Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals

Most known virulence determinants of Pseudomonas aeruginosa are remarkably conserved in this bacterium's core genome, yet individual strains differ significantly in virulence. One explanation for this discrepancy is that pathogenicity islands, regions of DNA found in some strains but not in others, contribute to the overall virulence of P. aeruginosa. Here we employed a strategy in which the virulence of a panel of P. aeruginosa isolates was tested in mouse and plant models of disease, and a highly virulent isolate, PSE9, was chosen for comparison by subtractive hybridization to a less virulent strain, PAO1. The resulting subtractive hybridization sequences were used as tags to identify genomic islands found in PSE9 but absent in PAO1. One 99-kb island, designated P. aeruginosa genomic island 5 (PAGI-5), was a hybrid of the known P. aeruginosa island PAPI-1 and novel sequences. Whereas the PAPI-1-like sequences were found in most tested isolates, the novel sequences were found only in the most virulent isolates. Deletional analysis confirmed that some of these novel sequences contributed to the highly virulent phenotype of PSE9. These results indicate that targeting highly virulent strains of P. aeruginosa may be a useful strategy for identifying pathogenicity islands and novel virulence determinants.     

Structure of Pseudomonas aeruginosa populations analyzed by single nucleotide polymorphism and pulsed-field gel electrophoresis genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.     

In silico comparison of pKLC102-like genomic islands of Pseudomonas aeruginosa

The genomic island pKLC102 first detected in Pseudomonas aeruginosa clone C strains can cross species barriers and exhibits the highest mobilization rate of a genomic island known to date. Homologous genomic islands of 81-108 kb in size were identified in the completely sequenced P. aeruginosa strains PA7, PA14, 2192, C3719 and PACS2, but not in strains PAO1 and LES. All pKLC102-like genomic islands are integrated in chromosomal tRNA(Lys) genes and share a syntenic set of more than 70 homologous ORFs, part of which are related to DNA replication or mobility genes. The conserved backbone has predilection sites for the uptake of island-specific gene cassettes. A major difference between the islands is the organization of the origin of replication oriV.     

Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis

Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.     

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.     

Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.     

Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.     

Genetic and phenotypic characterization of a Pseudomonas aeruginosa population with high frequency of genomic islands

Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a-n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs.     

Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.     

The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS

Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.     

Genetic and biochemical analyses of a eukaryotic-like phospholipase D of Pseudomonas aeruginosa suggest horizontal acquisition and a role for persistence in a chronic pulmonary infection model

Phospholipases D (PLDs) are virtually ubiquitous in eukaryotic organisms; however, they are relatively uncommon in prokaryotes. In this report, we demonstrate that the environmentally acquired, opportunistic pathogen Pseudomonas aeruginosa expresses PLD activity. A gene designated pldA was identified in the genomic database of P. aeruginosa PAO1 encoding a protein with significant homology to eukaryotic PLDs, but not to any prokaryotic PLDs. PldA is most homologous to PLDs from mammals and yeast. The pldA gene was cloned and shown to express an approximately 116 kDa protein with calcium-regulated PLD activity that is localized to the periplasm. Interestingly, not all strains of P. aeruginosa carry pldA. When present, pldA is always linked to an open reading frame (ORF), ORF4, and a gene (vgrA1) encoding a protein homologous to Vgr from Escherichia coli. Vgr proteins contain regularly repeated dipeptide motifs (valine-glycine repeats). In E. coli, genes encoding Vgr are associated with multicopy genetic elements designated Rhs (rearrangement hot-spots). P. aeruginosa PAO1 has 10 vgr homologues dispersed throughout its genome, but the copy number of these genetic elements varies considerably in different strains. Neither vgrA1 nor ORF4 is present in strains lacking pldA. Furthermore, sequences flanking vgrA1, pldA and ORF4 in the P. aeruginosa strains examined are highly conserved, suggesting a specific site of insertion. These and other data suggest that vgrA1, pldA and ORF4 constitute an approximately 7 kb mobile genetic element and that pldA was acquired horizontally, perhaps from a eukaryotic organism. Competition studies between a PldA knock-out mutant and the parental wild-type strain indicate that PldA contributes to the ability of P. aeruginosa PAO1 to persist in a chronic pulmonary infection model in rats.     

Mapping of the gene specifying aminoglycoside 3''-phosphotransferase II on the Pseudomonas aeruginosa chromosome.

We examined the aminoglycoside inactivation enzymes in Pseudomonas aeruginosa strains, seven clinical isolates and seven laboratory strains without plasmids. All strains were found to possess the enzyme aminoglycoside 3'-phosphotransferase II [APH(3')-II]. We isolated an APH(3')-II-deficient mutant from a PAO strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. By plasmid (FP5 or R68.45)-mediated conjugation, we determined the locus of the gene specifying the APH(3')-II between trp-6 and pro-82 on the PAO chromosome and designated this gene aphA. It was concluded that the intrinsic resistance of P. aeruginosa to kanamycins, neomycins, paromomycins, ribostamycin, and butirosins was due to this newly determined gene.     

Diversity of the abundant pKLC102/PAGI-2 family of genomic islands in Pseudomonas aeruginosa

The known genomic islands of Pseudomonas aeruginosa clone C strains are integrated into tRNA(Lys) (pKLC102) or tRNA(Gly) (PAGI-2 and PAGI-3) genes and differ from their core genomes by distinctive tetranucleotide usage patterns. pKLC102 and the related island PAPI-1 from P. aeruginosa PA14 were spontaneously mobilized from their host chromosomes at frequencies of 10% and 0.3%, making pKLC102 the most mobile genomic island known with a copy number of 30 episomal circular pKLC102 molecules per cell. The incidence of islands of the pKLC102/PAGI-2 type was investigated in 71 unrelated P. aeruginosa strains from diverse habitats and geographic origins. pKLC102- and PAGI-2-like islands were identified in 50 and 31 strains, respectively, and 15 and 10 subtypes were differentiated by hybridization on pKLC102 and PAGI-2 macroarrays. The diversity of PAGI-2-type islands was mainly caused by one large block of strain-specific genes, whereas the diversity of pKLC102-type islands was primarily generated by subtype-specific combination of gene cassettes. Chromosomal loss of PAGI-2 could be documented in sequential P. aeruginosa isolates from individuals with cystic fibrosis. PAGI-2 was present in most tested Cupriavidus metallidurans and Cupriavidus campinensis isolates from polluted environments, demonstrating the spread of PAGI-2 across habitats and species barriers. The pKLC102/PAGI-2 family is prevalent in numerous beta- and gammaproteobacteria and is characterized by high asymmetry of the cDNA strands. This evolutionarily ancient family of genomic islands retained its oligonucleotide signature during horizontal spread within and among taxa.     

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

Background

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.

Results

The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.

Conclusion

Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.     

Distribution of phage-resistant Pseudomonas aeruginosa strains

The sensitivity of a number of P. aeruginosa clinical strains to virulent bacteriophages has been studied. Phage-resistant strains have been found to constitute a considerable proportion among the tested P. aeruginosa strains. The strains under study fall into 19 groups differing in their sensitivity to the bacteriophages used in this investigation. The strains belonging to some groups are phenotypically identical to experimentally obtained P. aeruginosa phage-resistant mutants PAO. The use of bacteriophage mutants has made it possible to demonstrate that in most cases the resistance of P. aeruginosa natural strains to type phi k phages is due to disturbances in their adsorption, whereas their resistance to type phi m and phi mn phages is, seemingly, not linked with disturbances in their capacity for adsorption on the cell membranes of the bacteria.     

Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa

The yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain-dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hypertoxin-producing strain, PA103. The P. aeruginosa regA mutant strain, PA103-29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103-29 containing the regA locus cloned from the hypertoxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.     

IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.     

In vivo growth of Pseudomonas aeruginosa strains PAO1 and PA14 and the hypervirulent strain LESB58 in a rat model of chronic lung infection

Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.