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Published ahead of print on April 15, 2005, doi:10.1165/rcmb.2004-0352OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 56-64, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0352OC

Identification of DNA Markers for a Transmissible Pseudomonas aeruginosa Cystic Fibrosis Strain

Deborah A. Lewis, Andrew Jones, Julian Parkhill, David P. Speert, John R. W. Govan, John J. LiPuma, Stephen Lory, A. Kevin Webb and Eshwar Mahenthiralingam

Cardiff School of Biosciences, Cardiff University, Cardiff, Wales; Manchester Adult Cystic Fibrosis Centre, Wythenshawe Hospital, Southmoor Road, Manchester; Pathogen Sequencing Unit, The Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge; Department of Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh, Scotland, United Kingdom; Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada; Department of Pediatrics, University of Michigan, Ann Arbor, Michigan; and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Eshwar Mahenthiralingam, Ph.D, Senior Lecturer, Cardiff School of Biosciences, Main Building, Museum Avenue, Cardiff University, Cardiff, Wales CF10 3TL, UK. E-mail: MahenthiralingamE{at}cardiff.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
A number of transmissible Pseudomonas aeruginosa strains have been identified which potentially constitute an emerging threat to patients with cystic fibrosis (CF). We sought to identify DNA markers that were specific to a transmissible P. aeruginosa CF clone and evaluate these probes on a large collection of genotypically distinct P. aeruginosa strains. Using subtractive DNA hybridization, in combination with analysis using the P. aeruginosa PAO1 genome chip, DNA markers specific for or absent from the Manchester transmissible CF strain (MA) were identified. Five subtractive DNA hybridization markers (MA15, MA18, MA21, MA22, and MA30) were found to be specific to strain MA and were located within a novel 13,318-bp genomic island, designated the MA island. The MA island encoded 18 genes and consisted of two bacteriophage-like regions; one region encoded the MA-specific subtractive hybridization markers, while the other bacteriophage-like region contained a Vibrio cholera-like toxin gene. Probes MA15, MA18, MA21, MA22, and MA30 were all found to be specific to strain MA when a collection of 141 P. aeruginosa strains was examined by hybridization with each DNA marker. In contrast, a previously isolated DNA marker for the Liverpool transmissible CF strain, PS21, was not found to be specific, detecting two additional strain types in the collection screened. Both the Manchester and Liverpool strain types were not encountered in CF populations outside the United Kingdom. The MA genomic island and Vibrio cholera–like toxin gene within it constitute novel genetic factors associated with a transmissible P. aeruginosa strain and their role in pathogenesis remains to be determined.

Key Words: cystic fibrosis • transmission • identification • genomic island


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Patients with cystic fibrosis (CF) who acquire chronic Pseudomonas aeruginosa infections are at increased risk of morbidity and mortality. Recently, multiple antibiotic–resistant, highly transmissible strains of P. aeruginosa have been identified in populations with CF in the United Kingdom (1–3) and Australia (4, 5), but not in Canada (6). Emergence of transmissible strains has further exacerbated the problems faced by patients with CF by conferring a major infection control hazard in the United Kingdom (7, 8). The first major transmissible P. aeruginosa CF strain type was identified in Liverpool (2), and in addition to its ability to spread from one patient with CF to another, the strain was capable of: super-infection replacing infection with nonepidemic P. aeruginosa CF strains (9), spreading to relatives without CF (3), and causing increased morbidity among chronically infected patients with CF (10). A multiresistant transmissible strain type has also been identified at an adult CF treatment center in Manchester (1), where, although it is not specifically associated with raised morbidity, infection is associated with an increased treatment burden for infected patients compared with those who harbor their own unique strain (11). Nixon and coworkers (12) have also observed increased mortality among patients with CF associated with a transmissible P. aeruginosa clone in an Australian treatment center. A recent survey of 31 treatment centers in the United Kingdom demonstrated that transmissible strain types were present in 28 centers, with 28% of total number of patients with CF examined across all centers infected with a P. aeruginosa strain type that was shared with another patient (8). Although accurate identification of P. aeruginosa CF isolates can be achieved by use of selective media and standard biochemical analysis (13), differentiation of strains requires the application of DNA typing methods such as pulse-field gel electrophoresis (PFGE) (1, 2) or Random Amplified Polymorphic DNA (RAPD) fingerprinting (14). Although these genetic methods are highly discriminatory, they do not provide particularly rapid diagnosis of transmissible P. aeruginosa strains. Given the magnitude of the problem of transmissible strains in the United Kingdom (8), rapid diagnostic tests are urgently required to identify problematic strains and institute infection control precautions when indicated. This is also particularly important for clinics in which patient-to-patient spread of P. aeruginosa has not yet been documented (6).

Polymerase chain reaction (PCR) is a powerful tool for both identifying regions of DNA unique to particular bacterial strains (15, 16) and developing rapid diagnostic test for problematic strains (17). In conjunction with the increasing amount of genome sequence information available for the CF pathogens, molecular diagnostics represent an excellent means to develop strain specific assays (18). Spilker and colleagues (19) recently used a PCR method to develop diagnostic PCRs for the detection of P. aeruginosa in patients with CF. Although very useful for speeding up detection of a potential P. aeruginosa infection, such tests do not provide definitive identification of the strain type, which is important for the detection of transmissible strains. Parsons and coworkers (17) used a suppression subtractive hybridisation PCR procedure to identify a diagnostic probe, PS21, specific for the identification of the Liverpool epidemic P. aeruginosa CF strain, a highly problematic epidemic clone in the United Kingdom (2). Although a PCR diagnostic has been developed for the Liverpool strain (20), rapid detection of other transmissible strain types is currently not possible. In addition, we still understand very little about phenotypic or genotypic features which make these P. aeruginosa CF strains highly transmissible.

Here we describe the identification of strain-specific DNA markers for a problematic P. aeruginosa CF strain, the Manchester transmissible clone (1), designated as strain MA. Genetic differences between strain MA and genome-sequenced strain P. aeruginosa PAO1 (21) were also examined by using genomic microarrays. Identification and evaluation of DNA markers specific to, or absent from, strain MA were performed. In addition, a strain collection representative of P. aeruginosa–infected patients with CF in the United Kingdom, Canada, and the United States, was screened for the presence of the novel MA strain markers and existing probe PS21 for the Liverpool epidemic clone (17). A novel genomic island specific to strain MA, encoding bacteriophage-like regions and a Vibrio cholera toxin–like gene, is also described.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
P. aeruginosa Strain Collection and General Methods
A total of 141 P. aeruginosa genetically distinct strains were used in this study. 122 were from CF infection: the MA strain (8 isolates each from a different patient; 1); a prevalent CF strain, Clone C (1 isolate; 22); the Liverpool CF epidemic strain (2 isolates, G599 and P8959; 2, 17), typed CF strains drawn from previous epidemiologic studies (1, 6, 14), and 20 strains representative of 20 American patients with CF attending treatment centers in 14 different cities across 10 states. Nineteen strains were from patients without CF with urinary tract, wound, and ear infections; strain PA01 (21) was used as the genetic reference. Isolates C3719 and C3425 were chosen as representatives of strain MA for genomic comparison analysis because they were more susceptible to gentamicin, hence potentially allowing the antibiotic to be used as a selectable marker for genetic manipulation. Bacterial culture, genomic DNA isolation, and typing by PFGE and RAPD were performed as previously described (1, 6, 14) and strain genotype determined using computer software (GeneDirectory; Syngene Ltd, Cambridge, UK). Fingerprint similarity was evaluated by clustering using the unweighted pair-group method average (UPGMA) and calculation of the Dice coefficient; isolates possessing Dice coefficients greater than 0.70 were designated as a single strain type. Replicate isolates were only counted once as representative of a single strain type in the evaluation of probe specificity.

Genomic Comparison of P. aeruginosa Strains: Genes Absent in Strain MA
To determine if any of the genes present in the genome reference strain PAO1 were absent in strain MA, DNA from MA isolates C3719 and C3425 was hybridised to the P. aeruginosa GeneChip Microarrays (Affymetrix, Santa Clara, CA) exactly as described previously (23). Genes absent in strain MA were identified and bioinformatically characterized using the Pseudomonas genome project website (http://www.pseudomonas.com) and Artemis software (http://www.sanger.ac.uk/Software/Artemis/).

Genomic Comparison of P. aeruginosa Strains: Genes Unique to Strain MA
To determine which genes were present in strain MA but absent in the genome strain PAO1, the genomes of the two strains were compared using a PCR-based method for creating a subtractive hybridization library (17). MA isolate C3719 was used as a tester strain and subtractive hybridization performed against strain PAO1 as the driver strain. A subtractive hybridization clone library of MA-specific DNA was created using the PCR-Select bacterial genome subtraction kit following the manufacturer's instructions (BD Clontech UK, Basingstoke, UK), except for (1) using HaeIII to digest MA DNA to fragments of less than 3 kb and (2) increasing the extension time to 10 min in the final round of PCR to facilitate successful cloning of strain-specific fragments into a TA Cloning Vector (Invitrogen, Renfrew, UK). PCR amplification followed by restriction fragment length polymorphism (RFLP) analysis using the enzyme HaeIII was used to determine individuality of each clone. Unique clones were sequenced (MWG Biotech, Milton Keynes, UK) and compared with other sequences held in the National Centre for Biotechnology Information database (NCBI) using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov).

PCR Amplification
PCR primers (Table 1) were designed using the program Primer 3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). PCR was performed as described (16) with 50 ng of template DNA and 10 pmol of each primer added to a standard 25 µl reaction. Thermal cycling was performed on a Flexigene Thermal Cycler (Techne Ltd, Newcastle, UK) and conditions for all primer sets were as follows: an initial cycle of 94°C for 5 min followed by 30 cycles of 94°C for 30 s, annealing at the appropriate temperature (Table 1) for 30 s, and extension at 72°C for 60 s, with a final 10-min extension at 72°C. PCR products were analyzed by agarose gel electrophoresis as described (16).


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  		TABLE 1. Probes and pcr primers used in this study

 
Genomic DNA Arrays and DNA-DNA Hybridization
To determine the prevalence of DNA markers among the collection of P. aeruginosa strains replicate genomic DNA arrays were prepared exactly as described for the Burkholderia cepacia complex (16). Briefly, genomic DNA (~ 100 ng/µl) from each P. aeruginosa strain was aliquoted into 384-well plates (ABgene Ltd., Epsom, UK) and arrayed onto positively charged nylon membranes for use in DNA–DNA hybridization analysis as described (16). To determine if specific genes were associated with either CF strains or strains from other clinical infections, Principal Component Analysis (PCA) was performed using the statistical analysis software Minitab Release 14 (Minitab Inc., Coventry, UK). A data matrix listing strains and corresponding absence (scored as 0) or presence (scored as 1) of genes probed (see Table 1) was prepared; the matrix was then analyzed by PCA using strains and then gene probes as the variables.

Construction of a Cosmid Library of P. aeruginosa Strain MA and Nucleotide Sequence Analysis
A cosmid library of MA isolate C3719 was prepared using the shuttle vector, pSCOSPA1, a derivative of cosmid pSCOSBC1 (24) with the trimethoprim marker replaced by a gentamicin resistance cassette. A total of 960 cosmid clones were screened by Southern hybridization as described (16). Complete nucleotide sequence analysis of cosmid pa131 encoding the MA-specific probes was performed by the Pathogen Sequencing Unit at the Sanger Institute Wellcome Trust Genome Campus, Hinxton, Cambridge, UK, as described previously (25). The sequence of the genomic island within cosmid pa131 has been deposited in EMBL under accession number CR848688.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Genomic Comparison of P. aeruginosa Strains: Genes Absent in Strain MA
Increased virulence in bacterial pathogens is classically associated with acquisition of DNA by lateral gene transfer, for example with elements such as bacterial pathogenicity islands (26). However, in a few bacteria such as Shigella species, loss of DNA has also resulted in phenotypic alterations which considerably enhance infectivity and pathogenicity (27). To determine if similar genomic deletions had occurred in strain MA, its DNA was hybridized to the P. aeruginosa PAO1 GeneChip microarrays (23) to identify the extent of its genomic identity with strain PAO1. Out of the 5,549 nonredundant PAO1 genes on the array, 182 were absent in strain MA. These included the following types of genes: hypothetical genes of unknown function (n = 79); genes associated with genetic mobility (n = 29; phage, transposon, or plasmid-associated genes); genes linked to metabolism (n = 18); genes associated with transcriptional regulation (n = 14); genes associated with secreted factors (n = 12; including pyocin, pyoverdin, and type 4 fimbriae production); transport or membrane-linked genes (n = 11); genes associated with antimicrobial resistance (n = 2); and lipopolysaccharide biosynthesis genes (n = 17; serotype 05 in PAO1). Strain MA DNA did not hybridize to the additional lipopolysaccharide probes on the PAO1 microarray for serotypes 06 and 011. From the homology of the MA-specific probes described below, the serotype of strain MA was probably 03. In terms of other known P. aeruginosa virulence factors, the microarray data showed strain MA possessed an a-type flagellin gene and a homolog of exoS; correspondingly an exoU homolog was not detected in strain MA (23).

To investigate if any of the genomic deletions were specific to strain MA or other problematic CF clones, nine genes corresponding to the largest deletions and each of known putative function (PA0620, PA0724, PA0985, PA2032, PA2104, PA2220, PA2398, PA3506, and PA4554; Tables 1 and 2) were selected for further analysis. Seven of these MA deletions were also observed in a collection of 19 strains examined by Wolfgang and coworkers (23) using the PAO1 gene chip; however, deletion of PA2032 and PA4554 was not observed in the latter study (see Table 2).


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  		TABLE 2. Prevalence of strain MA deletions among p. AERUGINOSA collection

 
Prevalence of Strain MA Genomic Deletions in the P. aeruginosa Collection
The extent to which other P. aeruginosa strains shared the same deletions as strain MA was determined by hybridization of PCR probes for PA0620, PA0724, PA0985, PA2032, PA2104, PA2220, PA2398, PA3506, and PA4554 (Table 1), to the P. aeruginosa genomic DNA arrays. The hybridization results for gene PA0985 are shown in Figure 1 and the prevalence of deletions is summarized in Table 2. No deletions were found to be absolutely specific to strain MA. Seven of the nine genes absent from strain MA but present in P. aeruginosa PAO1 were also deleted a rate of 23% or greater among the 141 strains tested (Table 2). Two strain MA deletions, PA0724 and PA2032, were only shared by 6% or less of the strains: PA0724, a bacteriophage coat protein was present in 98% of the strains and PA2032, a transcriptional regulator-like gene, was encoded by 94% of strains. Both the Liverpool (2) and Melbourne epidemic (5) strains possessed genes homologous to the latter probes. The absence of homologs of PA2104 and PA3506 were negative genetic traits shared by strain MA, the Liverpool, and Melbourne epidemic strains; however, these genes were also absent in 74% and 78%, respectively, of P. aeruginosa strains examined (Table 2). Weak cross-hybridization was apparent for several of the PAO1-specific gene probes indicating the closely related homologs were present in many P. aeruginosa strains (see Figure 1). No statistically significant correlation between strain source and genomic deletion was observed by PCA analysis (data not shown).



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  		Figure 1. Southern hybridization of the P. aeruginosa probes to the genomic DNA array. Hybridization results for probes MA18, PA0985, and PS21, are shown respectively in panels A, B, and C. Positive signals highlighted by the black boxes indicate the location of the positive control strains for each hybridization (MA18 = MA strain C3719; PA0985 = genome strain PAO1; PS21 = Liverpool strain G599).

 
Genomic Comparison of P. aeruginosa Strains: Genes Unique to Strain MA
Subtractive hybridization of DNA from MA isolate C3719 against strain PAO1 was used to identify genes unique to strain MA. Thirty-three subtractive hybridization clones were obtained and screened initially by PCR-RFLP analysis to determine which inserts were unique. Of the 33 clones, 21 possessed unique RFLPs and were sequenced. Subtractive hybridization clones 4, 17, 26, 28, and 29 encoded DNA overlapping the same region and showed 99% identity to the P. aeruginosa serotype 03 putative O-antigen biosynthesis gene cluster (AF498414); these data suggest that the serotype of strain MA is O3. Clones 16, 25, and 32 also encoded DNA from the same genomic region that showed 100% identity to pilus gene cluster of P. aeruginosa strain PA14 (AY273871). Both of these overlapping groups of probes were not examined further as they showed significant homology to conserved P. aeruginosa genes and were unlikely to be specific as probes. The remaining 13 subtractive hybridization clones were all different from each other and showed no significant homology to any other sequences in the database (data not shown). PCR primers were designed to amplify DNA products from these novel regions of MA to enable their specificity for strain MA to be determined (see Table 1).

Prevalence of Putatively MA-Specific Genes in the P. aeruginosa Strain Collection
Prevalence of the novel putatively MA-specific clones was determined by DNA-hybridization analysis to the P. aeruginosa genomic DNA arrays as described above for the MA strain deletions. The hybridization results for subtractive hybridization probe MA18 are shown in Figure 1 (data not shown for other probes), and the prevalence data for all 13 MA probes examined in detail are summarized in Table 3. Strain MA probes MA3, MA12, and M27, identified by subtractive hybridization, hybridized to the greatest number of strains on the DNA array (Table 3; 40%, 54%, and 24%, respectively), indicating that the prevalence of these genetic regions in the species P. aeruginosa was high. Probes MA7, MA8, MA10, MA11, and MA14 hybridized to between 3.5 and 7% of strains on the array (Table 3). Probes MA7, MA8, and MA11 only hybridized to CF strains; however, when analyzed statistically this association was not shown to be significant (data not shown). Five subtractive hybridization probes were absolutely specific to strain MA, MA15, MA18, MA21, MA22, and MA30 (Table 3). These probes only hybridized to strain MA isolate C3719 (which was used specifically to isolate the DNAs by subtractive hybridization) and eight other clonal isolates from the Manchester epidemic which were included on the array as controls (Figure 1A). Hybridization signals were clear and no evidence of background or cross-hybridization was apparent with these MA-specific probes (Figure 1A). As with the genes deleted from strain MA, the genes identified by the subtractive hybridization procedure did not show any statistically significant prevalence patterns, except for the fact that strain MA (the source of the probes) always possessed a unique profile distinct from all the other P. aeruginosa strains examined (data not shown).


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  		TABLE 3. P. AERUGINOSA strains hybridizing to the strain MA subtractive hybridization clones, probe PS21 and PA131 gene 4

 
Each MA subtractive hybridization probe was also mapped to the P. aeruginosa strain MA cosmid library to determine if they were encoded within similar genomic region. The MA-specific probes (MA15, MA18, MA21, MA22, and MA30) all mapped to two overlapping cosmid clones (clones pa27 and pa131). In addition, restriction mapping of these cosmids demonstrated that all five probes were located within a single 30-kb region of the strain MA genome (data not shown). Three clusters of two probes (MA1 and MA3; MA7 and MA11; MA12 and MA27) mapped to one cosmid each, demonstrating that each member of the probe pairs was encoded within close proximity to the other.

The Liverpool Epidemic Strain Probe: PS21
Probe PS21 was described by Parsons and colleagues (17) as a specific diagnostic marker for the Liverpool CF epidemic strain (2). The efficacy of this probe was evaluated on the same P. aeruginosa strain collection as the MA probes. Probe PS21 demonstrated 97.8% specificity, detecting itself and three other P. aeruginosa strains in our collection (Figure 1C and Table 3). Weak cross-hybridization was also apparent on the genomic DNA array, indicating the presence of DNA closely related to the PS21 sequence in several other P. aeruginosa strains (Figure 1C).

The three strains detected by Southern hybridization with probe PS21 were further investigated to confirm the presence of the Liverpool marker and re-investigate their genotypic identity. Each of the strains was tested by PCR with the Liverpool probe primers and found to be positive (data not shown). Each amplification product also demonstrated the same RFLP as the Liverpool strain amplicon when digested with the enzyme HaeIII (data not shown), confirming their genetic identity. Clonality of the three positive strains was then examined by PFGE genetic fingerprinting (Figure 2). Strain E597 from a Sheffield patient with CF (28) demonstrated considerable identity with the Liverpool strain (Figure 2; 0.76 Dice coefficient), indicating that it was a close clonal relative of the Liverpool strain. Strain E579, from an Edinburgh patient with CF, and strain C6166, from a Canadian patient with CF, were each genotypically distinct strains not related to either the Liverpool or MA epidemic clones (Figure 2) or any other strain in our collection. Hybridization of the PS21 probe to the PFGE digested DNA also indicated that marker was encoded on a different genomic fragment for strains E579 and C6166 (data not shown).



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  		Figure 2. PFGE fingerprint analysis of strain detected by the Liverpool epidemic strain probe PS21. PFGE fingerprints obtained after restriction digestion with XbaI were evaluated by computer software. A dendogram indicating the relatedness of each strain is shown on the left and the PFGE fingerprint pattern for each strain are shown on the right. Strain names and their clonal derivation (LIV = Liverpool; MA = Manchester) are shown on the right.

 
MA Strain Diagnostic Probes
The primary objective of this study was to develop a rapid diagnostic probe for the detection of strain MA. The collection of 141 P. aeruginosa strains was evaluated by PCR with all five MA-specific probes (MA15, 18, 21, 22, and 30). Each MA probe was found to have 100% specificity for strain MA (data not shown). Because of its optimal size for PCR (308 bp), probe MA15 was further evaluated as a PCR diagnostic, by multiplexing it with the universal 16S rRNA gene primers, UNI2 and UNI5 (29). These rRNA gene primers were included in the PCR as an amplification control because they are robust, detecting all proteobacterial DNA (29) and if negative would indicate reaction failure rather than a false-negative with the MA15 PCR. With this multiplex PCR, probe MA15 once again demonstrated 100% specificity for the MA strain lineage (Figure 3).



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  		Figure 3. Multiplex PCR of the MA strain probe MA15 with and the universal 16S rRNA gene primers, UNI2 and UNI5. Multiplex PCR was performed to ensure that samples negative for the MA-specific probes were capable of supporting PCR amplification. Lanes are as follows: M, molecular size markers (relevant fragment sizes are indicated on the left); 1, 2, 9, and 10, MA strains C3719, C3425, C3667, and C3652, respectively; 3, strain E597; 4, strain E579; 5, C6166; 6 and 7, Liverpool CF epidemic strain; 8, PAO1.

 
Identity of MA-Specific Markers
Preliminary sequence analysis of the five MA-specific subtractive hybridization clones (MA15, 18, 21, 22, and 30) did not identify any signficant matches to the genetic databases (data not shown). Cosmid clone pa131, which encoded these markers, was subjected to complete sequence analysis to determine the identity of this region. The cosmid carried a 41,704-bp insert P. aeruginosa strain MA DNA, of which nearly two thirds was highly homologous to the PAO genome. However, inserted at a serine tRNA (between homologs of PAO1 genes PA1013 and PA1014) was a genomic island, 13,318 bp in size, flanked by 84-bp direct repeats and encoding 18 genes (Figure 4). The island, designated the MA island, possessed a low GC content (55.3% GC) compared with both the cosmid insert (62.7% GC) and the PAO1 genome (66.5% GC) average, and was composed of two cryptic bacteriophage-like regions. Several phenotypic attributes of the MA island corroborated the bioinformatic analysis, suggesting that it was cryptic and incapable of excision or transfer. Probe PCR and hybridization data demonstrated that the phage genes within the MA probe region of the island (Figure 4) were stably inserted in the genome of strain MA and its clonal relatives (Figure 1 and Table 3). In addition, serial laboratory passage (10 culture passages) and growth at 42°C (100 colonies screened by PCR) failed to destabilize this DNA region in strain MA and its clonal derivatives (data not shown).



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  		Figure 4. Features of the MA strain-specific genomic island. A graphic representation of the genetic features of the MA island is shown. A shows a GC content plot of the region with the mean content of cosmid pa131 shown by the horizontal line (calculated over a window size of 500 bases). The gene content of island is shown in B with genes (arrows) on the forward and reverse open reading frames shown above and below (respectively) a scale bar marked at every kilobase of DNA. Flanking direct repeats and the downstream tRNA are shown on the scale bar. Homologous genes are shaded as follows: bacteriophage Pf1-like genes (black); genes similar to those encoding conserved hypothetical proteins in Bacteriodes species (dark gray); and the cryptic integrase (light gray). The MA probe and toxin probe regions are also indicated.

 
At the right end of the MA island was a phage-like region (47.9% GC; seven genes; Figure 4) with a cryptic integrase (gene 17, Figure 4) and several conserved hypothetical coding sequences. The MA-specific probes (Table 3) all mapped to this low GC region (Figure 4) with probes MA15, 21, and 22, spanning regions within genes 12, 13, and 14, and MA18 and 30 spanning gene 14. Gene 12 possessed no significant database similarities. Gene 13 was similar to the putative transcriptional regulator in bacteriophage Wphi, and gene 14 was homologous to a conserved hypothetical protein in the Bacteroides thetaiotaomicron genome. The upstream portion of the MA island encoded a copy of a filamentous phage (61.24% GC; 11 genes) with several genes homologous to those within P. aeruginosa phage Pf1 (Figure 4). Within this region, gene 4 encoded a protein highly homologous to the V. cholera Zonular occludens type toxin (Zot; Table 3 and Figure 4). Zot is encoded by bacteriophages that integrate into the genome of toxigenic V. cholera and enables such strains to disrupt the cellular tight junctions in the intestinal lining (30). While the major cholera toxin produces the most severe symptoms of diarrhea associated with this pathogen, the Zot toxin is also associated with significant V. cholera diarrheagenicity (30). A search of the DNA databases revealed that both P. aeruginosa strain PAO1 and PA-14 possessed close homologs of the Zot-like gene present on the MA island. No homologs of the major cholera toxin were found in the latter P. aeruginosa genomes and none of the subtractive hybridization clones encoded DNA homologous to it. To determine the prevalence of toxin gene among our P. aeruginosa strain collection, a PCR probe (pa131 gene 4; Table 1) was designed and hybridized to the DNA arrays. Close homologs of the toxin gene were detected in 33% of the P. aeruginosa strains, with the 91% of the positive strains recovered from CF infection (Table 3). Although clearly not specific to transmissible CF strains, the toxin gene was present in both the Liverpool strain and strain MA, but absent in the Melbourne epidemic clone.


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Identifying highly transmissible CF strains of P. aeruginosa promptly is critical to a rapid response infection control policy for reducing the spread of these strains (7). Transmissible strains may also be more virulent, causing greater treatment requirements and an accelerated decline in spirometry and nutritional status (8, 10). Phenotypic identification of transmissible strains is not possible and extensive molecular epidemiologic surveillance is normally required for their detection (1, 3). We used comparative molecular genomic techniques to successfully identify several PCR probes which on the basis of the extensive P. aeruginosa strain panel investigated are specific for strain MA. These strain-specific genetic probes may be used to analyze isolates cultured from patients with CF, and can even be applied directly to CF sputum to detect the presence of the MA strain (E. Mahenthiralingam, unpublished data). Several diagnostic PCRs for the detection of transmissible B. cepacia complex strains have been developed and had considerable impact on reducing the spread of these problematic CF pathogens (15, 16, 29). The analogous application of the MA-specific probes we have developed will greatly assist in the implementation of infection control measures needed to control the emerging problem of transmissible P. aeruginosa strains.

Using subtractive hybridization to identify DNA specific to strain MA and genomic hybridization to the P. aeruginosa PAO1 microarray to identify DNA absent from the strain MA, we have been able to characterize several genotypic features of this transmissible strain lineage. Although 33 DNA clones were initially identified from the subtractive hybridization procedure, only five were found to have absolute specificity for the strain MA when tested by both DNA hybridization (Table 3) and PCR (Figure 3). The fact the MA-specific probes detected by the subtractive hybridization procedure was found to be part of a low GC content genomic island is not surprising, as pathogen-specific DNA is frequently associated with such entities in other bacteria (26).

Several genomic islands have now been defined in P. aeruginosa CF strains (31). The PAGI-1 island encoded by the urinary tract strain X245409 is associated with possible detoxification of oxygen radicals in pathogenic strains (32), while the flagellin glycosylation island possessed by strain PAK is required for production of the flagellum, a well-characterized P. aeruginosa virulence factor (33). Compative hybridization of strain MA to the PAO1 GeneChip microarrays demonstrated that it lacked genes homologous to those encoded by both the PAGI-1 and flagellin glycosylation islands (data not shown). Insertion of the PAGI-1 island in P. aeruginosa strain X25409 was associated with a corresponding deletion of genes homologous to PA2218 through PA2222 (32). An analogous but slightly larger deletion was also present in strain MA, spanning genes homologous to PA2218 through PA2228. The absence of the PAGI-1 island in strain MA suggests a novel island may be present at this location. One of the genes lost as part of this deletion, the transcriptional regulator PA2220, was absent in 60% of the strains examined (Table 2), suggesting that this region may also be a common P. aeruginosa insertion point for genomic islands acquired through horizontal gene transfer.

Interestingly, the majority of the genomic deletions observed by Wolfgang and coworkers (23) were also shared by strain MA and most of the strains within the collection examined herein (Table 2). Wolfgang and colleagues (23) showed that the deletion of genes around PA2220 was common (79% of strains; Table 2). In addition, they were able to show that in different P. aeruginosa strains, a variety of islands including novel ExoU toxin encoding segments may insert at the PA0985 locus, another region that was absent in strain MA and in 81% of P. aeruginosa strains examined in this study (Table 2). Overall, these data showing that large numbers of strains possess the same genomic deletions suggest that P. aeruginosa as a species possesses a relatively conserved genome content, with chromosomal inversions and island insertions contributing to the mosaic genome organization that underpins the genotypic diversity seen in individual strains.

The MA island was composed of two phage-like regions, and only the downstream phage region (Figure 4) appeared to be absolutely specific for strain MA. The upstream region of the MA island encoded genes that were highly homologous to the P. aeruginosa Pf1 filamentous bacteriophage that was isolated 30 yr ago (34). The presence of a Zot toxin (30) gene, gene 4 (Figure 4), in this region was intriguing, especially since although it occurred in only about one third of P. aeruginosa strains, it was more frequently encoded in CF strains (Table 3). The functional and clinical ramifications of this toxin in terms of P. aeruginosa strain virulence during respiratory infection remain to be determined. Its presence in the Liverpool clone (associated with increased morbidity; 10) and strain MA (linked to increased treatment burdens; 11) suggest that this toxin and the rest of the MA island are worthy of further systematic study.

Parsons and coworkers (17) identified DNA markers and developed a PCR that appeared to be specific for the Liverpool CF strain. The Liverpool strain probe, PS21 (17), was evaluated alongside the MA probes, and detected three other strains in our collection of P. aeruginosa strains by both hybridization (Figure 1) and PCR (data not shown). One of these strains demonstrated close genotypic identity with the Liverpool strain when examined by PFGE (strain E597; Figure 2), corroborating the recent results of Edenborough and colleagues (28), who had shown that it was a member of this transmissible lineage. However, the two other PS21-positive strains, E579 and C6166, were completely distinct from the Liverpool strain (Figure 2). The identification of these distinct Liverpool marker–positive strains demonstrates that the PS21 probe can no longer be considered specific for this epidemic strain lineage. Strain E579 was recovered from an individual patient with CF in Edinburgh, Scotland. Strain C6166 was isolated from another individual patient with CF in Vancouver, Canada. There was no epidemiologic evidence to indicate the potential spread of these strains in either the Vancouver or Edinburgh CF populations. This observation further emphasizes the importance of considering the epidemiologic relatedness among patients before inferring patient-to-patient spread of genetically related isolates.

In addition to its multiresistance and transmissibility, strain MA has a number of unusual phenotypic features including being nonpigmented, nonmotile, and possessing a unique and rare pyocin type (1). The comparative analysis of genome content performed with the P. aeruginosa PAO1 microarrays demonstrated that strain MA possessed over 96% gene conservation, with the latter strain corroborating the previous findings on this species (23). The lack of genes for pyoverdin biosynthesis in strain MA may explain its lack of pigmentation; however, none of the other absent genes were easily correlated to its other phenotypic features. Indeed, strain MA appeared to possess the genes required for flagellum expression and biosynthesis, hence loss of these genes could not explain its nonmotile phenotype. Recent studies suggest that downregulation of flagellum expression occurs as a result of exposure to CF lung airway fluids (35), suggesting that strain MA may be an ancestral CF strain that has adapted to this infection niche and is probably not a new environmental strain that has emerged as problematic strain in Manchester. Few systematic genetic differences were observed between CF strains and the non-CF strains examined in this study (Table 2 and 3). However, the absence of DNA homologous to the MA-specific probes, MA7, MA8, and MA11 in non-CF strains is worthy of further investigation.

In summary, we have developed specific molecular diagnostic probes for strain MA and have shown that this strain is rare, and probably does not occur in two CF populations (Canada and the United States) outside the United Kingdom. These probes will aid the rapid diagnosis and the implementation of appropriate clinical procedures for patients with CF infected with this problematic strain. The genome content of strain MA was also shown not to be highly distinct from P. aeruginosa strain PAO1, although the functional significance of the unique MA genomic island remains to be fully determined. We have also shown that the PS21 probe for the Liverpool epidemic strain (17) is not absolutely specific and strains positive for this PCR marker should be further evaluated by typing analysis before they are assigned to this transmissible lineage. The probes developed for strain MA show 100% specificity for this transmissible strain type within the 141 P. aeruginosa genetically distinct strains examined, a much larger number of samples than the 24 non-Liverpool isolates examined in the latter study (17). However, their reliability in clinical diagnosis will need be tested by continued epidemiologic surveillance.


   Acknowledgments
 
The authors are grateful to Maureen Campbell, and Catherine Doherty for performing the bacteriological and strain typing analysis; Nicola Morris and Nora Sabbuba kindly provided strains from urinary tract infection. The authors thank Matthew Wolfgang, Theodora Vatopoulou, and Gemma Braidley for assistance with the analysis of P. aeruginosa PAO1 genetic loci; Angela Marchbank for her technical assistance with hybridization analysis; and Carol Churcher, Karen Brooks, and the Sanger Institute Core teams for sequencing cosmid pa131. They acknowledge John Fry for assistance with the statistical analysis.


   Footnotes
 
This work was supported by grants from the UK Cystic Fibrosis Trust (grant number PJ503) and Canadian Cystic Fibrosis Foundation (D.P.S.). The Affymetrix GeneChip P. aeruginosa Genome Arrays were obtained and subsidized via the Cystic Fibrosis Genomics Program (http://cfgenomics.unc.edu/index.htm) sponsored by Cystic Fibrosis Foundation Therapeautics Inc.

Conflict of Interest Statement: D.A.L. has no declared conflicts of interest; A.J. has no declared conflicts of interest; J.P. has no declared conflicts of interest; D.P.S. has no declared conflicts of interest; J.R.W.G. has no declared conflicts of interest; J.J.L. has no declared conflicts of interest; S.L. has no declared conflicts of interest; A.K.W. has no declared conflicts of interest; and E.M. has no declared conflicts of interest.

Received in original form November 10, 2004

Received in final form March 24, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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