprLDANIEL DE VOS,1 ANTONIO LIM, JR.,1 JEAN-PAUL PIRNAY,2 MARC STRUELENS,3

CHRISTIAN VANDENVELDE,4 LUC DUINSLAEGER,2 ALAIN VANDERKELEN,2

AND PIERRE CORNELIS1*

Flanders Interuniversity Institute for Biotechnology, Instituut voor Moleculaire Biologie, Vrije Universiteit Brussel,B-1640 Sint-Genesius-Rode,1 Brandwonden Centrum2 en Bloedbank,4 Militair Hospitaal Koningin

Astrid, B-1120, Brussels, and Laboratoire de Microbiologie, Hopital UniversitaireErasme, Universite Libre de Bruxelles, B-1070, Brussels,3 Belgium

Received 11 September 1996/Returned for modification 9 December 1996/Accepted 6 February 1997

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, wasdesigned and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI openreading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) inclinical material. A collection of reference strains including 20 different species of fluorescent pseudomonadswas tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n 5 150),including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the otherfluorescent pseudomonads. All other bacteria tested (n 5 15) were negative by the amplification test. The lowerdetection level for P. aeruginosa was estimated to be 102 cells/ml. Preliminary evaluation on testing skin biopsyspecimens from patients with burns (n 5 14) and sputum samples from cystic fibrosis patients (n 5 49) andother patients (n 5 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplexPCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens.Further evaluation of its specificity in longitudinal clinical studies is warranted.

Bacterial infections in burn wound patients are common andare difficult to control. Sepsis as a consequence is common andthe sepsis is often fatal (12, 16). In recent decades, followingthe introduction of antibiotic therapy, Pseudomonas aeruginosahas emerged as one of the most problematic gram-negativebacteria in modern hospital settings; this organism is increas-ingly isolated as a nosocomial pathogen, resulting in high mor-bidity and mortality rates. Burn patients, mechanically venti-lated patients, and cystic fibrosis (CF) patients are particularlysusceptible to P. aeruginosa infections (9, 16, 21, 22, 25). Thisgram-negative microorganism is inherently resistant to com-mon antibiotics and even survives in antiseptics (1, 9, 14, 16, 18,20, 22). There is a need for sensitive and specific tests whichare more rapid than culture.

The outer membrane proteins of P. aeruginosa play impor-tant roles in the interaction of the bacterium with the environ-ment (7). The inherent resistance of P. aeruginosa to antibioticsis, at least for many antibiotics, the consequence of the pres-ence of specific outer membrane proteins that have been im-plicated in efflux transport systems or that affect cell perme-ability (14, 18). Our group has cloned and molecularlycharacterized two outer membrane lipoprotein genes, oprI andoprL (2, 13). We and others also showed that oprI is conservedamong members of fluorescent pseudomonads (3, 24). Thepresent report describes the development of a multiplex (M-

PCR) assay for the detection of fluorescent pseudomonads andthe identification of P. aeruginosa from clinical samples on thebasis of the simultaneous amplification of the oprI and oprLgenes.

MATERIALS AND METHODS

Strains and culture conditions. In order to assess the specificity of the M-PCRfor P. aeruginosa, we used the same batch of representative bacteria describedearlier (3). They comprised representatives of fluorescent pseudomonads, non-fluorescent pseudomonads, and gram-negative and gram-positive bacteria (seeTable 1). All bacteria were obtained from the LMG-culture collection (Labora-torium voor Microbiologie, University of Ghent, Ghent, Belgium). The Luria-Bertani medium was purchased from Gibco-BRL-Life Technologies, and theBacto Casamino Acids medium (CAA) was purchased from Difco. The CAAmedium contained 5 g of Casamino Acids per liter, to which was added 1.18 g ofK2HPO4 z 3H2O and 0.25 g of MgSO4 z 7H2O per liter of demineralized water.P. aeruginosa and the other bacteria were grown at 37°C, while the other fluo-rescent pseudomonads were grown at 28°C.

Clinical samples. In this study we included as direct clinical material skinbiopsy specimens (14 samples) from infected and noninfected burn wound pa-tients and expectorations from patients with CF (49 samples) and patients with-out CF (19 samples). Skin biopsy specimens (about 10 mg) were aseptically takenby a surgeon and put in preweighed and autoclave-sterilized microcentrifugetubes (Eppendorf), one for bacterial culture (26) and one for PCR, and trans-ferred to the laboratory. Once the microcentrifuge tube containing the biopsyspecimen arrived in the laboratory, 500 ml of the following buffer was added: 50mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.5% Tween 20 (nonionic Taq-compatible detergent). The sample was first boiled in a water bath for 10 min,and then proteinase K (Boehringer Mannheim) at 200 mg/ml was added. Thetube was further incubated in a water bath at 65°C for 40 min. After deactivationof the proteinase K by heat treatment (10 min in a boiling water bath), the tubewas centrifuged at 13,000 rpm in a microcentrifuge (Eppendorf), and 10 ml of thesupernatant was taken as template for the M-PCR and was used at the followingdilutions: 1/1, 1/10, 1/100, 1/500, and 1/1,000 (the dilutions were made withdistilled, filter-sterilized and autoclaved water). Expectorations were obtainedfrom CF patients attending the Erasmus Hospital CF clinic, and as negativesamples, expectorations from non-CF patients known on the basis of culture

* Corresponding author. Mailing address: Flanders InteruniversityInstitute for Biotechnology, Instituut voor Moleculaire Biologie, VrijeUniversiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode,Belgium. Phone: 32.2.3590239. Fax: 32.2.3590390. E-mail: pcornel@vub.ac.be.

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results not to be infected with P. aeruginosa were included. The sputum speci-mens were Gram stained and examined under an oil immersion microscope inorder to select specimens for culture according to following criteria: the presenceof fewer than 25 squamous epithelial cells and more than 25 polymorphonuclearcells per 3100 power field. A purulent portion of the sample was taken with asterile cotton swab and was inoculated onto two Columbia agar plates, each ofwhich was supplemented with 5% sheep blood (BBL, Becton Dickinson, Erem-bodegem, Belgium), and the plates were incubated at 35°C, with one plateincubated under a normal atmosphere and the second plate, which was alsosupplemented with chocolate agar Polyvitex, and bacitracin (50 IU/ml), wasincubated in a 5% CO2 atmosphere. A third inoculum was incubated at 35°C ona MacConkey II agar plate (BBL, Becton Dickinson). The different agar plateswere incubated for 48 h and examined daily; plates showing no growth werereincubated for another 24 to 48 h at 30°C under a normal atmosphere. Theexpectorations, obtained after chest physiotherapy, were stored at 4°C for up to3 weeks, while template DNA was obtained either by phenol-chloroform extrac-tion or simply by boiling (1 to 5 ml of sputum sample diluted in 500 ml of distilled,filter-sterilized, and autoclaved water, of which 20 ml was used directly as a PCRtemplate).

PCR. The PCR was completed in adapted PCR microcentrifuge tubes accord-ing to the thermocycler used. The PCR mixture (100 ml final volume) contained37 ml of distilled, filter-sterilized, and autoclaved water, 10 ml of 103 PCR buffer(500 mM-KCl, 100 mM Tris-HCl [pH 8.4], 15 mM MgCl2), 10 ml of a deoxynucle-otide mixture (dGTP, dTTP, dATP, and dCTP; 2 mM each), 10 ml of dilutedtemplate DNA, 10 ml of each primer (30 pmol), and 2 U of enzyme (0.2 U whenGoldstar polymerase was used). As enzyme we used different DNA polymerasessuch as AmplitaQ from Perkin-Elmer, Expand Taq from Boehringer, and Gold-star from Eurogentec (Seraing, Liege, Belgium). For the Goldstar polymerase weused the manufacturer’s accompanying standard buffer. When using the TechneGene-E PCR apparatus, the reaction mixture was overlaid with 70 ml of mineraloil (Sigma) in order to avoid evaporation. Two sets of primers were used in thisM-PCR; one set (primers PS1 and PS2) consisted of primers corresponding tothe beginning and the end of the open reading frame of the oprI gene (3), whilethe other set of primers (primers PAL1 and PAL2) was designed to amplify theopen reading frame of the oprL gene (EMBL accession no. Z50191) (13). Theprimers were ordered from Pharmacia-Biotech (Pharmacia Benelux, Roosend-aal, The Netherlands) and had the following sequences: PS1, 59-ATGAACAACGTTCTGAAATTCTCTGCT-39 (a 27-mer corresponding to the beginning ofthe open reading frame of oprI); PS2, 59-CTTGCGGCTGGCTTTTTCCAG-39(a 21-mer corresponding to the end of the open reading frame of oprI); PAL1,59-ATGGAAATGCTGAAATTCGGC-39 (a 21-mer corresponding to the begin-ning of the open reading frame of oprL); and PAL2, 59-CTTCTTCAGCTCGACGCGACG-39 (a 21-mer corresponding to the end of the open reading frame ofoprL). A reaction mixture containing all the ingredients except the template wasmade. Different thermocyclers were used in order to compare the amplificationprotocol and to determine whether the program could be used in differentlaboratory facilities. The different thermocyclers were New Brunswick TechneTC-1, New Brunswick Techne Gene-E, New Brunswick Techne Pro-Gene, andPerkin-Elmer 2400. The amplification program was set at 25 to 30 cycles ofdenaturation at 94°C for 40 s (Techne TC-1 and Techne Gene-E) or 20 s (TechnePro-Gene and Perkin-Elmer 2400). The annealing was done at 57°C with thesame time settings mentioned above, while the extension was carried out at 72°Cfor 50 s (Techne TC-1 and Techne Gene-E) and 20 s, respectively (TechnePro-Gene and Perkin-Elmer 2400). One-tenth (10 ml) of the reaction mixturewas put on an agarose gel of 1.5% (wt/vol) for electrophoresis and visualizationof the product after staining with ethidium bromide on a UV transilluminator.All experiments included a positive control with P. aeruginosa PAO-1 DNA (3)and a negative control without DNA to check for eventual carry over contami-nants. When the PCR was performed in order to amplify only one lipoproteingene, only one set of primers was used and the volume of the PCR mixture wasadjusted accordingly. In order to avoid contamination and false-positive results,the measures proposed by Kwok and Higuchi (11) were taken. Samples wereprepared in a separate room, and sterile disposable gloves, sterile apyrogenicwater (Inflac; B. Brown Pharma, Diegem, Belgium), and positive displacementpipettes with sterile filter tips were systematically used.

Determination of lower detection limit. A serial dilution was made from abacterial culture (PAO-1) grown to the mid-logarithmic phase in order to lookfor the lowest number of bacteria needed to give a positive signal in the PCR.Dilutions to 10215 were made in physiological water. As an inoculum, 100 ml wasplated out, in triplicate, onto Luria-Bertani agar plates and the iron-limited CAAmedium (to allow for the visualization of pyoverdine production). The agarplates were incubated overnight at 37°C, and the colonies were counted in orderto estimate the number of CFU per dilution tube. A total of 10 ml was taken fromeach dilution tube as a template for a parallel PCR. A similar experiment wasdone with a reconstituted sample of a sterile skin biopsy specimen, and the sameresult was found.

Nucleotide sequence accession numbers. The oprI sequence has been givenEMBL accession no. X13748, and the oprL sequence has been given EMBLaccession no. Z50191.

RESULTS

Figure 1 shows that the M-PCR, as visualized after agarosegel electrophoresis, is specific for P. aeruginosa (lane 2), sincetwo amplification bands of 249 bp (oprI) and 504 bp (oprL)were observed when a P. aeruginosa colony lysate was used asthe template for the PCR. Lanes 3 and 4 show the result of asimilar PCR with only primers PS1 and PS2 corresponding tooprI (lane 3) and with primers PAL1 and PAL2 correspondingto oprL (lane 4). Other fluorescent pseudomonads such asPseudomonas fluorescens (lane 7) are detected by the presenceof only one band corresponding to the 249-bp open readingframe of oprI (3). Other bacteria such as Stenotrophomonasmaltophilia (previously known as Pseudomonas maltophilia)(lane 8), Escherichia coli (lane 9), and the gram-positive bac-terium Staphylococcus aureus (lane 10) did not react in theM-PCR, confirming previous observations (3, 24). As shown inlanes 5 and 6, a positive amplification after M-PCR was ob-tained for skin biopsy specimens from two burn patients knownto be infected with P. aeruginosa. Figure 2 shows that identicalresults were obtained whether the M-PCR was carried out withPAO-1 colony lysates (lane 2), with PAO-1 DNA (lane 3), ordirectly with sputa from P. aeruginosa-colonized CF patients(lanes 4 and 5). Table 1 presents the results of the PCR am-plifications for different fluorescent and nonfluorescent pseu-domonads as well as for other bacteria. The data in Table 1confirm that oprI can be amplified from all members of the flu-orescent pseudomonads (3, 24), while the oprL band can only beamplified from P. aeruginosa, but that it can be amplified con-sistently. Indeed, among the 150 isolates of P. aeruginosatested, none was found to be negative for oprL amplification(Table 1). The results were identical for all thermocyclers andpolymerases used, providing that the time settings were adapt-ed. In the Perkin-Elmer system, we were able to reduce the

FIG. 1. M-PCR after agarose gel electrophoresis. Lanes 1 and 12, molecularsize marker (bacteriophage l PstI), expressed in kilobases; lane 2, PAO-1 colonylysate M-PCR; lane 3, PAO-1 colony lysate oprI PCR; lane 4, PAO-1 colonylysate oprL PCR; lanes 5 and 6, skin biopsy specimen M-PCR (1:1,000 templatedilution); lane 7, P. fluorescens M-PCR; lane 8, S. maltophilia M-PCR; lane 9, E.coli M-PCR; lane 10, S. aureus M-PCR; lane 11, negative control.

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amplification time to 40 min, resulting in a total proceduretime of 130 min for sample preparation, amplification, and gelanalysis.

A clear PCR signal was observed up to the dilution showingbetween 20 and 30 CFU/plate, indicating a sensitivity down totwo to three bacteria per PCR sample (10 ml).

Table 2 presents the M-PCR results obtained with clinicalsamples such as skin biopsy specimens and expectorations fromCF or non-CF patients in comparison with those of culture. Allculture-positive samples were also positive by the M-PCR.However, several culture-negative skin biopsy specimens andexpectorations from CF patients and other patients were pos-itive by the M-PCR. The specificity of the M-PCR versus thatof culture for this study is 74%. Chart review of bacteriologicalresults for patients with a positive M-PCR result but a negativeculture result for P. aeruginosa showed one CF patient knownto be previously colonized with P. aeruginosa (presumably afalse-negative culture) and one patient with a heavy growth ofS. maltophilia from the three specimens tested.

A dilution of 1/500 to 1/1,000 of the clinical sample, espe-cially in the case of skin biopsy specimens, was found to giveconsistent results for the amplification of the two genes.

DISCUSSION

The aim of the present study was to develop a rapid andsimple technique for the specific detection and identification ofP. aeruginosa in clinical specimens. Although the classical mi-crobiological techniques currently in use for P. aeruginosa de-tection and identification are satisfactory in most situationsand remain necessary for drug susceptibility testing, morerapid tests may be useful in some specific situations. Theseinclude diagnosis in critically ill patients such as patients withburns, patients at risk for nosocomial pneumonia like mechan-ically ventilated patients, and CF patients at risk for chronicinfections with P. aeruginosa, for whom early diagnosis of ini-

tial colonization could be essential (8, 9, 29). Both target genesused in this M-PCR (oprI and oprL) were detected in all P.aeruginosa strains tested, including mucoid strains and pyover-dine-negative isolates. This can be of importance because the

FIG. 2. Results of M-PCR for colony lysate, purified DNA extract, and crudeexpectorations. Lane 1, bacteriophage l PstI molecular size marker (kilobases); lane2, PAO-1 colony lysate M-PCR; lane 3, PAO-1 DNA M-PCR; lanes 4 and 5, M-PCRfor 1:500-diluted crude expectoration from a CF patient; lane 6, negative control;lane 7, PAO-1 DNA, oprI PCR; lane 8, PAO-1 DNA, oprL PCR.

TABLE 1. Amplification results of the M-PCR of representatives ofthe family Pseudomonadaceae, some members of the familyEnterobacteriaceae, and some other bacteria based on the

two lipoprotein genes oprI and oprL of P. aeruginosa

Bacterial species(no. of isolates tested)a

PCR result foroprI (bandof 249 bp)

PCR result foroprL (bandof 504 bp)

Pseudomonas aeruginosa (150) 1 1Pseudomonas agarici 1 2Pseudomonas alcaligenes (3) 1 2Pseudomonas asplenii 1 2Pseudomonas aureofaciens 1 2Pseudomonas chlororaphis 1 2Pseudomonas cichorii 1 2Pseudomonas corrugata 1 2Pseudomonas fluorescens (29) 1 2Pseudomonas fragi 1 2Pseudomonas fuscovaginae 1 2Pseudomonas marginalis (2) 1 2Pseudomonas mendocina 1 2Pseudomonas oleovorans 1 2Pseudomonas pseudoalcaligenes 1 2Pseudomonas putida (3) 1 2Pseudomonas reptilovora 1 2Pseudomonas stutzeri 1 2Pseudomonas syringae 1 2Pseudomonas taetrolens 1 2Pseudomonas tolaasii 1 2Burkholderia cepacia 2 2Pseudomonas diminuta 2 2Stenotrophomonas maltophilia (3) 2 2Ralstonia solanacearum 2 2Acinetobacter sp. 2 2Alcaligenes eutrophus 2 2Azotomonas macrocytogenes 2 2Comamonas acidovorans 2 2Escherichia coli 2 2Klebsiella aerogenes 2 2Proteus sp. 2 2Serratia marcescens 2 2Providencia sp. 2 2Salmonella sp. 2 2Staphylococcus aureus 2 2

a No number indicates that only one isolate was tested.

TABLE 2. Results of culture for P. aeruginosa and M-PCR assaywith sputum specimen from CF and non-CF patients and

skin biopsy specimens from burn patients

Specimen type andculture result

No. of samples

M-PCR positive Total

Skin biopsy specimenPositive 7 7Negative 2 7

CF patient expectorationPositive 40 40Negative 4 9

Non-CF patient expectorationNegative 3 19

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isolation criteria for P. aeruginosa are still often based on theproduction of pyoverdine, although the bacterial morphologyof P. aeruginosa is highly versatile. Besides the detection ofP. aeruginosa, this M-PCR also detected other fluorescentpseudomonads through the amplification of the oprI gene (3,24).

The specificity of the oprL PCR for P. aeruginosa was ratherunexpected given the presence of an immunologically relatedpeptidoglycan-associated lipoprotein in other members of flu-orescent pseudomonads (17); however, when comparing thesequences corresponding to the beginning of the open readingframes of the P. aeruginosa (59-ATGGAAATGCTGAAATTCGGC-39) (13) and the Pseudomonas putida (59-ATGGAAATGCTGAAGTTTGGT-39) (23) oprL genes, three mismatches(in boldface) were discovered, with the last one correspondingto the last base of primer PAL1 (a T in place of a C), explainingwhy the amplification did not take place in the case of P.putida. In this study we tested three different strains of P.putida, namely, biotype A (strain LMG 2257) and B (strainLMG 1247) strains and a field isolate (isolate AS6) from awheat rhizosphere, and found no amplification of oprL usingour primers. Furthermore, as indicated in Table 1, among the20 species of fluorescent pseudomonads tested, none reactedpositively in the PCR for oprL. It would be interesting to testwhether, with primers based on the P. putida sequence (23), anamplification of oprL can take place for other species of fluo-rescent pseudomonads. The simultaneous detection of otherfluorescent pseudomonads can be important since fluorescentpseudomonads other than P. aeruginosa, such as Pseudomonasstutzeri, Pseudomonas fluorescens, Pseudomonas mendocina,and P. putida, have sometimes been claimed to be involved inopportunistic infections (5, 19). In the infrequent case of acoinfection with P. aeruginosa and another fluorescent pseudo-monad species, the PCR pattern would probably correspond tothe P. aeruginosa pattern.

The data presented here indicate that the PCR techniquebased on the detection of oprI and oprL is very sensitive andprovides results within 2 h. All sample material except thebiopsy specimen was processed by only a short boiling step.The M-PCR detected as few as two to three bacteria, corre-sponding to 102 CFU/ml, even in the presence of a differentDNA background (e.g., skin biopsy specimens). This level ofsensitivity is in the range those found in other studies describedby others who used a nested PCR based on the algD gene (15).The specificity of the oprI and oprL PCR assay was high incomparison with routine culture when it was based on testinggenomic DNA from a collection of bacterial strains but ap-peared much lower in comparison with routine culture (74%)when it was based on a preliminary clinical evaluation. Theremay be several explanations for these discrepancies. Theseinclude false-positive M-PCR results due to sample or ampli-con carryover contamination or to the presence of nonspecifichomologous target sequences in other bacteria. Alternatively,the discrepancies can reflect true P. aeruginosa infection with afalse-negative culture result due to sample overgrowth by otherbacteria or to the presence of noncultivable organisms (e.g., asa result of antimicrobial therapy) or auxotrophic mutations inthe organism (27). DNA contamination was an unlikely expla-nation because all standard precautions of good laboratorypractices were taken, as were specific measures to avoid car-ryover contamination, which in our case was never observed innegative controls (11). The eventual nonspecific reaction byrelated organisms, e.g., S. maltophilia, is very unlikely since weconfirmed during this study the results, already obtained pre-viously by Saint Onge et al. (24) and by us (3), that the targetsequence could not be amplified from S. maltophilia type

strains (LMG 958 and ATCC 13637) or clinical isolates. Fi-nally, the probability of a false-negative culture result wasillustrated for one clinically colonized CF patient with a spec-imen positive for P. aeruginosa by M-PCR and negative for P.aeruginosa by culture. To resolve these discrepancies, to assessthe clinical relevance of these findings, and to study in moredetail the specificity of this M-PCR test with clinical material,a longitudinal study with follow-up testing of samples, in du-plicate, by culture and by PCR would be required. This testcould also be considered a potential monitoring or early warn-ing system for hydrotherapy facilities, and other medical careinstruments which can be reservoirs or vehicles for P. aerugi-nosa (4, 6, 10, 20, 28).

In conclusion, this proposed M-PCR assay should be furtherevaluated for its clinical relevance as well as for its routineapplicability for the diagnosis of P. aeruginosa infection inspecific situations.

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