WO1997001647A2

WO1997001647A2 - Dna sequences for identifying highly transmissible lineages of pseudomonas (burkholderia) cepacia

Info

Publication number: WO1997001647A2
Application number: PCT/US1996/011132
Authority: WO
Inventors: Richard Goldstein
Original assignee: Trustees Of Health & Hospitals Of City Of Boston Inc
Current assignee: Trustees Of Health & Hospitals Of City Of Boston Inc
Priority date: 1995-06-28
Filing date: 1996-06-28
Publication date: 1997-01-16
Anticipated expiration: 1997-12-28
Also published as: WO1997001647A3; AU6404196A

Abstract

The present invention provides DNA based fingerprints and DNA sequences for indentifying highly transmissible lineages of Pseudomonas cepacia. More specifically, the present invention provides genetic band patterns or DNA based fingerprints for identifying highly transmissible lineages of Pseudomonas cepacia produced by: (a) ribotyping analyses, i.e., the determination of restriction fragment length polymorphisms (RFLPs) associated with the multicopy RNA operon (rrn); and (b) pulsed field gel electrophoresis (PFGE)-based resolution of chromosomal macro-RFLP patterns. Also provided are unique primer oligonucleotide sequences and DNA probes derived from variants of a gene, encoding a 17-KDa major subunit pilin protein (cb1A) of a cystic fibrosis-associated Pseudomonas cepacia. The invention also discloses methods and diagnostic kits for identifying highly transmissible lineages of Pseudomonas cepacia that utilize the above DNA based fingerprints, oligonucleotide primers and DNA probes. Another object of this invention is to provide isolated DNA molecules encoding a 17-KDa major subunit pilin protein of certain cystic fibrosis-associated Pseudomonas cepacia strains, as well as recombinant DNA molecules, transformed hosts and methods for the production of that protein. Also contemplated are antibodies to the 17-KDa major subunit pilin protein.

Description

DNA SEQUENCES FOR IDENTIFYING HIGHLY TRANSMISSIBLE LINEAGES OF PSEUDOMONAS (BURKHOLDERIA) CEPACIA

FIELD OF THE INVENTION This invention relates generally to the detection of highly transmissible strains of Pseudomonas (Burkholderia) cepacia, * and particularly to the use of DNA based fingerprints and sequences for identifying such epidemic lineages.

BACKGROUND OF THE INVENTION

Pseudomonas cepacia is an aerobic, gram- negative bacillus with an ubiquitous distribution in soil and water. In recent years this organism has emerged as an important pathogen among cystic fibrosis (CF) patients. CF patients with respiratory colonization or infection with P. cepacia have higher morbidity and mortality than those CF patients not infected by this organism. While the significant increase in P. cepacia infection suggests epidemic spread ' , the source and transmissibility of

* In view of recent molecular phylogenic analyses, the bacterium Pseudomonas cepacia has been renamed Burkholderia cepacia . However, in order to be consistent with the terminology of the literature cited in the specification, the original nomenclature is maintained herein. 9

P. cepacia remains controversial . Nonetheless, given the potentially grave consequence of P. cepacia infection, stringent infection control policies have been adopted, many CF camps in North America have been closed and all but one lung transplant center have ceased to accept P. cepacia-infected CF patients as transplant candidates.

The epidemiology of P. cepacia infection has been previously examined by both ribotyping 1' 9 and pulsed-field gel electrophoresis (PFGE)-based resolution of chromosomal macro-restriction fragment

3 9 10 profiles ' ' . Comprehensive studies applying both methods generated two very different conclusions regarding clonality, persistence, and transmissibility. One study in the UK (Western General Hospital,

Edinburgh) , found a clonal relationship among isolates

3 from 13 patients over six years . In contrast, during an eight year period at a US CF center (UNC Hospitals,

Chapel Hill) , not a single identical or closely-related strain was found among 23 infected clinic and lung

9 transplant patients . Serial isolate analysis further confirmed this picture, typically demonstrating persistent infection by a single strain.

There also existed an isolate collection from another CF center (Hospital for Sick Children, Toronto) where there was anecdotal evidence for an epidemic of

P. cepacia . Although the isolates were not characterized for genetic-relatedness, they had been uniformly resolved to express peritricious, giant cable (Cbl)-like pili that specifically bound to CF mucin and

4 11 airway epithelial cells '

As the cblA pilin subunit gene encoded by all

15 of the Toronto isolates was the first adhesin pilus subunit gene characterized for P. cepacia, a subsequent hybridization-based survey for the presence of cblA was carried out on multiple isolates from seven other CF centers in the US (Chapel Hill NC, Jackson MS, Norfolk VA, Cleveland OH, Phila. PA, New York NY) and Europe (Edinburgh) as well as clinical and environmental strains. All of these isolates were cblA except for one isolate from a CF center in Jackson Mississippi.

By examining the genetic-relatedness of cblA and cblA strains, we have discovered the emergence of a highly transmissible lineage, seemingly adapted for efficient transmission in the CF population. Resolved genetic markers uniquely associated with this lineage, which may be used to rapidly identify its presence, are of immediate practical importance in CF centers in both Europe and North America.

SUMMARY OF THE INVENTION

It is an object of this invention to provide chromosomal restriction fragment length polymorphism (RFLP) patterns (generically referred to as DNA based fingerprints) and DNA sequences for identifying highly transmissible lineages of Pseudomonas cepacia .

More specifically, the present invention provides DNA based fingerprints for identifying highly transmissible lineages of Pseudomonas cepacia produced by: (a) ribotyping analyses, i.e., the determination of RFLPs associated with the multicopy RNA operon(rrn); and (b) pulsed field gel electrophoresis (PFGE)-based resolution of chromosomal macro-RFLP patterns. Another object of this invention is to provide isolated DNA molecules encoding a 17-KDa major subunit pilin protein (cblA) of cystic fibrosis- associated, epidemic and non-epidemic Pseudomonas cepacia strains. Also contemplated is the recombinant production of polypeptides using the isolated DNA molecules. The recombinant polypeptides are then used to produce antibodies for use in methods for identifying epidemic strains of Pseudomonas cepacia .

It is also an object of this invention to provide unique primer oligonucleotide sequences derived from cblA gene variants for use in polymerase chain reaction (PCR)-based methods for detection of highly transmissible strains of Pseudomonas cepacia .

It is also an object of this invention to provide DNA probes derived from unique regions of variant cblA gene sequences for detection of highly transmissible strains of Pseudomonas cepacia .

It is also an object of this invention to provide methods for identifying highly transmissible lineages of Pseudomonas cepacia that utilize the above DNA based fingerprints, oligonucleotide primers and DNA probes.

It is a further object of this invention to provide diagnostic kits for identifying highly transmissible strains of Pseudomonas cepacia .

Brief Description of the Dra inπs

Figs 1 A-C together depict RFLPs of 17 P. cepacia isolates cited in the specification. Lane order for the 17 isolates is maintained in all three figures. Isolate numbers of examined strains appear at the top of the figure immediately above each lane. Subscript letters preceding isolate number indicate CF center from which P. cepacia (PC) strain was isolated: PC_E, Edinburgh Scotland; PC_MS, Jackson MS; PC_N , Chapel Hill NC; PC_NY, New York NY; PC_0H, Cleveland OH; PC_T, Toronto Canada, and C_V / Norfolk VA.

Fig. IA. : PFGE-resolved Spel RFLPs. As

9 described previously , samples were prepared and restriction fragments separated by pulsed field gel electrophoresis with a CHEF Mapper system (BIO-RAD) through 1% agarose using a field strength of 6 V/cm and an initial and final pulse time of 1.2 sec and 54 sec, respectively. Fragment sizes were determined using a λ concatenate ladder. Bar-code format translation of chromosomal fingerprint profiles was made using a

Macintosh Quadra 950 running Gene Construction Kit

(Texto) . Fragments below 100 kbp are not shown. In the latter range, Toronto and Edinburgh isolates displayed in lanes 1-8 had two identical fragments (60 kbp and 48 kbp) . Other isolates (lanes 9-17) had polymorphic sets of three to six fragments in this lower range.

Fig. IB: rrn EcoRI RFLPs. Southern blot hybridization methods were as we described

, 9, 26 . 32„ . . . . previously using a P-labeled rn the entire rrnB operon of E. coli K12. Fig. IC: cJJA hybridization analysis of EcoRI generated RFLPs. This was accomplished by stripping bound rrn probe from the membrane used in

Fig. IB, followed by hybridization with a previously

4 26 ddee:scribed cblA gene probe using standard methods ' 27

Fig. 2: rrn-RFLP based phylogenic tree of representative isolates from patients at seven CF centers in North America (Chapel Hill NC, Jackson MS, Norfolk VA, Cleveland OH, Philadelphia PA, New York NY, Toronto Ontario) and Europe (Edinburgh) plus environmental and clinical (non-CF) sources. All cited isolates are described in the text and in the Methods section. Indicated isolate number is followed by

+ source (CF, environmental or clinical) . cblA± , cblAi , isolate (s) that encode the cblA gene (Fig. IC) and express adhesin Cbl pili (Fig. 3) . cblA-t_ , identical 501 bp sequence carried by Toronto and Edinburgh CF center isolates (Fig. 4) ; cblA₂ , polymorphic 501 bp sequence carried by Jackson

Mississippi CF center isolate PC_MS_2323 (Fig. 4) .

Number above each branch indicates the percentage of time each was joined together under bootstrap

14 analysis (confidence intervals less than 10 have been omitted for clarity) . The lineages included in this tree are representative of the larger sample of isolates collected. Multiple CF patient serial isolates of an identical rrn RFLP profile have not been included as they do not affect the tree topology.

However, multiple isolates from Toronto (PC_T_5, PC_T_7) and Edinburgh (PC_E_1359,PC_E_2315) CF centers are noted because further analysis by DNA sequence revealed that the cblA genes encoded by these four isolates are identical (Fig. 4) . The remaining 13 and 11 isolates, respectively, from each of these two CF centers are members of the indicated epidemic lineage based on 100% correlation of their rrn RFLP profiles with those of the prototypic patterns of the Toronto/Edinburgh isolates shown in Fig. IB.

Fig. 3: Transmission electron micrograph of Toronto epidemic strain PC_T_7 expressing Cbl adhesin pili. High resolution was achieved with a JOEL 100 CX electron microscope as pre- 11 electron microscope as previously described . Bar in lower right corner, 0.1 μm

Fig. 4: Identical 501 bp sequence (top-most line) of the cblA structural gene encoded by 2 prototypic Toronto epidemic isolates (PC_T_7 and PC_T_5) and 2 prototypic Edinburgh isolates (PC_E_2315 and PC_E_ 1359) compared to the variant cblA sequence carried by the single Jackson Mississippi CF center isolate PC^g. 2323 (lower line) .

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention is directed to the identification of highly transmissible strains of Pseudomonas cepacia using DNA based fingerprints. More specifically, such identification

SUBST ESHEET(RUL!2S is accomplished by the use of fingerprints produced by: (a) ribotyping analyses, i.e., the determination of restriction fragment length polymorphisms (RFLPs) associated with the multicopy RNA operon (rrn) ; and (b) pulsed field gel electrophoresis (PFGE)-based resolution of chromosomal macro-RFLP patterns.

The techniques for ribotyping are known to those of skill in the art. Generally, DNA samples from bacterial strains are isolated, digested with a restriction endonuclease such as EcoRI and separated by agarose gel electrophoresis. The DNA fragments from the agarose gel are then transferred to nitrocellulose membranes and probed with either radiolabeled or chemiluminescent E. coli ribosomal RNA {rRNA) probes. The RFLPs detected by hybridization with the E. coli rRNA probes are then analysed to categorize the bacterial strains according to their distinctive bands of rRNA encoding DNA (i.e., ^MDNA fingerprints").

Similarly, the skilled artisan would also be familiar with the published methods for PFGE-based resolution of chromosomal macro-RFLP patterns. Such methods involve immobilization of bacteria in agarose plugs after which the bacteria are lysed and the DNA is digested with an infrequently (rare) cutting restriction endonuclease such as Spel . The agarose plugs with the digested DNA are then subjected to transverse alternating field electrophoresis in agarose gels using commercially available equipment. The gels are generally stained with ethidium bromide or cyber- green and the resulting chromosomal RFLPs are analysed preferably after computer-generated translation of the RFLP profiles into bar code format using commercially available equipment and software. Also well known to those skilled in the art are the standard criteria for the statistical analysis of the ribotypes and the chromosomal RFLP profiles obtained using the above methods. Using these statistical criteria, bacterial strains may be compared and their genetic relationships characterized. In the present invention, the determination of whether a given strain of Pseudomonas cepacia is highly transmissible is made by comparing its ribotypic and chromosomal RFLP profiles to those of a known strain of highly transmissible lineage.

In another aspect of the present invention, isolated DNA molecules encoding a 17-KDa major subunit pilin protein (cblA) of cystic fibrosis-associated, epidemic and non-epidemic Pseudomonas cepacia strains are provided. The genes encoding the cblA protein are isolated and sequenced by standard techniques. The cblA genes isolated from epidemic and non-epidemic strains of Pseudomonas cepacia enabled for the first time the comparison of those variant genes and the identification of the differences in their nucleotide sequences.

The isolated DNA molecules are then used for the recombinant production of the 17-kDa major subunit pilin protein (cblA) of cystic fibrosis-associated, epidemic and non-epidemic Pseudomonas cepacia strains. As such, the present invention also contemplates recombinant DNA molecules containing the above DNA molecules and unicellular hosts transformed with those recombinant DNA molecules. The recombinant polypeptides and their fragments are then used for the production of antibodies that can distinguish epidemic and non-epidemic strains of Pseudomonas cepacia in

SUBSTTTiπi SHEET (RULE 2δ) standard immunologic assays such as ELISA, radioimmunoassay and western blots. The methods for recombinant protein production, protein purification and generation of antibodies are well within the purview of the ordinary skilled artisan.

This invention also provides unique primer oligonucleotide sequences derived from cblA gene variants for use in polymerase chain reaction (PCR)- based methods for detection of highly transmissible strains of Pseudomonas cepacia . These unique primers are usually synthesized using standard procedures following identification of desirable nucleotide sequences based on the comparison of the cblA gene sequences of epidemic and non-epidemic strains of Pseudomonas cepacia . The PCR techniques contemplated by and used in the present invention are well known. Essentially, the primers, DNA from the bacterial strain to be tested and a thermostable PCR enzyme are mixed and the reaction carried out according to established procedure in a thermocycler block. The PCR products are then analyzed generally by electrophoretic separation.

The invention also provides DNA probes derived from unique regions of variant cblA gene sequences that may be used in standard hybridization based assays such as colony hybridization or Southern blot transfers to detect highly transmissible strains of Pseudomonas cepacia .

The present invention also contemplates the use of the above DNA based fingerprints, oligonucleotide primers, DNA hybridization probes, polypeptides and antibodies in diagnostic kits for the

BSΠΠJTE SHEET Buna) detection of highly transmissible lineages of Pseudomonas cepacia .

In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

Materials and Methods Bacterial isolates:

133 Pseudomonas cepacia isolates were obtained from the following sources: (i) sixty-five isolates from patients at the University of North Carolina Cystic Fibrosis Center (1985 through 1993) including 17 clinic and 5 transplant patients, 4 of whom were infected transfers from other distant

9 11 locations as described previously ' . Those cited in the Figures include isolates PC_NC_566, PC_NC_1711, PC_NC_

1845, PC_NC_1910, PC_NC_1948, PC_NC_2008, PC_NC_2028, PC_NC_ 2211, PC_NC_2225 and PC_NC_2747, each from different local clinic patients, and four isolates from infected transfer patients (PC_NY_792 from New York, New York, PC_VA_1963 from Norfolk Virginia, PC_OH_2034 from Cleveland Ohio and the cblA isolate PC_MS_2323 from Jackson Mississippi) ; (ii) 8 ATCC clinical and environmental isolates: human endocarditis

13945, human urinary tract PC_ATCC_17765, human bronchial PC^cc-²⁵⁶⁰⁹' human tibia fracture PC_ATCC_ 27515, forest soil (Trinidad) PC_ATCC_17759, onion PC_ATCC_25416 (the ATCC P. cepacia type strain), soil (California) PC_ATCC_29352, cornfield soil (New Jersey)

SUBSTTTUTE SHEET (RULE 26) 9 11 PC_ATCC_39277, all as described previously ' ; (iii) 15 c--ZA isolates from 15 CF center clinic patients at the

Hospital for Sick Children (1987 through 1988),

Toronto. Those cited in the Figures include isolates

4 11 PC_T_5, PC_T_7, PC_T_19, PC_T_25 ' ; (iv) two isolates,

PC_E_SBC27 and PC_E_SBC29, from two CF patients at

Western General Hospital (Edinburgh Scotland) that were not associated with epidemic transmission within this

3 4 11 CF center ' ' ; (v) four isolates from clinic patients at Western General Hospital (1989 through

1990) , Edinburgh, all which had been associated with

₃ epidemic transmission within this CF center . Those cited in the Figures include isolates PC_E_509, PC_E_

1359, PC_E_1392 and PC_E_2315; (vi) ten isolates from 10

2 patients at a Philadelphia Pennsylvania CF center .

Those cited in the Figures include isolates PCp_A_535, PC_PA_542 and PCp_A_544; (vii) twenty-four isolates from

24 patients at the Rainbow Babies and Children's

. 17 Hospital, Cleveland Ohio including cited isolate PC_OH_524, PC_OH_525 and PC_OH_2034; and(viii) 5 bronchial isolates from 5 ventilator patients at Boston City

Hospital (Massachusetts) including cited isolate C^.

3137.

Restriction Fragment Length Polymorphism Analyses Ribotyping:

Chromosomal DNA was prepared using the following procedure. Overnight cultures were diluted 10-fold in 10 ml of LB and incubated at 37°C until they reached mid-log phase. Cells were pelleted, washed twice with 0.9% NaCL, and then resuspended in cold TE (10 mM Tris-HCl, 10 mM EDTA [pH 8.0]). Lysozyme was added to a final concentrations of 20 mg/ml, and the solution was incubated at 37°C for 30 min. Proteinase K (20 mg/ml in TE; Sigma) and sodium dodecyl sulfate were added to final concentration of 20 μg/ml and 0.1%, respectively. The lysates were incubated overnight at 50°C. Sarcosine-free acid (Sigma) was added to a final concentration of 2%, and the solution was mixed gently.

The DNA was then purified by cesium chloride-ethidium

20 bromide equilibrium density gradient centrifugation.

EcoRI rRNA {rrn) restriction fragment length polymorphisms (RFLPs) for phylogenetic analyses were analyzed by using an rrnB probe spanning the entire

28 rrnB operon of Escherichia coli K-12. Plasmid DNA

29 was purified by the alkaline lysis method , followed by cesium chloride-ethidium bromide gradient

20 centrifugation. The DNA restriction fragment used to generate the probe was separated by horizontal slab gel electrophoresis in 0.8% low-melting point agarose. The relevant restriction fragment from a slice of the gel was radiolabeled in the agarose by random

32 oligonucleotide priming with [ - P.dCTP (Dupont, NEN

30 Research Products) .

The chromosomal DNA prepared according to the above-described method was restricted with JEcoRl and the fragments were separated by agarose gel electrophoresis. Following electrophoresis, restriction fragments were transferred to nitrocellulose membranes and hybridized with the rrnB probe described above. Hybridized bands were visualized by autoradiography and the banding patterns

9 26 were analysed using standard methods. '

Pulsed-field gel electrophoresis (PFGE) : Cells were grown to early log phase in LB, harvested by centrifugation, washed with 1:1 TE buffer, resuspended in 1 ml of 10:1 TE buffer, and mixed with 1.2 volumes of melted 1% InCert agarose (Bio-Rad) in TE buffer. The mixture was dispensed into 120-μl insert molds (Pharmacia) and allowed to solidify on ice. Plugs were sliced and incubated in 20 μg of lysozyme per ml at 37°C for 12 h. The lysozyme buffer was replaced with ESP buffer (0.5 MEDTA [pH9], 1% sarcosyl, 200 μg of proteinase K [Sigma] per ml) , and the plugs were incubated at 37°C for 5 h and then washed with TE buffer for 4 h at 37°C. Single plug slices were incubated with Spel (Boehringer Mannheim) in restriction enzyme buffer for 2 h. Restriction fragments were separated by PFGE using a CHEF Mapper system (Bio-Rad) through 1% agarose (Bio-Rad; Molecular Biology Certified grade) in 0.5 X TBE (1 X TBE is 0.9 M Tris-HCl, 0.9 M boric acid, and 1.0 mM EDTA) . Electrophoresis was performed for 24h in 0.5 X TBE buffer at 14°C, with a field strength of 200 V (6V/cm) and initial and final pulse times of 1.2 and 54 s, respectively. Lambda concatemers were used as DNA size standards. The gels were stained with ethidium bromide or cyber-green and photographed under UV transillumination with a Polaroid camera. Fragment sizes were determined, and the computer-generated translation of chromosomal fingerprint profiles was made into bar code format by using a Macintosh Quadra 950 and Gene Construction Kit (Texto) software.

Statistical Analysis of Ribotypes and PFGE Patterns:

According to established criteria for P.

24 cepacia , strains were assigned to the same ribotype when comparison of sizes of hybridizing fragments revealed 3 or fewer bands differing between the two patterns under comparison.

Standard criteria were used for comparing

23 PFGE chromosomal fingerprints. Briefly, PFGE patterns were considered different (i) when they had the same number of DNA fragments but when the size of at least one band varied by more than I standard deviation (5%), (ii) when they exhibited a different number of DNA fragments, or (iii) when the sum of the sizes of the differing bands in the first PFGE pattern did not correspond to that of the differing DNA fragments in the second PFGE pattern.

Quantitative pairwise comparison of both types of RFLP patterns was accomplished using the Dice coefficient of similarity calculated as D = 2n_χy/ (n^ + n₂), where n^ is the total number of DNA fragments from strain X, n₂ is the total number from strain Y, and n_χv

12 the number of fragments identical in the two strains '

25 . The coefficient of similarity for two PFGE RFLPs

D>0.90 represents closely related strains, while unrelated strains have D<0.60. Intervening values, remarkably, were not observed, and values between 0.5

9 and 0.6 are rare . For rrn RFLPs, given that P. cepacia strains typically display 7-10 distinct hybridizing bands, the shared ribotype (above) would correspond to D=0.79 to 0.85. Comparisons between mean values were performed by Student's t test using a

Systat program (Systat Inc.) . Amplification and Sequencing of the cblA Gene:

9 Using previously described methods , CsCl

20 equilibrium density gradient purified chromosomal DNA was isolated from the two prototypic Toronto epidemic isolates (PC_T.7 and PC_T_5) , the two prototypic Edinburgh epidemic isolates (PC_E_2315 and PC_E_1359) and the single Jackson Mississippi CF center isolate PC_MS_2323.

From each of these cblA probe-positive chromosomes, the

21 cblA gene was PCR amplified using a DNA thermocycler

(Perkin-Elmer) with a GeneAmp PCR Core Reagents Kit

{ibid.). Based on the previously determined sequence of the c-lA gene encoded by isolate PCrp_74, sense and antisense primers used for these reactions were 5'- CCAAAGGACTAACCCA-3' and 5^»ACGCGATGTCCATCACA-3', respectively. PCR reactions were: cycle 1, 2 min at 94°C, 2 min at 37°C, 1 min at 72°C. The remaining 29 cycles were: 1 min at 9 °C, 1 min at 45°C, 1 min at 72°C, followed by 7 min extension at 72°C. PCR products were electrophoresed through 0.8% agarose and for each a single band was observed with ethidium bromide staining. Bands were electroeluted into DEAE membrane (Schleicher & Schuell) and cloned with a TA Cloning Kit

(Invitrogen) . DNA sequences were determined by the

22 Sanger dideoxy method with the same primers used for

PCR amplication (above) . Five PCR-amplified c lA gene clones of PC_MS_2323 were generated, three of which were sequenced for confirmatory purpose, with no variation resolved.

PCR Amplification Detection, Using Unique Primer Oligonucleotide Sequences Derived From cblA Gene Variants:

Method A: This method was based upon three 'sets' of VARIANT EXTERNAL (downstream to 3' end of cblA gene) anti-sense PCR primers and COMMON INTERNAL sense primers (at 5' end of C- JA gene) . Each set specifically amplifies only one or another of the 3 known variant cblA gene sequences, including that uniquely associated with the Toronto/Edinburgh (T/E) epidemic clone. • To amplify only T/E* epidemic lineage clones the following primers were used to generate a 573 base pair (bp) PCR product: common sense primer:

1 ATGCT GAAAT ACGTT CCGAT

20** variant anti-sense primer: 573

ATGGT TTTTC AGGA GT

558***

* T/E (Toronto/Edinburgh) : the sough^ after, highly transmissible, epidemically spread cblA clone, for example isolates PC_T-5 clone, PC_T-7, PC_E-1359, PC_E-2315.

** 1-20: in accord with conserved region of cblA gene sequence, numbered as in Fig. 4.

*** 558-573: unique sequence beginning 57 bp (base pairs) downstream of 3' end of T/E clone cblA gene, i.e. end of cblA gene sequence is bp No. 501, thus 501 + 57 = 558.

RULE 26 To amplify only MS_p-type* (non-epidemic) the following primers were used to generate a 573 base pair (bp) PCR product: common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20** variant anti-sense primer: 573 AGGAT TTCCA AAGGA GT

557*** • To amplify only MS_w-type**** (non-epidemic) the following primers were used to generate a 573 base pair (bp) PCR product: common sense primer:

1 ATGCT GAAAT ACGTT CCGAT

20* variant anti-sense primer: 573

TGACT TCCGA AGGAC TACT

* MSp-type: non-epidemic, negligible transmissibility cblA strain from Mississippi, for example isolate PC_MS-2323.

** In accord with conserved region of cblA gene sequence, numbered as Fig. 4.

*** 557-573: unique sequence beginning 56 bp downstream of 3' end of MSp-type cblA gene, i.e. end of cblA gene sequence is bp No. 501, thus 501 + 56 = 557.

**** MS_w-type: anothe^ non-epidemic, negligible transmissibility cblA strain from Mississippi.

H E 26 555*

Method B:

This method was based upon three 'sets' of VARIANT EXTERNAL (downstream to 3' end of cblA gene) anti-sense PCR primers and VARIANT INTERNAL sense primers (at 5' end of cblA gene) . Each set specifically amplifies only one or another of the 3 known variant cblA gene sequences, including that uniquely associated with the Toronto/Edinburgh (T/E) epidemic clone. To amplify only T/E epidemic lineage clones the following primers were used to generate a 331 base pair (bp) PCR product: variant sense primer: 243 GACTG CCCCG GCTTT GAA

260** variant anti-sense primer: 573

ATGGT TTTTC AGGAG T 558***

• To amplify only MSp-type (non-epidemic) the following primers were used to generate a 331 base pair PCR product:

* 555-573: unique sequence beginning 54 bp downstream of 3' end of MS_w-type cblA gene, i.e. end of c-JA gene sequence is bp No. 501, thus 501 + 54 = 555.

** 243-260: in accord with cblA gene sequence numbering in Fig. 4 for epidemic isolates PC_T-5, PC_T-7, PC_E-1359, PC_E-2315.

*** 558-573: unique sequence beginning 57 bp (base pairs) downstream of 3' end of T/E clone cblA gene, i.e. end of c-biA gene sequence is bp No. 501, thus 501 + 57 = 558. variant sense primer : 243

GGCCG AGCCG GCGCT GAA

260* variant anti-sense primer:

573

AGGAT TTCCA AAGGA GT

557** • To amplify only MS_w-type (non-epidemic) the following primers were used to generate a 331 base pair PCR product: variant sense primer: 243

GACCG CTCCG TCGCT CAA 260*** variant anti-sense primer: 573

TGACT TCCGA AGGAC TACT

555****

* 243-260: in accord with cblA gene sequence numbering in Fig. 4 for non-epidemic Mississippi isolate PC_MS-2323.

** 557-573: unique sequence beginning 56 bp downstream of 3' end of MSp-type cblA gene, i.e., end of b-ZA gene sequence is bp No. 501, thus 501 + 56 = 557.

*** 243-260: this (MS_w-type) is a recently discovered variant c-blA gene sequence carried by another non- epidemic strain of negligible transmissibility from Mississippi.

**** 555-573: unique sequence beginning 54 bp downstream of 3' end of MS_w-type c-blA gene, i.e. end of c-bJA gene sequence is bp No. 501, thus 501 + 54 = 555. Method C:

This method was based upon three 'sets' of Internal (within cbJA gene) PCR primers. Each set specifically amplifies only one or another of the 3 known variant cblA gene sequences, including that uniquely associated with the T/E epidemic clone.

METHOD Cl: was based on 'common' sense primer and variant antisense primers.:

• To amplify only T/E epidemic lineage clones the following primers were used to generate a 427 base pair (bp) PCR product: common sense primer: 1

ATGCT GAAAT ACGTT CCGAT 20* variant anti-sense primer: 427

CTTCG ACCTT CTTCT GACC

409**

* 1-20: in accord with conserved region of c-bIA gene sequence, numbering as in Fig. 4.

** 427-409: in accord with cblA gene sequence numbering in Fig. 4 uniquely for clonal epidemic isolates PC_T-5, PC_T-7, PC_E-1359, PC_E-2315. To amplify only MSp-type (non-epidemic) the following primers were used to generate a 427 base pair (bp) PCR product: common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20** variant anti-sense primer: 427 CAGCG ACAGT TTTCT GGCC

409*

• To amplify only MS -type (non-epidemic) the following primers were used to generate a 427 base pair (bp) PCR product: common sense primer:

1 ATGCT GAAAT ACGTT CCGAT

20** variant anti-sense primer: 427

CAGCG ACAGT TTTCT GGCC

409***

* 427-409: in accord with cb-ZA gene sequence numbering in Fig. 4 uniquely for non-epidemic Mississipi isolate PC_MS-2323.

*** 427-409: this (MS_w-type) is a recently discovered variant c-bIA gene sequence carried by a non-epidemic strain of negligible transmissibility from Mississippi.

' METHOD C2: was based on variant sense primers and variant antisense primers.:

• To amplify only T/E epidemic lineage clones the following primers were used to generate a 185 base pair (bp) PCR product: variant sense primer: 243

GACTG CCCCG GCTTT GAA

260* variant anti-sense primer:

427

CTTCG ACCTT CTTCT GACC

409** To amplify only MSp-type (non-epidemic) the following primers were used to generate a 185 base pair (bp) PCR product: variant sense primer: 243

GGCCG AGCCG GCGCT GAA 260*** variant anti-sense primer: 427

CTCCG GCCGT CTTCT GTTC

* 243-260: in accord with cblA gene sequence numbering in Fig. 4 for epidemic isolates PC_T-5, PC_T-7, PC_E-1359, PC_E-2315.

** 427-409: in accord with cblA gene sequence numbering in Fig. 4 for epidemic isolates PC_T-5, PC_T-7, PC_E-1359, PC_E-2315.

*** 243-260: in accord with cblA gene sequence numbering in Fig. 4 for non-epidemic Mississippi isolate PC_MS-2323. 409^<

• To amplify only MS -type (non-epidemic) the following primers were used to generate a 185 base pair (bp) PCR product: variant sense primer:

243

GACCG CTCCG TCGCT CAA

260** variant anti-sense primer: 427

CAGCG ACAGT TTTCT GGCC

409***

* 427-409: in accord with αb-ZA gene sequence numbering in Fig. 4 for non-epidemic Mississippi isolate PC_MS-2323.

** 243-260: this (MS_w-type) is a recently discovered variant cbJA gene sequence carried by another non- epide ic strain of negligible transmissibility from Mississippi.

*** 427-409: this (MS_w-type) is a recently discovered variant cblA gene sequence carried by another non- epidemic strain of negligible transmissibility from Mississippi. The probability of the random occurrence of 16- to 19-base long sequences, such as the specific primers employed in any of the above methods, is once every 4 „¹⁶ /(or ca. one i•n 1 ι r0s ⁹ s) 4t-o 4/.19 /(or ca. one in 10 11) bp x 2. Given that the size of the P. cepacia genome is ca. 7 x 10 bp, the chance occurrence would be remote.

In order to determine whether a given sample contains a transmissible strain of P. cepcia any one of the above pairs of primers corresponding to the T/E epidemic lineage is used in standard PCR reactions with DNA from the bacterial strain to be tested.

Following PCR amplification, the reaction products are analyzed for the presence of the appropriate size PCR product using standard methods.

For example, in Method A (when the pair of primers for the T/E epidemic lineage is used) , the detection of a 573 bp PCR product by electrophoresis on an agarose gel would indicate that the sample contains a strain of the highly transmissible T/E. Whereas, the absence of such a PCR product would indicate otherwise. Similarly in Methods B, Cl and C2, the presence of a strain of the highly transmissible T/E would be confirmed by PCR products of 331 bp, 427 bp and 185 bp, respectively.

Hybridization-specific detection, based on 'probes' derived from unique regions of variant cblA gene sequences:

Each unique probe sequence shown below can be generated by restriction endonuclease double digestion of one or another of the three cblA gene variants, based on:

(i) common Smal site at bp (base pair) 273-278 (C CCGGG) . flanking 3' end of cb-ZA probe

(ii) common BsrI site at bp 371-376 (AAC TGG) . flanking 5' end of cblA probe

the c lA-specific DNA probes:

• Hybridization with only T/E epidemic lineage clones

Unique T/E cblA gene 'probe' sequence:

273

C CCGGG CGCGG CGGAA ATTCC GCTGT CGGTC AAGCT TGGCG AAACC GAGCT GACCA CCACG GCCGC GACGC TGAAG ACCGC AGAGC TCTTC CCCGG CGAAC TGG

376*

* 273-376: in accord with cblA gene sequence numbering in Fig. 4 for epidemic isolates PC_T-5, PC_T-7, PC_E-1359, PC_E-2315. Hybridization with only MSp-type (non-epidemic) strains

Unique MSP-type cblA gene 'probe' sequence: 273 C CCGGG CGCCA AGGAG ATTCC GCTGG CGGTC AAGCT

GGGCA CCACC GCGCT GAGCA CCACG GCGAC GACGC TGAAG GCGTC GGAGA TCTTT ACCGG CGAAC TGG

376*

Hybridization with only MSW-type (non-epidemic) strains

Unique MS_w-type cb-ZA gene 'probe' sequence: 273

C CCGGG TGCGG CCGAA ATTCC GTTGG CTGTC AAACT GGGCG ACACC GAGTT GAGCA CGACG TCGGC GACCC TGAAG GCCGC GGAAC TCTTT CCCGG CGAAC TGG

376**

The probes for the T/E lineage disclosed above can be used in standard hybridization methods, such as colony hybridization assays or Southern blot transfer, to detect the presence of the highly transmissible P. cepacia strain of the T/E lineage.

* 273-376: in accord with cblA gene sequence numbering in Fig. 4 for non-epidemic Mississippi isolate PC_MS-2323.

** 273-376: this (MS -type) is a recently discovered variant cb-ZA gene sequence carried by another non- epidemic strain of negligible transmissibility from Mississippi. RESULTS

The above-described methods were used to characterize the epidemiological-relatedness of the 15 Toronto isolates expressing mucin-binding Cbl pili. To investigate the genetic relationship between these isolates and those found elsewhere, we included clinical and environmental isolates as well as 78 strains from the seven other CF centers cited above that were c-b-ZA . At the time the report of RFLP- identical P. cepacia isolates transmitted among patients at an Edinburgh CF center appeared and we

3 obtained the involved strains to include in this phylogenetic characterization. See Fig. IA and IB.

Profiles in lanes 9-17 of both figures depict typical polymorphic patterns resolved for isolates from different CF centers. For these isolates mean D (Dice

12 coefficient of similarity) for any pair by PFGE- resolved chromosomal macro-RFLP profile was 0.14±0.07

(Fig. IA) , a level of diversity not significantly different from that found previously among eight independently isolated ATCC environmental and clinical

9 control isolates . A similar degree of chromosomal

RFLP variability was found between the other CF- associated isolates from the seven CF centers (results not shown) , confirming that these are epidemiologically distinct strains with RFLP variability not significantly different from that of the random collection of ATCC strains (0.l>p>0.05) . Despite the

9 lower discriminatory power of ribotyping , a similar degree of phylogenic relationship among these CF- associated isolates is apparent in Fig. IB. The heterogeneity of the RFLP profiles of the isolates from the seven CF centers (e.g. lanes 9-17, Fig. IA and IB) is similar to that described in our previous study involving multiple isolates from 23

9 patients at the UNC CF center . This degree of variability contrasts markedly with the two closely- related, conserved RFLP patterns found for the 15 c JA encoding Toronto CF center isolates (lanes 1-4, Fig. IA and IB) . Here, by examining both PFGE and ribotype RFLP profiles, the coefficient of similarity amongst the Toronto isolates proved to be very high, with PFGE D = 0.95+0.03 and rrn D = 0.87+0.09. This contrasted with (a) the mean D values amongst isolates from the other seven CF centers which was very low (e.g. lanes 9-17, Fig. IA and IB), and (b) the mean D between the Toronto isolates and the other CF center isolates which was also very low: PFGE D = 0.20+0.07, rrn D = 0.39±0.09. These findings strongly suggest that all 15 of the Toronto CF center isolates were members of a unique lineage associated with an epidemic.

Displayed in lanes 5-8 of Fig. IA and IB are

P. cepacia PFGE and ribotype RFLP profiles of isolates

₃ from CF patients at the Edinburgh CF center . The RFLP profiles displayed in Fig. IA and IB support this picture, as D for any analyzed pair by either type of

RFLP profile was very high (PFGE D = 0.98±0.02, rrn D =

1.0). Further, pair-wise comparison of the Edinburgh strains to the closely-related Toronto CF center strains (lanes 1-4 of Fig. IA and IB) likewise produced robust D values (PFGE D = 0.97+0.03, rrn D =

0.90±0.04), strongly suggesting that the Edinburgh and

Toronto isolates are members of the same unique lineage, despite the Atlantic Ocean barrier. Based on ribosomal RNA {rrn) RFLP profiles, phylogenic relationships of the 133 isolates described above and in the Methods section were determined with

13 the neighbor-joining method . Confidence intervals on the tree topology were estimated by bootstrapping

14 analysis . See Fig. 2. The resultant phylogenic tree indicates that: (i) the cluster of Toronto and

Edinburgh isolates comprise a single, clonally-related lineage, only distantly related to all other isolates, and (ii) the remaining, independently isolated strains from other CF centers are as distantly-related to one another as they are either to the Toronto/Edinburgh clusters or to the independently isolated non-CF clinical and environmental strains. We then examined the epidemic Edinburgh

4 isolates to see if, like the Toronto strains , they expressed Cbl pili and encoded the cb-ZA gene.

Phenotypic survey using electron microscopy revealed that these highly transmissible strains expressed appendage pili that were structurally equivalent to

+ 4 tthhose expressed by all of the cb-ZA Toronto isolates ' 11

See Fig. 3. Genotypic survey was carried out by stripping rrn-probe from an EcoRI chromosomal digest membrane (Fig. IB) followed by hybridization with a cb-ZA probe. Fig. IC (lanes 1-8) indicates that four highly transmissible Edinburgh isolates as well as the closely-related Toronto clones encode cblA.

Because the implications of our studies have the potential to directly affect clinical management of some 70,000 CF patients in Europe and North America, we used DNA sequence analysis to test the RFLP-based conclusions that isolates from Toronto and Edinburgh were clonal. Primers were synthesized from the cb-ZA sequence of the Toronto isolate PC_T_74 and used for PCR-based amplification of the cbJA gene from isolates to be characterized. Resultant PCR products were then cloned and sequenced (see Methods) . Complete cblA sequences were thus obtained from isolates with the two slightly variant though closely-related RFLP profiles typical of the 15 Toronto CF center isolates (Fig. IA and IB, lanes 1-4), the two slightly variant though closely-related RFLP profiles typical of the 13

Edinburgh CF center isolates (Fig. IA and IB, lanes 5- 8) , and the significantly variant Jackson Mississippi CF-associated strain PC_MS_2323 (Fig. IA and IB, lane 17) . Comparison of these five sequences indicated that the chro osomally-encoded, 501 bp cb-ZA pilin subunit structural gene carried by the closely-related isolates from the Toronto and Edinburgh CF centers was invariant in sequence. In contrast, the cblA gene encoded by the distantly-related Jackson Mississippi strain PC_MS_2323 exhibited polymorphism at the sequence level, with changes in 60 bp of the 501 bp sequence (88% identity; see Figure 4) . The perfect conservation of the cb-ZA pilin sequence among isolates from multiple patients over four years at the Toronto and Edinburgh centers is precisely what would be expected for epidemic transmission of a highly infectious clone. Likewise, variability of cblA encoded by the Mississippi CF center isolate is in accord with that expected for a distantly-related isolate.

CONCLUSIONS

Consideration of these findings leads us to conclude: (i) isolates of P. cepacia axe not equally transmissible between CF patients; rather, there exists at least one significantly divergent, highly transmissible clonal lineage plus numerous moderately heterogeneous lineages of negligible transmissibility (see Fig. 2), and (ii) the highly transmissible lineage identified is responsible for epidemics at North American and British CF centers. This was most likely due to an as yet unidentified transatlantic transmission, possibly summer camp attendance. Based solely on rrn RFLP profiles or anecdotal evidence, additional reports exist suggesting the occurrence of P. cepacia transmission at CF centers

2 17 in Philadelphia and Cleveland , respectively. We characterized strains involved with both of these putative outbreaks (see Methods section) and found that by neither ribotype or macro-chromosomal RFLPs profile did the prototypic RFLP fingerprints of the putatively epidemic strains from either center appear similar to one another (mean D ≤ 0.3), nor to the unique, highly transmissible lineage involved with the Toronto and Edinburgh CF patients. Nonetheless, highly conserved RFLP profiles (mean D _> 0.85) within the individual outbreaks did support a picture of epidemic transmission within each of the two centers. When these isolates were further characterized, hybridization-based survey for the presence of the cblA pilin gene proved negative for all 35 of the involved strains. These results suggest that there may exist P. cepacia lineages of high transmissibility other than the cbJA clone that we have identified.

While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic disclosures can be altered to provide other embodiments which utilize the methods and compositions of this invention.

To the extent the following references include protocols or materials employed in the methods discussed herein, they are incorporated herein by reference.

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Claims

We Claim:

1. A method for detecting in a sample the presence of a strain of a transmissible lineage of Pseudomonas cepacia comprising the step of: analyzing the sample for restriction fragment length polymorphisms (RFLPs) linked to a strain known to be of a transmissible lineage of Pseudomonas cepacia .

2. The method according to claim 1 wherein the sample is analyzed by ribotyping.

3. The method according to claim 1 wherein the sample is analyzed by DNA fingerprinting using pulsed field gel electrophoresis.

. The method according to any one of claims 1-3 wherein the strain known to be of a transmissible lineage of Pseudomonas cepacia expresses cable adhesin type II pili.

5. The method according to claim 4 wherein the strain of Pseudomonas cepacia is of the highly transmissible Toronto/Edinburgh lineage.

6. The method according to claim 5 wherein the strain is selected from the group consisting of: PC_T_5, PC_T_7, PC_T_19, PC_T_25, PC_E_509, PC_E_1359, PC_E_1392 and PC_E_2315.

7. A method for detecting in a sample the presence of a strain of a transmissible lineage of Pseudomonas cepacia by polymerase chain reaction (PCR) using one or more pairs of oligonucleotide primers, said primers having nucleotide sequences identical to portions of a gene encoding a 17 kDa major subunit pilin protein of the cable adhesin type II _> cepacia pili.

8. A method for detecting in a sample the presence of a strain of a transmissible lineage of Pseudomonas cepacia by polymerase chain reaction (PCR) using one or more pairs of oligonucleotide primers selected from the group consisting of:

(a) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer:

573

ATGGT TTTTC AGGA GT

558;

(b) variant sense primer: 243

GACTG CCCCG GCTTT GAA

260 variant anti-sense primer: 573 ATGGT TTTTC AGGAG T

558; (c) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer:

427

CTTCG ACCTT CTTCT GACC

409; and

(d) variant sense primer: 243

GACTG CCCCG GCTTT GAA

260 variant anti-sense primer: 427 CTTCG ACCTT CTTCT GACC

409.

9. A method for detecting in a sample the presence of a strain of a transmissible lineage of Pseudomonas cepacia by DNA hybridization using a DNA probe having the DNA sequence: 273

C CCGGG CGCGG CGGAA ATTCC GCTGT CGGTC AAGCT TGGCG AAACC GAGCT GACCA CCACG GCCGC GACGC TGAAG ACCGC AGAGC TCTTC CCCGG CGAAC TGG 376.

B ET

10. A pair of oligonucleotide primers selected from the group consisting of:

(a) common sense primer: 1 ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer: 573

ATGGT TTTTC AGGA GT 558;

(b) variant sense primer: 243

GACTG CCCCG GCTTT GAA

260 variant anti-sense primer:

573

ATGGT TTTTC AGGAG T

558;

(c) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer: 427 CTTCG ACCTT CTTCT GACC

409; and (d) variant sense primer: 243

GACTG CCCCG GCTTT GAA

260 variant anti-sense primer:

427

CTTCG ACCTT CTTCT GACC

409.

11. A pair of oligonucleotide primers selected from the group consisting of:

(a) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer:

573

AGGAT TTCCA AAGGA GT

557;

(b) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer: 573 TGACT TCCGA AGGAC TACT

555;

(c) variant sense primer: 243

GGCCG AGCCG GCGCT GAA 260 variant anti-sense primer: 573

AGGAT TTCCA AAGGA GT

557;

(d) variant sense primer:

243

GACCG CTCCG TCGCT CAA

260 variant anti-sense primer: 573

TGACT TCCGA AGGAC TACT

555;

(e) common sense primer: 1 ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer: 427

CTCCG GCCGT CTTCT GTTC 409;

(f) common sense primer: 1

ATGCT GAAAT ACGTT CCGAT

20 variant anti-sense primer:

427

CAGCG ACAGT TTTCT GGCC

409; (g) variant sense primer: 243

GGCCG AGCCG GCGCT GAA

260 variant anti-sense primer:

427

CTCCG GCCGT CTTCT GTTC

409; and

(h) variant sense primer :

243

GACCG CTCCG TCGCT CAA

260 variant anti-sense primer: 427

CAGCG ACAGT TTTCT GGCC

409.

12. A DNA probe having the DNA sequence: 273

376.

13. A DNA probe having a DNA sequence selected from the group consisting of: (a) C CCGGG CGCCA AGGAG ATTCC GCTGG CGGTC AAGCT GGGCA CCACC GCGCT GAGCA CCACG GCGAC GACGC TGAAG GCGTC GGAGA TCTTT ACCGG CGAAC TGG; and

(b) C CCGGG TGCGG CCGAA ATTCC GTTGG CTGTC AAACT GGGCG ACACC GAGTT GAGCA CGACG TCGGC GACCC

TGAAG GCCGC GGAAC TCTTT CCCGG CGAAC TGG.

14. A diagnostic kit comprising at least one pair of oligonucleotide primers of claim 10.

15. A diagnostic kit comprising the DNA probe of claim 12.

16. An isolated DNA molecule encoding the 17 kDa major subunit pilin protein of the cable adhesin type II _{# cepacia} pilus.

17. The DNA molecule according to claim 15 having a DNA sequence selected from the two DNA sequences depicted in Figure 4.

18. A recombinant DNA molecule comprising a DNA molecule of claim 16.

19. A unicellular host transformed with a recombinant DNA molecule according to claim 18.

20. A method of producing a polypeptide comprising the step of culturing a unicellular host transformed with a recombinant DNA molecule according to claim 18.

21. A polypeptide produced according to the method of claim 20.

22. An antibody directed against a polypeptide of claim 21.

23. A diagnostic kit comprising a polypeptide of claim 21.

24. A diagnostic kit comprising an antibody of claim 22.