Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Prophages are Infrequently Associated With Antibiotic Resistance in Pseudomonas aeruginosa Clinical Isolates.

Antimicrobial resistance (AMR) is a significant obstacle to the treatment of bacterial infections, including in the context of Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF). Lysogenic bacteriophages can integrate their genome into the bacterial chromosome and are known to promote genetic transfer between bacterial strains. However, the contribution of lysogenic phages to the incidence of AMR is poorly understood. Here, in a set of 187 clinical isolates of Pseudomonas aeruginosa collected from 82 patients with CF, we evaluate the links between prophages and both genomic and phenotypic resistance to five anti-pseudomonal antibiotics: tobramycin, colistin, ciprofloxacin, meropenem, aztreonam, and tazobactam. We find that P. aeruginosa isolates contain on average 3.06 +/-1.84 (SD) predicted prophages. We find no significant association between the number of prophages per isolate and the mean inhibitory concentration (MIC) for any of these antibiotics. We then investigate the relationship between particular prophages and AMR. We identify a single lysogenic phage that is associated with phenotypic resistance to the antibiotic tobramycin. Consistent with this association, we identify AMR genes associated with resistance to tobramycin in these strains and find that they are not encoded directly on prophage sequences. These findings suggest that prophages are infrequently associated with the AMR genes in clinical isolates of P. aeruginosa .

Re-assessing thermal response of schistosomiasis transmission risk: Evidence for a higher thermal optimum than previously predicted.

The geographical range of schistosomiasis is affected by the ecology of schistosome parasites and their obligate host snails, including their response to temperature. Previous models predicted schistosomiasis' thermal optimum at 21.7°C, which is not compatible with the temperature in sub-Saharan Africa (SSA) regions where schistosomiasis is hyperendemic. We performed an extensive literature search for empirical data on the effect of temperature on physiological and epidemiological parameters regulating the free-living stages of S. mansoni and S. haematobium and their obligate host snails, i.e., Biomphalaria spp. and Bulinus spp., respectively. We derived nonlinear thermal responses fitted on these data to parameterize a mechanistic, process-based model of schistosomiasis. We then re-cast the basic reproduction number and the prevalence of schistosome infection as functions of temperature. We found that the thermal optima for transmission of S. mansoni and S. haematobium range between 23.1-27.3°C and 23.6-27.9°C (95% CI) respectively. We also found that the thermal optimum shifts toward higher temperatures as the human water contact rate increases with temperature. Our findings align with an extensive dataset of schistosomiasis prevalence in SSA. The refined nonlinear thermal-response model developed here suggests a more suitable current climate and a greater risk of increased transmission with future warming for more than half of the schistosomiasis suitable regions with mean annual temperature below the thermal optimum.

Re-assessing thermal response of schistosomiasis transmission risk: evidence for a higher thermal optimum than previously predicted.

The geographical range of schistosomiasis is affected by the ecology of schistosome parasites and their obligate host snails, including their response to temperature. Previous models predicted schistosomiasis' thermal optimum at 21.7 °C, which is not compatible with the temperature in sub-Saharan Africa (SSA) regions where schistosomiasis is hyperendemic. We performed an extensive literature search for empirical data on the effect of temperature on physiological and epidemiological parameters regulating the free-living stages of S. mansoni and S. haematobium and their obligate host snails, i.e., Biomphalaria spp. and Bulinus spp., respectively. We derived nonlinear thermal responses fitted on these data to parameterize a mechanistic, process-based model of schistosomiasis. We then re-cast the basic reproduction number and the prevalence of schistosome infection as functions of temperature. We found that the thermal optima for transmission of S. mansoni and S. haematobium range between 23.1-27.3 °C and 23.6-27.9 °C (95 % CI) respectively. We also found that the thermal optimum shifts toward higher temperatures as the human water contact rate increases with temperature. Our findings align with an extensive dataset of schistosomiasis prevalence in SSA. The refined nonlinear thermal-response model developed here suggests a more suitable current climate and a greater risk of increased transmission with future warming for more than half of the schistosomiasis suitable regions with mean annual temperature below the thermal optimum.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates impacts quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous bacterial virulence phenotypes. Most work on Pf phages has focused on strain Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf strains infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, virulence factor production, and virulence. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates, but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.

Climatic, land-use and socio-economic factors can predict malaria dynamics at fine spatial scales relevant to local health actors: Evidence from rural Madagascar.

While much progress has been achieved over the last decades, malaria surveillance and control remain a challenge in countries with limited health care access and resources. High-resolution predictions of malaria incidence using routine surveillance data could represent a powerful tool to health practitioners by targeting malaria control activities where and when they are most needed. Here, we investigate the predictors of spatio-temporal malaria dynamics in rural Madagascar, estimated from facility-based passive surveillance data. Specifically, this study integrates climate, land-use, and representative household survey data to explain and predict malaria dynamics at a high spatial resolution (i.e., by Fokontany, a cluster of villages) relevant to health care practitioners. Combining generalized linear mixed models (GLMM) and path analyses, we found that socio-economic, land use and climatic variables are all important predictors of monthly malaria incidence at fine spatial scales, via both direct and indirect effects. In addition, out-of-sample predictions from our model were able to identify 58% of the Fokontany in the top quintile for malaria incidence and account for 77% of the variation in the Fokontany incidence rank. These results suggest that it is possible to build a predictive framework using environmental and social predictors that can be complementary to standard surveillance systems and help inform control strategies by field actors at local scales.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Filamentous Bacteriophages and the Competitive Interaction between Pseudomonas aeruginosa Strains under Antibiotic Treatment: a Modeling Study.

Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work has implicated Pf bacteriophages, nonlytic filamentous viruses produced by Pa, in the chronicity and severity of Pa infections. Pf phages act as structural elements in Pa biofilms and sequester aerosolized antibiotics, thereby contributing to antibiotic tolerance. Consistent with a selective advantage in this setting, the prevalence of Pf-positive (Pf+) bacteria increases over time in these patients. However, the production of Pf phages comes at a metabolic cost to bacteria, such that Pf+ strains grow more slowly than Pf-negative (Pf-) strains in vitro. Here, we use a mathematical model to investigate how these competing pressures might influence the relative abundance of Pf+ versus Pf- strains in different settings. Our model suggests that Pf+ strains of Pa cannot outcompete Pf- strains if the benefits of phage production falls onto both Pf+ and Pf- strains for a majority of parameter combinations. Further, phage production leads to a net positive gain in fitness only at antibiotic concentrations slightly above the MIC (i.e., concentrations for which the benefits of antibiotic sequestration outweigh the metabolic cost of phage production) but which are not lethal for Pf+ strains. As a result, our model suggests that frequent administration of intermediate doses of antibiotics with low decay rates and high killing rates favors Pf+ over Pf- strains. These models inform our understanding of the ecology of Pf phages and suggest potential treatment strategies for Pf+ Pa infections. IMPORTANCE Filamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf- strains of Pa in different settings. Our results identify conditions likely to favor Pf+ strains and thus antibiotic tolerance. This study contributes to a better understanding of the unique ecology of filamentous phages in both environmental and clinical settings and may facilitate improved treatment strategies for combating antibiotic tolerance.