Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa.
ABSTRACT: Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of causing a variety of life-threatening human infections. The genetic basis for preferential infection of certain immunocompromised patients or individuals with cystic fibrosis by P. aeruginosa is not understood. To establish whether variation in the genomic repertoire of P. aeruginosa strains can be associated with a particular type of infection, we used a whole-genome DNA microarray to determine the genome content of 18 strains isolated from the most common human infections and environmental sources. A remarkable conservation of genes including those encoding nearly all known virulence factors was observed. Phylogenetic analysis of strain-specific genes revealed no correlation between genome content and infection type. Clusters of strain-specific genes in the P. aeruginosa genome, termed variable segments, appear to be preferential sites for the integration of novel genetic material. A specialized cloning vector was developed for capture and analysis of these genomic segments. With this capture system a site associated with the strain-specific ExoU cytotoxin-encoding gene was interrogated and an 80-kb genomic island carrying exoU was identified. These studies demonstrate that P. aeruginosa strains possess a highly conserved genome that encodes genes important for survival in numerous environments and allows it to cause a variety of human infections. The acquisition of novel genetic material, such as the exoU genomic island, through horizontal gene transfer may enhance colonization and survival in different host environments.
Project description: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.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen and is associated with nosocomial infections. Its ability to thrive in a broad range of environments is due to a large and diverse genome of which its accessory genome is part. The objective of this study was to characterize P. aeruginosa strains isolated from children who developed bacteremia, using pulse-field gel electrophoresis, and in terms of its genomic islands, virulence genes, multilocus sequence type, and antimicrobial susceptibility. Our results showed that P. aeruginosa strains presented the seven virulence genes: toxA, lasB, lecA, algR, plcH, phzA1, and toxR, a type IV pilin alleles (TFP) group I or II. Additionally, we detected a novel pilin and accessory gene, expanding the number of TFP alleles to group VI. All strains presented the PAPI-2 Island and the majority were exoU+ and exoS+ genotype. Ten percent of the strains were multi-drug resistant phenotype, 18% extensively drug-resistant, 68% moderately resistant and only 3% were susceptible to all the antimicrobial tested. The most prevalent acquired ?-Lactamase was KPC. We identified a group of ST309 strains, as a potential high risk clone. Our finding also showed that the strains isolated from patients with bacteremia have important virulence factors involved in colonization and dissemination as: a TFP group I or II; the presence of the exoU gene within the PAPI-2 island and the presence of the exoS gene.
Project description:One of the hallmarks of the Gram-negative bacterium Pseudomonas aeruginosa is its ability to thrive in diverse environments that includes humans with a variety of debilitating diseases or immune deficiencies. Here we report the complete sequence and comparative analysis of the genomes of two representative P. aeruginosa strains isolated from cystic fibrosis (CF) patients whose genetic disorder predisposes them to infections by this pathogen. The comparison of the genomes of the two CF strains with those of other P. aeruginosa presents a picture of a mosaic genome, consisting of a conserved core component, interrupted in each strain by combinations of specific blocks of genes. These strain-specific segments of the genome are found in limited chromosomal locations, referred to as regions of genomic plasticity. The ability of P. aeruginosa to shape its genomic composition to favor survival in the widest range of environmental reservoirs, with corresponding enhancement of its metabolic capacity is supported by the identification of a genomic island in one of the sequenced CF isolates, encoding enzymes capable of degrading terpenoids produced by trees. This work suggests that niche adaptation is a major evolutionary force influencing the composition of bacterial genomes. Unlike genome reduction seen in host-adapted bacterial pathogens, the genetic capacity of P. aeruginosa is determined by the ability of individual strains to acquire or discard genomic segments, giving rise to strains with customized genomic repertoires. Consequently, this organism can survive in a wide range of environmental reservoirs that can serve as sources of the infecting organisms.
Project description:Pseudomonas aeruginosa is an important opportunistic pathogen in hospitals, responsible for various infections that are difficult to treat due to intrinsic and acquired antibiotic resistance. Here, 20 epidemiologically unrelated strains isolated from patients in a general hospital over a time period of two decades were analyzed using whole genome sequencing. The genomes were compared in order to assess the presence of a predominant clone or sequence type (ST). No clonal structure was identified, but core genome-based single nucleotide polymorphism (SNP) analysis distinguished two major, previously identified phylogenetic groups. Interestingly, most of the older strains isolated between 1994 and 1998 harbored exoU, encoding a cytotoxic phospholipase. In contrast, most strains isolated between 2011 and 2016 were exoU-negative and phylogenetically very distinct from the older strains, suggesting a population shift of nosocomial P. aeruginosa over time. Three out of 20 strains were ST235 strains, a global high-risk clonal lineage; these carried several additional resistance determinants including aac(6')Ib-cr encoding an aminoglycoside N-acetyltransferase that confers resistance to fluoroquinolones. Core genome comparison with ST235 strains from other parts of the world showed that the three strains clustered together with other Brazilian/Argentinean isolates. Despite this regional relatedness, the individuality of each of the three ST235 strains was revealed by core genome-based SNPs and the presence of genomic islands in the accessory genome. Similarly, strain-specific characteristics were detected for the remaining strains, indicative of individual evolutionary histories and elevated genome plasticity.
Project description:Virulent strains of Pseudomonas aeruginosa are often associated with an acquired cytotoxic protein, exoenzyme U (ExoU) that rapidly destroys the cell membranes of host cells by its phospholipase activity. Strains possessing the exoU gene are predominant in eye infections and are more resistant to antibiotics. Thus, it is essential to understand treatment options for these strains. Here, we have investigated the resistance profiles and genes associated with resistance for fluoroquinolone and beta-lactams. A total of 22 strains of P. aeruginosa from anterior eye infections, microbial keratitis (MK), and the lungs of cystic fibrosis (CF) patients were used. Based on whole genome sequencing, the prevalence of the exoU gene was 61.5% in MK isolates whereas none of the CF isolates possessed this gene. Overall, higher antibiotic resistance was observed in the isolates possessing exoU. Of the exoU strains, all except one were resistant to fluoroquinolones, 100% were resistant to beta-lactams. 75% had mutations in quinolone resistance determining regions (T81I gyrA and/or S87L parC) which correlated with fluoroquinolone resistance. In addition, exoU strains had mutations at K76Q, A110T, and V126E in ampC, Q155I and V356I in ampR and E114A, G283E, and M288R in mexR genes that are associated with higher beta-lactamase and efflux pump activities. In contrast, such mutations were not observed in the strains lacking exoU. The expression of the ampC gene increased by up to nine-fold in all eight exoU strains and the ampR was upregulated in seven exoU strains compared to PAO1. The expression of mexR gene was 1.4 to 3.6 fold lower in 75% of exoU strains. This study highlights the association between virulence traits and antibiotic resistance in pathogenic P. aeruginosa.
Project description:Pseudomonas aeruginosa uses its type III secretion system to inject the effector proteins ExoS and ExoU into eukaryotic cells, which subverts these cells to the bacterium's advantage and contributes to severe infections. We studied the environmental reservoirs of exoS+ and exoU+ strains of P. aeruginosa by collecting water, soil, moist substrates and plant samples from environments in the Chicago region and neighbouring states. Whole-genome sequencing was used to determine the phylogeny and type III secretion system genotypes of 120 environmental isolates. No correlation existed between geographic separation of isolates and their genetic relatedness, which confirmed previous findings of both high genetic diversity within a single site and the widespread distribution of P. aeruginosa clonal complexes. After excluding clonal isolates cultured from the same samples, 74 exoS+ isolates and 16 exoU+ isolates remained. Of the exoS+ isolates, 41 (55%) were from natural environmental sites and 33 (45%) were from man-made sites. Of the exoU+ isolates, only 3 (19%) were from natural environmental sites and 13 (81%) were from man-made sites (p?<?0.05). These findings suggest that man-made water systems may be a reservoir from which patients acquire exoU+ P. aeruginosa strains.
Project description:BACKGROUND: Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains. METHODS: Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE. RESULTS: A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P?0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased. CONCLUSIONS: The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.
Project description:BACKGROUND:Pseudomonas aeruginosa (PA) is one of the most common and serious causes of healthcare-associated bacteremia. The emergence and dissemination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) PA strains pose a major clinical concern. ST235-PA is a high-risk clone which shows a high capacity to acquire antibiotic resistance. Here we describe the first autochthonous New Delhi metallo-?-lactamase (NDM)-producing Pseudomonas aeruginosa ST235 identified in Italy. CASE PRESENTATION:In October 2019, a patient residing in an elderly health care and rehabilitation facility, was hospitalized and died from sepsis caused by an XDR-PA. The strain belonged to the high-risk clone sequence type ST235. Whole genome sequencing (WGS) revealed the presence of genes encoding NDM-1 and multiple ?-lactamases, many clinically significant multidrug efflux pump complexes and also the virulence gene ExoU, which is associated with a high cytotoxic phenotype. CONCLUSIONS:Few strains of NDM-1-PA have been identified worldwide, all belonging to ST235. The combination of ST235 and ExoU is a predictor of highly unfavorable prognosis. The potential spread of these high-risk clones in healthcare settings is worrisome because treatment options are limited. Early identification of high-risk clones could help in outbreaks investigation and infections control.
Project description:The diversification of microbial populations may be driven by many factors including adaptation to distinct ecological niches and barriers to recombination. We examined the population structure of the bacterial pathogen Pseudomonas aeruginosa by analyzing whole-genome sequences of 739 isolates from diverse sources. We confirmed that the population structure of P. aeruginosa consists of two major groups (referred to as Groups A and B) and at least two minor groups (Groups C1 and C2). Evidence for frequent intragroup but limited intergroup recombination in the core genome was observed, consistent with sexual isolation of the groups. Likewise, accessory genome analysis demonstrated more gene flow within Groups A and B than between these groups, and a few accessory genomic elements were nearly specific to one or the other group. In particular, the exoS gene was highly overrepresented in Group A compared with Group B isolates (99.4% vs. 1.1%) and the exoU gene was highly overrepresented in Group B compared with Group A isolates (95.2% vs. 1.8%). The exoS and exoU genes encode effector proteins secreted by the P. aeruginosa type III secretion system. Together these results suggest that the major P. aeruginosa groups defined in part by the exoS and exoU genes are divergent from each other, and that these groups are genetically isolated and may be ecologically distinct. Although both groups were globally distributed and caused human infections, certain groups predominated in some clinical contexts.
Project description:Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.