Evolutionary diversification of Pseudomonas aeruginosa in an artificial sputum model.

BACKGROUND: During chronic lung infections of cystic fibrosis patients Pseudomonas aeruginosa populations undergo extensive evolutionary diversification. However, the selective drivers of this evolutionary process are poorly understood. To test the effects of temperate phages on diversification in P. aeruginosa biofilms we experimentally evolved populations of P. aeruginosa for approximately 240 generations in artificial sputum medium with or without a community of three temperate phages. RESULTS: Analysis of end-point populations using a suite of phenotypic tests revealed extensive phenotypic diversification within populations, but no significant differences between the populations evolved with or without phages. The most common phenotypic variant observed was loss of all three types of motility (swimming, swarming and twitching) and resistance to all three phages. Despite the absence of selective pressure, some members of the population evolved antibiotic resistance. The frequency of antibiotic resistant isolates varied according to population and the antibiotic tested. However, resistance to ceftazidime and tazobactam-piperacillin was observed more frequently than resistance to other antibiotics, and was associated with higher prevelence of isolates exhibiting a hypermutable phenotype and increased beta-lactamase production. CONCLUSIONS: We observed considerable within-population phenotypic diversity in P. aeruginosa populations evolving in the artificial sputum medium biofilm model. Replicate populations evolved both in the presence and absence of phages converged upon similar sets of phenotypes. The evolved phenotypes, including antimicrobial resistance, were similar to those observed amongst clinical isolates from cystic fibrosis infections.

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms.

Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote within-host evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as free-floating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.

Temperate phages enhance pathogen fitness in chronic lung infection.

The Liverpool Epidemic Strain (LES) is a polylysogenic, transmissible strain of Pseudomonas aeruginosa, capable of superinfecting existing P. aeruginosa respiratory infections in individuals with cystic fibrosis (CF). The LES phages are highly active in the CF lung and may have a role in the competitiveness of the LES in vivo. In this study, we tested this by competing isogenic PAO1 strains that differed only by the presence or absence of LES prophages in a rat model of chronic lung infection. Lysogens invaded phage-susceptible populations, both in head-to-head competition and when invading from rare, in the spatially structured, heterogeneous lung environment. Appreciable densities of free phages in lung tissue confirmed active phage lysis in vivo. Moreover, we observed lysogenic conversion of the phage-susceptible competitor. These results suggest that temperate phages may have an important role in the competitiveness of the LES in chronic lung infection by acting as anti-competitor weapons.

Polylysogeny magnifies competitiveness of a bacterial pathogen in vivo.

The rise of next generation sequencing is revealing a hidden diversity of temperate phages within the microbial community. While a handful of these phages have been well characterized, for the vast majority, the role of phage carriage, and especially multiple phage carriage, is poorly understood. The Liverpool epidemic strain of Pseudomonas aeruginosa is an aggressive pathogen in cystic fibrosis lung infections that has recently been found to contain several unique prophages within its genome. Here, we experimentally investigate the role of two of these phages in vivo, using an insect model of infection. We find that while no benefit is conferred by phage carriage in single bacterial infections, phages confer a large fitness advantage during mixed infections by mediating bacteria-bacteria competition. Differences between the two phages appeared to be associated with the rate at which the competitor acquired the phage, and therefore resistance. However, the advantage was greatest in the polylysogen, carrying both phages. These findings suggest that the LES phages may play an important role in host invasions and more generally show that the carriage of multiple phages may itself be beneficial by hindering the spread of resistance in rival bacterial populations.

Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections.

Pseudomonas aeruginosa is the most common bacterial pathogen infecting the lungs of cystic fibrosis (CF) patients. The transmissible Liverpool epidemic strain (LES) harbours multiple inducible prophages (LESϕ2; LESϕ3; LESϕ4; LESϕ5; and LESϕ6), some of which are known to confer a competitive advantage in an in vivo rat model of chronic lung infection. We used quantitative PCR (Q-PCR) to measure the density and dynamics of all five LES phages in the sputa of 10 LES-infected CF patients over a period of 2 years. In all patients, the densities of free-LES phages were positively correlated with the densities of P. aeruginosa, and total free-phage densities consistently exceeded bacterial host densities 10-100-fold. Further, we observed a negative correlation between the phage-to-bacterium ratio and bacterial density, suggesting a role for lysis by temperate phages in regulation of the bacterial population densities. In 9/10 patients, LESϕ2 and LESϕ4 were the most abundant free phages, which reflects the differential in vitro induction properties of the phages. These data indicate that temperate phages of P. aeruginosa retain lytic activity after prolonged periods of chronic infection in the CF lung, and suggest that temperate phage lysis may contribute to regulation of P. aeruginosa density in vivo.

Differential infection properties of three inducible prophages from an epidemic strain of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the most common bacterial pathogen infecting the lungs of patients with cystic fibrosis (CF). The Liverpool Epidemic Strain (LES) is transmissible, capable of superseding other P. aeruginosa populations and is associated with increased morbidity. Previously, multiple inducible prophages have been found to coexist in the LES chromosome and to constitute a major component of the accessory genome not found in other sequenced P. aerugionosa strains. LES phages confer a competitive advantage in a rat model of chronic lung infection and may, therefore underpin LES prevalence. Here the infective properties of three LES phages were characterised. RESULTS: This study focuses on three of the five active prophages (LESφ2, LESφ3 and LESφ4) that are members of the Siphoviridae. All were induced from LESB58 by norfloxacin. Lytic production of LESφ2 was considerably higher than that of LESφ3 and LESφ4. Each phage was capable of both lytic and lysogenic infection of the susceptible P. aeruginosa host, PAO1, producing phage-specific plaque morphologies. In the PAO1 host background, the LESφ2 prophage conferred immunity against LESφ3 infection and reduced susceptibility to LESφ4 infection. Each prophage was less stable in the PAO1 chromosome with substantially higher rates of spontaneous phage production than when residing in the native LESB58 host. We show that LES phages are capable of horizontal gene transfer by infecting P. aeruginosa strains from different sources and that type IV pili are required for infection by all three phages. CONCLUSIONS: Multiple inducible prophages with diverse infection properties have been maintained in the LES genome. Our data suggest that LESφ2 is more sensitive to induction into the lytic cycle or has a more efficient replicative cycle than the other LES phages.

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung.

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa.

Phage production in response to antibiotics varied among four isolates of a Pseudomonas aeruginosa cystic fibrosis (CF) epidemic strain. Whereas ciprofloxacin induced higher levels of phage production, other CF-relevant antibiotics led to reduced production. We detected free phages directly in CF patient sputum samples by both plaque (40% positive) and PCR (76% positive) assays. Our observations suggest that the choice of antibiotics could influence the number of free phages within the CF lung environment.

HIV protease inhibitors are substrates for OATP1A2, OATP1B1 and OATP1B3 and lopinavir plasma concentrations are influenced by SLCO1B1 polymorphisms.

OBJECTIVE: OATP1B1 and OATP1B3 are major hepatic drug transporters whilst OATP1A2 is mainly located in the brain but is also located in liver and several other organs. These transporters affect the distribution and clearance of many endobiotics and xenobiotics and have been reported to have functional single nucleotide polymorphisms (SNPs). We have assessed the substrate specificities of these transporters for a panel of antiretrovirals and investigated the effects of SNPs within these transporters on the pharmacokinetics of lopinavir. METHODS: SLCO1A2, SLCO1B1 and SLCO1B3 were cloned, verified and used to generate cRNA for use in the Xenopuslaevis oocyte transport system. Using the oocyte system, antiretrovirals were tested for their substrate specificities. Plasma samples (n=349) from the Liverpool therapeutic drug monitoring registry were genotyped for SNPs in SLCO1A2, SLCO1B1 and SLCO1B3 and associations between SNPs and lopinavir plasma concentrations were analysed. RESULT: Antiretroviral protease inhibitors, but not non-nucleoside reverse transcriptase inhibitors, are substrates for OATP1A2, OATP1B1 and OATP1B3. Furthermore, ritonavir was not an inhibitor of OATP1B1. The 521T>C polymorphism in SLCO1B1 was significantly associated with higher lopinavir plasma concentrations. No associations were observed with functional variants of SLCO1A2 and SLCO1B3. CONCLUSION: These data add to our understanding of the factors that contribute to variability in plasma concentrations of protease inhibitors. Further studies are now required to confirm the association of SLCO1B1 521T>C with lopinavir plasma concentrations and to assess the influence of other polymorphisms in the SLCO family.

Intrinsic apoptotic pathways of gingival epithelial cells modulated by Porphyromonas gingivalis.

Porphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase-3. The anti-apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriae-deficient mutants and a naturally occurring non-fimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real-time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis-infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP-1 and cIAP-2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro-survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.

LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe(2+) ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.