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Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations
Abstract and Figures
Chronic respiratory infection by Pseudomonas aeruginosa contributes significantly to the morbidity and mortality associated with cystic fibrosis (CF). Using a series of phenotypic and genotypic tests on collections of 40 isolates per sputum sample, we analysed fluctuations within sputum populations of the P. aeruginosa Liverpool epidemic strain (LES) during pulmonary exacerbations. For each of three patients, three sequential sputum samples were analysed: (1) on presentation with exacerbation at the Regional Adult Cystic Fibrosis Unit, Liverpool; (2) a few days into intravenous antibiotic treatment; (3) when the patient had recovered. Fluctuations were observed in morphotype distribution, the production of virulence-associated quorum-sensing-dependent exoproducts (the phenazine compound pyocyanin and the elastase LasA), antibiotic susceptibility profiles and levels of auxotrophy. PCR assays were used to screen isolates for the presence of novel regions of the LES genome (islands and prophages) and to detect free phages. In one patient there was an increase in the prevalence of the LESGI-5 genomic island during the sampling period from 10 to 97.5 % carriage. LES phages 2-4 were detected in either the majority or all sputum samples tested, indicating widespread phage activity during the sampling period. The results of this study are indicative that significant fluctuations occur within P. aeruginosa populations during short periods of pulmonary exacerbation and intravenous antibiotic therapy.
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... Deletions of more than 1000 bp have been observed in other lineages (in 10 out of 12 lineages in the study of Klockgether and colleagues), with the size of deleted regions reaching 188 kb [6,7,11,17]. Here again, these deletions were shown to affect both core and accessory genomes, as prophages and genomic islands were shown to be partially or totally lost during P. aeruginosa adaptation to the CF environment [11,27,28,31,[33][34][35]. Notably, the genomic islands PAPI-1 and the P. aeruginosa genomic island-2 (PAGI-2) were found either excised or impacted by deletions in CF isolates [27,28]. ...
... In the case of longitudinal genomic studies, the sequencing of a single strain per time point is an important limitation and provides only a restricted fraction of the different evolutionary paths that the bacterium has taken. This issue obviously feeds through to all genotypic and phenotypic characterizations of CF P. aeruginosa strains, but is increasingly taken into account for sequencing studies and the determination of antibioticresistance profiles [34,45,68,69]. ...
Pseudomonas aeruginosa is one of the main microbial species colonizing the lungs of cystic fibrosis patients and is responsible for the decline in respiratory function. Despite the hostile pulmonary environment, P. aeruginosa is able to establish chronic infections thanks to its strong adaptive capacity. Various longitudinal studies have attempted to compare the strains of early infection with the adapted strains of chronic infection. Thanks to new ‘-omics’ techniques, convergent genetic mutations, as well as transcriptomic and proteomic dysregulations have been identified. As a consequence of this evolution, the adapted strains of P. aeruginosa have particular phenotypes that promote persistent infection.
... We next evaluated the proposed method 2SigFinder on P. aeruginosa LESB58 genome, whose genomic islands have been explored widely [38][39][40]. There are currently 6 prophage gene clusters and 5 annotated pathogenicity islands in P. aeruginosa LESB58 [38,41,42]. ...
... We next evaluated the proposed method 2SigFinder on P. aeruginosa LESB58 genome, whose genomic islands have been explored widely [38][39][40]. There are currently 6 prophage gene clusters and 5 annotated pathogenicity islands in P. aeruginosa LESB58 [38,41,42]. We applied 2SigFinder to identify the genomic islands in the P. aeruginosa LESB58 genome, where transformed window is 4, eye window is 5, neighbourhood size is 4 and long window size is 100, using 256 core features and 4 dynamic features, with 4 iterations in IST-LFS and 4 iterations in ILST-DSFS, and 0.05 standard error. ...
Background:
Genomic islands are associated with microbial adaptations, carrying genomic signatures different from the host. Some methods perform an overall test to identify genomic islands based on their local features. However, regions of different scales will display different genomic features.
Results:
We proposed here a novel method "2SigFinder ", the first combined use of small-scale and large-scale statistical testing for genomic island detection. The proposed method was tested by genomic island boundary detection and identification of genomic islands or functional features of real biological data. We also compared the proposed method with the comparative genomics and composition-based approaches. The results indicate that the proposed 2SigFinder is more efficient in identifying genomic islands.
Conclusions:
From real biological data, 2SigFinder identified genomic islands from a single genome and reported robust results across different experiments, without annotated information of genomes or prior knowledge from other datasets. 2SigHunter identified 25 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats from 27 Pathogenicity, 1 tRNA, 2 Virulence and 2 Repeats, and detected 101 Phage and 28 HEG out of 130 Phage and 36 HEGs in S. enterica Typhi CT18, which shows that it is more efficient in detecting functional features associated with GIs.
... Tout d'abord, même si le patient est colonisé par une souche de départ, l'évolution ne va pas suivre le même schéma pour tous les clones découlant de cette souche. La sous-population de ce pathogène va alors prendre différentes directions d'évolution et une population hétérogène sera alors retrouvée dans le poumons, avec différents clones présents en différentes proportions en fonction des conditions, ce qui est particulièrement le cas lors des infections chroniques à Pa (Fothergill et al., 2010) mais a également été observé pour d'autres bactéries comme Burkholderia multivorans par exemple (Silva et al., 2011). Une caractéristique souvent observée dans ces populations est l'hypermutabilité. ...
Afin de mieux lutter contre les infections bactériennes dans le cadre des infections pulmonaires chez les personnes atteintes de mucoviscidose, il est important de mieux comprendre les interactions entre pathogènes. Nous nous sommes intéressés aux interactions entre Pseudomonas aeruginosa, pathogène infectant de manière chronique les patients atteints de mucoviscidose, et d’autres pathogènes retrouvés chez ces patients au travers de la bataille que ces bactéries se livrent pour l’approvisionnement en fer. Le fer est un élément clé pour la croissance bactérienne, mais il est très peu biodisponible dans les fluides de l’hôte. Les bactéries produisent alors des sidérophores pour lier le fer dans le milieu extracellulaire et permettre son import dans la bactérie. P. aeruginosa produit deux sidérophores, la pyoverdine et la pyochéline. Au cours de l’infection chronique à P. aeruginosa, la production de ces sidérophores varie, en même temps que la virulence des souches. Afin de mieux discerner l’importance des sidérophores dans l’import en fer et dans ces mécanismes de virulence, nous avons étudié les interactions entre des isolats de P. aeruginosa issus d’un patient unique, mais prélevé à différents temps de l’infection chronique et 5 autres pathogènes. Nous avons d’abord mieux caractérisé la régulation de la production de pyochéline, moins bien étudiée, puis développé un outil pour différencier des bactéries en co-cultures grâce à un marqueur fluorescent. Nous avons ainsi pu mieux spécifier l’importance de ces sidérophores en tant que facteurs de virulence et en tant que facteur d’import du fer. La baisse de leur production au cours de l’infection chronique permet aux autres bactéries de mieux proliférer et à des interactions bactériennes de s’établir. Enfin, nous avons également établi un lien entre certains traitements antibiotiques, récurrents chez ces patients, et l’évolution de la production de sidérophores chez P. aeruginosa.
... DNA viral sequencing of CF sputum has revealed a CF phageome distinct from the phageome of people without CF [199]. Studies investigating the CF airway virome have revealed widespread genomic rearrangements and phage activity in the CF airway during periods of exacerbation and intravenous antibiotic treatment [200]. Important CF pathogens including, P. aeruginosa, S. aureus, and B. cenocepacia, have been shown to contain antimicrobial inducible prophages in their accessory genome [201,202]. ...
Pseudomonas aeruginosa is one of the most dominant pathogens in cystic fibrosis (CF) airway disease and contributes to significant inflammation, airway damage, and poorer disease outcomes. The CF airway is now known to be host to a complex community of microorganisms, and polymicrobial interactions have been shown to play an important role in shaping P. aeruginosa pathogenicity and resistance. P. aeruginosa can cause chronic infections that once established are almost impossible to eradicate with antibiotics. CF patients that develop chronic P. aeruginosa infection have poorer lung function, higher morbidity, and a reduced life expectancy. P. aeruginosa adapts to the CF airway and quickly develops resistance to several antibiotics. A perplexing phenomenon is the disparity between in vitro antimicrobial sensitivity testing and clinical response. Considering the CF airway is host to a diverse community of microorganisms or ‘microbiome’ and that these microorganisms are known to interact, the antimicrobial resistance and progression of P. aeruginosa infection is likely influenced by these microbial relationships. This review combines the literature to date on interactions between P. aeruginosa and other airway microorganisms and the influence of these interactions on P. aeruginosa tolerance to antimicrobials.
... Resistance against ciprofloxacin occurs most commonly through mutations in the DNA gyrase subunits GyrA and GyrB or the topoisomerase subunits ParC/ParD but also through the overexpression of efflux (Yang et al., 2015). Mutations in gyrA and gyrB are commonly identified in CF associated infections and are likely to arise during prolonged antibiotic treatment with ciprofloxacin (Fothergill et al., 2010;Marvig et al., 2015). The most frequently observed mutations in both CF and non-CF isolates, as well as in in vitro experiments, are the substitution of an amino acid at position 83 in GyrA from threonine to isoleucine and an amino acid substitution of serine to leucine at position 87 in ParC (Higgins et al., 2003;Lee et al., 2005;Bruchmann et al., 2013). ...
P. aeruginosa is classified as a priority one pathogen by the World Health Organisation, and new drugs are urgently needed, due to the emergence of multidrug-resistant (MDR) strains. Antimicrobial-resistant nosocomial pathogens such as P. aeruginosa pose unwavering and increasing threats. Antimicrobial stewardship has been a challenge during the COVID-19 pandemic, with a majority of those hospitalized with SARS-CoV2 infection given antibiotics as a safeguard against secondary bacterial infection. This increased usage, along with increased handling of sanitizers and disinfectants globally, may further accelerate the development and spread of cross-resistance to antibiotics. In addition, P. aeruginosa is the primary causative agent of morbidity and mortality in people with the life-shortening genetic disease cystic fibrosis (CF). Prolonged periods of selective pressure, associated with extended antibiotic treatment and the actions of host immune effectors, results in widespread adaptive and acquired resistance in P. aeruginosa found colonizing the lungs of people with CF. This review discusses the arsenal of resistance mechanisms utilized by P. aeruginosa, how these operate under high-stress environments such as the CF lung and how their interconnectedness can result in resistance to multiple antibiotic classes. Intrinsic, adaptive and acquired resistance mechanisms will be described, with a focus on how each layer of resistance can serve as a building block, contributing to multi-tiered resistance to antimicrobial activity. Recent progress in the development of anti-resistance adjuvant therapies, targeting one or more of these building blocks, should lead to novel strategies for combatting multidrug resistant P. aeruginosa. Anti-resistance adjuvant therapy holds great promise, not least because resistance against such therapeutics is predicted to be rare. The non-bactericidal nature of anti-resistance adjuvants reduce the selective pressures that drive resistance. Anti-resistance adjuvant therapy may also be advantageous in facilitating efficacious use of traditional antimicrobials, through enhanced penetration of the antibiotic into the bacterial cell. Promising anti-resistance adjuvant therapeutics and targets will be described, and key remaining challenges highlighted. As antimicrobial stewardship becomes more challenging in an era of emerging and re-emerging infectious diseases and global conflict, innovation in antibiotic adjuvant therapy can play an important role in extending the shelf-life of our existing antimicrobial therapeutic agents.
... In addition to causing oxidative stress to the lung, PYO is directly toxic to cilia, upregulates interleukin (IL)-8 activity, causes cellular senescence [78][79][80][81][82] and inactivates α 1 -antitrypsin, an important component of the endogenous antiprotease shield, contributing to protease/antiprotease imbalance within the lung. PYO is regulated by the P. aeruginosa quorum sensing (QS) system [77] and levels have been directly correlated with prognosis [83] and frequency of pulmonary exacerbations [84]. ...
Objectives
Cystic fibrosis (CF) lung disease is characterised by mucus stasis, chronic infection and inflammation, causing progressive structural lung disease and eventual respiratory failure. CF airways are inhabited by an ecologically diverse polymicrobial environment with vast potential for interspecies interactions, which may be a contributing factor to disease progression. Pseudomonas aeruginosa and Aspergillus fumigatus are the most common bacterial and fungal species present in CF airways respectively and coinfection results in a worse disease phenotype.
Methods
In this review we examine existing expert knowledge of chronic co-infection with P. aeruginosa and A. fumigatus in CF patients. We summarise the mechanisms of interaction and evaluate the clinical and inflammatory impacts of this co-infection.
Results
P. aeruginosa inhibits A. fumigatus through multiple mechanisms: phenazine secretion, iron competition, quorum sensing and through diffusible small molecules. A. fumigatus reciprocates inhibition through gliotoxin release and phenotypic adaptations enabling evasion of P. aeruginosa inhibition. Volatile organic compounds secreted by P. aeruginosa stimulate A. fumigatus growth, while A. fumigatus stimulates P. aeruginosa production of cytotoxic elastase.
Conclusion
A complex bi-directional relationship exists between P. aeruginosa and A. fumigatus , exhibiting both mutually antagonistic and cooperative facets. Cross-sectional data indicate a worsened disease state in coinfected patients; however, robust longitudinal studies are required to derive causality and to determine whether interspecies interaction contributes to disease progression.
... Bacterial genome sequences obtained from multiple isolates collected longitudinally provide additional important information about adaptive processes in individual patients. The existence of co-evolving strains and sub-lineages of the same strain, and their rapid and dynamic tournover 85,86 , within the same patient complicates our understanding of genetic ...
Intense genome sequencing of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) airways has shown inefficient eradication of the infecting bacteria, as well as previously undocumented patient-to-patient transmission of adapted clones. However, genome sequencing has limited potential as a predictor of chronic infection and of the adaptive state during infection, and thus there is increasing interest in linking phenotypic traits to the genome sequences. Phenotypic information ranges from genome-wide transcriptomic analysis of patient samples to determination of more specific traits associated with metabolic changes, stress responses, antibiotic resistance and tolerance, biofilm formation and slow growth. Environmental conditions in the CF lung shape both genetic and phenotypic changes of P. aeruginosa during infection. In this Review, we discuss the adaptive and evolutionary trajectories that lead to early diversification and late convergence, which enable P. aeruginosa to succeed in this niche, and we point out how knowledge of these biological features may be used to guide diagnosis and therapy.
... We identified four studies where phage detection was reported from sputum, and they all involved dilution, chemical lysis, and filtration to remove the cellular components. [43][44][45][46] Further steps to purify the phage or viral component of the sputum are taken using phenol/chloroform 45 or cesium chloride. 46 We have sought to provide a resource on the optimal methods for the collection and processing of sputum samples from patients with CF for microbial DNA studies, with a particular emphasis on Pf phage and Pseudomonas. ...
Background:
There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult.
Materials and Methods:
In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum.
Results:
Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies.
Conclusion:
These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF.
... Some prophages in the CF metagenome described here may result from antibiotic induction, as antibiotic treatment of CF patients contributes to phage induction in bacteria (Fothergill et al., 2010). Phages contribute to the development of antimicrobial resistance in environmental bacteria by transferring genes encoding antibiotic resistance (Colomer-Lluch et al., 2011). ...
Bacteriophages, viruses infecting bacteria, are uniformly present in any location where there are high numbers of bacteria, both in the external environment and the human body. Knowledge of their diversity is limited by the difficulty to culture the host species and by the lack of the universal marker gene present in all viruses. Metagenomics is a powerful tool that can be used to analyse viral communities in their natural environments. The aim of this study was to investigate diverse populations of uncultured viruses from clinical (a sputum of patient with cystic fibrosis, CF) and environmental samples (a sludge from a dairy food wastewater treatment plant) containing rich bacterial populations using genetic and metagenomic analyses. Metagenomic sequencing of viruses obtained from these samples revealed that the majority of the metagenomic reads (97-99%) were novel when compared to the NCBI protein database using BLAST. A large proportion of assembled contigs were assignable as novel phages or uncharacterised prophages, the next largest assignable group being single-stranded eukaryotic virus genomes. Sputum from a cystic fibrosis patient contained DNA typical of phages of bacteria that are traditionally involved in CF lung infections and other bacteria that are part of the normal oral flora. The only eukaryotic virus detected in the CF sputum was Torque Teno virus (TTV). A substantial number of assigned sequences from dairy wastewater could be affiliated with phages of bacteria that are typically found in the soil and aquatic environments, including wastewater. Eukaryotic viral sequences were dominated by plant pathogens from the Geminiviridae and Nanoviridae families, and animal pathogens from the Circoviridae family. Antibiotic resistance genes were detected in both metagenomes suggesting phages could be a source for transmissible antimicrobial resistance. Overall, diversity of viruses in the CF sputum was low, with 89 distinct viral genotypes predicted, and higher (409 genotypes) in the wastewater.
Function-based screening of a metagenomic library constructed from DNA extracted from dairy food wastewater viruses revealed candidate promoter sequences that have ability to drive expression of GFP in a promoter-trap vector in Escherichia coli. The majority of the cloned DNA sequences selected by the assay were related to ssDNA
xvi
circular eukaryotic viruses and phages which formed a minority of the metagenome assembly, and many lacked any significant homology to known database sequences.
Natural diversity of bacteriophages in wastewater samples was also examined by PCR amplification of the major capsid protein sequences, conserved within T4-type bacteriophages from Myoviridae family. Phylogenetic analysis of capsid sequences revealed that dairy wastewater contained mainly diverse and uncharacterized phages, while some showed a high level of similarity with phages from geographically distant environments.
With the improving survival of cystic fibrosis (CF) patients and the advent of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) therapy, the clinical spectrum of this complex multisystem disease continues to evolve. One of the most important clinical events for patients with CF in the course of this disease is acute pulmonary exacerbation (PEx). Clinical and microbial epidemiology studies of CF PEx continue to provide important insight into the disease course, prognosis, and complications. This work has now led to several large-scale clinical trials designed to clarify the treatment paradigm for CF PEx. The primary goal of this review is to provide a summary and update of the pathophysiology, clinical and microbial epidemiology, outcome and treatment of CF PEx, biomarkers for exacerbation, and the impact of highly effective modulator therapy on these events moving forward.
aeruginosa to undergo the typical adaptation to the intractable mu- coid form in the CF lung. These findings indicate that gene activation in bacteria by toxic oxygen radicals, similar to that found in plants and mammalian cells, may serve as a defence mechanism for the bacteria. This suggests that mucoid conversion is a response to oxygen radical exposure and that this response is a mechanism of defence by the bacteria. This is the first report to show that PMNs and their oxygen radicals can cause this phenotypic and genotypic change which is so typical of the intractable form of P. aeruginosa in the CF lung. These findings may provide a basis for the development of anti-oxidant and anti-inflammatory therapy for the early stages of infection in CF patients.
During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled
gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients
chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate
that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening
of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of
the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined
for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays.
Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding
that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying
mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-l-homoserine lactone.
Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution.
CF-Marseille is multidrug resistant, has a hetero-Glycopeptide-Intermediate resistance S. aureus phenotype, grows on Cepacia agar with intense orange pigmentation and has a thickened cell wall. Phylogenetic analyses using Complete Genome Hybridization and Multi Locus VNTR Assay showed that CF-Marseille was closely related to strain Mu50, representing vancomycin-resistant S. aureus. Analysis of CF-Marseille shows a similar core genome to that of previously sequenced MRSA strains but with a different genomic organization due to the presence of specific mobile genetic elements i.e. a new SCCmec type IV mosaic cassette that has integrated the pUB110 plasmid, and a new phage closely related to phiETA3. Moreover this phage could be seen by electron microscopy when mobilized with several antibiotics commonly used in CF patients including, tobramycin, ciprofloxacin, cotrimoxazole, or imipenem. Phylogenetic analysis of phenotypically similar h-GISA in our study also suggests that CF patients are colonized by polyclonal populations of MRSA that represents an incredible reservoir for lateral gene transfer.
In conclusion, we demonstrated the emergence and spreading of a new isolate of MRSA in CF patients in Marseille, France, that has probably been selected in the airways by antibiotic pressure. Antibiotic-mediated phage induction may result in high-frequency transfer and the unintended consequence of promoting the spread of virulence and/or antibiotic resistance determinants. The emergence of well-adapted MRSA is worrying in such population chronically colonized and receiving many antibiotics and represents a model for emergence of uncontrollable super bugs in a specific niche.
This article was reviewed by Eric Bapteste, Pierre Pontarotti, and Igor Zhulin. For the full reviews, please go to the Reviewers' comments section.
A surprising example of interspecies competition is the production by certain bacteria of hydrogen peroxide at concentrations that are lethal for others. A case in point is the displacement of Staphylococcus aureus by Streptococcus pneumoniae in the nasopharynx, which is of considerable clinical significance. How it is accomplished, however, has been a great mystery, because H(2)O(2) is a very well known disinfectant whose lethality is largely due to the production of hyperoxides through the abiological Fenton reaction. In this report, we have solved the mystery by showing that H(2)O(2) at the concentrations typically produced by pneumococci kills lysogenic but not nonlysogenic staphylococci by inducing the SOS response. The SOS response, a stress response to DNA damage, not only invokes DNA repair mechanisms but also induces resident prophages, and the resulting lysis is responsible for H(2)O(2) lethality. Because the vast majority of S. aureus strains are lysogenic, the production of H(2)O(2) is a very widely effective antistaphylococcal strategy. Pneumococci, however, which are also commonly lysogenic and undergo SOS induction in response to DNA-damaging agents such as mitomycin C, are not SOS-induced on exposure to H(2)O(2). This is apparently because they are resistant to the DNA-damaging effects of the Fenton reaction. The production of an SOS-inducing signal to activate prophages in neighboring organisms is thus a rather unique competitive strategy, which we suggest may be in widespread use for bacterial interference. However, this strategy has as a by-product the release of active phage, which can potentially spread mobile genetic elements carrying virulence genes.
Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.
Chronic infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) and certain strains are more transmissible and virulent than others. Of these, the Liverpool Epidemic Strain (LES) is highly transmissible and cross infection has been reported between patients with CF and healthy non-CF relatives. However, the risk of transmission from humans to animals is unknown. The first report of interspecies transmission of the LES strain of P aeruginosa from an adult patient with CF to a pet cat is described. This development further complicates the issue of infection control policies required to prevent the spread of this organism.
BACKGROUND
Pseudomonas aeruginosa may undergo a phenotypic change from the wild (prototrophic) type to an auxotrophic phenotype in the course of respiratory infection in patients with cystic fibrosis. The clinical significance of this is unclear. A study was undertaken to investigate whether the presence of auxotrophs of P aeruginosa in the sputum of patients with cystic fibrosis correlated with severity of respiratory disease, and whether increased sputum concentrations of amino acids were associated with the emergence of these forms.
METHODS
Sixty adult patients with cystic fibrosis, colonised by P aeruginosa, were recruited and baseline clinical data including lung function were recorded. Serial sputum samples were obtained before, during, and after infective exacerbations where possible. These samples were used for routine microbiological culture, assessment of auxotrophy of P aeruginosa, measurement of amino acid content, and neutrophil elastase assay.
RESULTS
Auxotrophy was common in patients with cystic fibrosis and 20 (33%) had a mean percentage auxotroph count of more than 50% total cfu/ml. The mean percentage auxotroph count was inversely correlated with forced expiratory volume in one second (FEV1; τ = –0.194, p = 0.031). The median sputum amino acid concentration of the group was 12.5 mmol/l (range 0.13–40.6). The mean amino acid concentration in 33 subjects during infective exacerbations was 18.2 mmol/l (95% CI 15.1 to 21.3) compared with 12.3 mmol/l (95% CI 9.8 to 14.8) when well (p = 0.001). The amino acid content of sputum was inversely correlated with FEV1 (τ = –0.253, p = 0.005).
CONCLUSIONS
P aeruginosa frequently exhibits auxotrophy in patients with cystic fibrosis, particularly in those with severe underlying pulmonary disease. The sputum amino acid content of patients with cystic fibrosis is high during infective exacerbations and correlates with pulmonary disease severity.
Study objective
To determine the relationship between the antibiotic susceptibility of Pseudomonas aeruginosa isolated from the sputum of patients with cystic fibrosis (CF) and the patient’s response to parenteral antibiotic administration, we performed a retrospective analysis using data from patients in the placebo arm of a phase 3 trial of tobramycin solution for inhalation. All patients were chronically infected with P aeruginosa. Seventy-seven of the 262 patients receiving placebo experienced a pulmonary exacerbation during the trial for which they received therapy with IV tobramycin and ceftazidime. The susceptibility of the P aeruginosa isolates to ceftazidime and tobramycin was determined at trial enrollment by broth microdilution.
Design
The clinical response to combination antibiotic therapy was assessed by analyzing differences in spirometry before and after antibiotic administration. The FEV1 percent predicted at the first visit after the conclusion of antibiotic administration was compared to the FEV1 percent predicted prior to antibiotic therapy. The results were analyzed both descriptively and by regression analyses.
Results
The conditions of 54 patients improved, and those of 9 patients worsened, and in 14 patients there was no change in FEV1 with antibiotic administration. No correlation was observed between the susceptibility of P aeruginosa to tobramycin or ceftazidime and clinical response. Only the three following variables were observed to significantly correlate with FEV1 after antibiotic treatment on regression analysis: FEV1 prior to treatment (p < 0.0001); number of days elapsed between the previous FEV1 measurement and the initiation of IV antibiotic therapy (p < 0.002); and the number of days elapsed between the determination of the minimum inhibitory concentration and the initiation of IV therapy (p < 0.03). No significant trends were observed between the antibiotic susceptibility of P aeruginosa isolates and treatment outcomes.
Conclusion
While lack of statistical significance for a trend between bacterial susceptibilities and the response to parenteral antibiotic administration does not mean that no such trend exists, the precision of the confidence intervals allows us to conclude that even if isolate antibiotic susceptibilities affect outcome, the impact would be small and not clinically relevant.
Studies on cultured cells and in infection models have shown that cell density-dependent quorum-sensing (QS) controls many of the known virulence factors of Pseudomonas aeruginosa. However, it is less clear what role QS plays in chronic human lung infections associated with cystic fibrosis (CF). The involvement of QS in biofilm development, crucial to the establishment of long-term infections, suggests a role in the early stages of infection. However, the accumulation of QS mutants during chronic CF infections has been taken to indicate that any role diminishes thereafter. Here, we discuss the evidence for a continuing role for QS in P. aeruginosa CF infections, including QS activity in CF sputa and CF-relevant effects of QS-regulated products, such as pyocyanin. Bacterial population behaviour in CF is complex, and the exact roles of QS remains unclear. Therapeutic strategies directed against QS suggest that a greater understanding of bacterial populations during infection would be a valuable research goal from a clinical perspective.