Warming alters life-history traits and competition in a phage community.

Host-parasite interactions are highly susceptible to changes in temperature due to mismatches in species thermal responses. In nature, parasites often exist in communities, and responses to temperature are expected to vary between host-parasite pairs. Temperature change thus has consequences for both host-parasite dynamics and parasite-parasite interactions. Here, we investigate the impact of warming (37°C, 40°C, and 42°C) on parasite life-history traits and competition using the opportunistic bacterial pathogen Pseudomonas aeruginosa (host) and a panel of three genetically diverse lytic bacteriophages (parasites). We show that phages vary in their responses to temperature. While 37°C and 40°C did not have a major effect on phage infectivity, infection by two phages was restricted at 42°C. This outcome was attributed to disruption of different phage life-history traits including host attachment and replication inside hosts. Furthermore, we show that temperature mediates competition between phages by altering their competitiveness. These results highlight phage trait variation across thermal regimes with the potential to drive community dynamics. Our results have important implications for eukaryotic viromes and the design of phage cocktail therapies.IMPORTANCEMammalian hosts often elevate their body temperatures through fevers to restrict the growth of bacterial infections. However, the extent to which fever temperatures affect the communities of phages with the ability to parasitize those bacteria remains unclear. In this study, we investigate the impact of warming across a fever temperature range (37°C, 40°C, and 42°C) on phage life-history traits and competition using a bacterium (host) and bacteriophage (parasite) system. We show that phages vary in their responses to temperature due to disruption of different phage life-history traits. Furthermore, we show that temperature can alter phage competitiveness and shape phage-phage competition outcomes. These results suggest that fever temperatures have the potential to restrict phage infectivity and drive phage community dynamics. We discuss implications for the role of temperature in shaping host-parasite interactions more widely.

Introducing differential RNA-seq mapping to track the early infection phase for Pseudomonas phage ɸKZ.

As part of the ongoing renaissance of phage biology, more phage genomes are becoming available through DNA sequencing. However, our understanding of the transcriptome architecture that allows these genomes to be expressed during host infection is generally poor. Transcription start sites (TSSs) and operons have been mapped for very few phages, and an annotated global RNA map of a phage - alone or together with its infected host - is not available at all. Here, we applied differential RNA-seq (dRNA-seq) to study the early, host takeover phase of infection by assessing the transcriptome structure of Pseudomonas aeruginosa jumbo phage ɸKZ, a model phage for viral genetics and structural research. This map substantially expands the number of early expressed viral genes, defining TSSs that are active ten minutes after ɸKZ infection. Simultaneously, we record gene expression changes in the host transcriptome during this critical metabolism conversion. In addition to previously reported upregulation of genes associated with amino acid metabolism, we observe strong activation of genes with functions in biofilm formation (cdrAB) and iron storage (bfrB), as well as an activation of the antitoxin ParD. Conversely, ɸKZ infection rapidly down-regulates complexes IV and V of oxidative phosphorylation (atpCDGHF and cyoABCDE). Taken together, our data provide new insights into the transcriptional organization and infection process of the giant bacteriophage ɸKZ and adds a framework for the genome-wide transcriptomic analysis of phage-host interactions.

Resistance Evolution against Phage Combinations Depends on the Timing and Order of Exposure.

Phage therapy is a promising alternative to chemotherapeutic antibiotics for the treatment of bacterial infections. However, despite recent clinical uses of combinations of phages to treat multidrug-resistant infections, a mechanistic understanding of how bacteria evolve resistance against multiple phages is lacking, limiting our ability to deploy phage combinations optimally. Here, we show, using Pseudomonas aeruginosa and pairs of phages targeting shared or distinct surface receptors, that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance. Whereas sequential exposure allowed bacteria to acquire multiple resistance mutations effective against both phages, this evolutionary trajectory was prevented by simultaneous exposure, resulting in quantitatively weaker resistance. The order of phage exposure determined the fitness costs of sequential resistance, such that certain sequential orders imposed much higher fitness costs than the same phage pair in the reverse order. Together, these data suggest that phage combinations can be optimized to limit the strength of evolved resistances while maximizing their associated fitness costs to promote the long-term efficacy of phage therapy.IMPORTANCE Globally rising rates of antibiotic resistance have renewed interest in phage therapy where combinations of phages have been successfully used to treat multidrug-resistant infections. To optimize phage therapy, we first need to understand how bacteria evolve resistance against combinations of multiple phages. Here, we use simple laboratory experiments and genome sequencing to show that the timing and order of phage exposure determine the strength, cost, and mutational basis of resistance evolution in the opportunistic pathogen Pseudomonas aeruginosa These findings suggest that phage combinations can be optimized to limit the emergence and persistence of resistance, thereby promoting the long-term usefulness of phage therapy.

PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics.

We investigate the effect of bacteriophage infection and antibiotic treatment on the coordination of swarming, a collective form of flagellum- and pilus-mediated motility in bacteria. We show that phage infection of the opportunistic bacterial pathogen Pseudomonas aeruginosa abolishes swarming motility in the infected subpopulation and induces the release of the Pseudomonas quinolone signaling molecule PQS, which repulses uninfected subpopulations from approaching the infected area. These mechanisms have the overall effect of limiting the infection to a subpopulation, which promotes the survival of the overall population. Antibiotic treatment of P. aeruginosa elicits the same response, abolishing swarming motility and repulsing approaching swarms away from the antibiotic-treated area through a PQS-dependent mechanism. Swarms are entirely repelled from the zone of antibiotic-treated P. aeruginosa, consistent with a form of antibiotic evasion, and are not repelled by antibiotics alone. PQS has multiple functions, including serving as a quorum-sensing molecule, activating an oxidative stress response, and regulating the release of virulence and host-modifying factors. We show that PQS serves additionally as a stress warning signal that causes the greater population to physically avoid cell stress. The stress response at the collective level observed here in P. aeruginosa is consistent with a mechanism that promotes the survival of bacterial populations.IMPORTANCE We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen Pseudomonas aeruginosa Phage-infected P. aeruginosa subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes P. aeruginosa to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase P. aeruginosa resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2.

The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the development of alternative antibacterial strategies. Phage therapy has been proven successful for the treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological, genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was 94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were identified for each phage by mass spectrometry and were used to substantiate the function of the predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low multiplicity of infection levels and had good performance both on preventing biofilm formation and eradicating preformed biofilms. They were also stable over a wide range of temperature and pH values, supporting their potential use in the treatment of P. aeruginosa infections.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Excisionase in Pf filamentous prophage controls lysis-lysogeny decision-making in Pseudomonas aeruginosa.

Pf filamentous prophages are prevalent among clinical and environmental Pseudomonas aeruginosa isolates. Pf4 and Pf5 prophages are integrated into the host genomes of PAO1 and PA14, respectively, and play an important role in biofilm development. However, the genetic factors that directly control the lysis-lysogeny switch in Pf prophages remain unclear. Here, we identified and characterized the excisionase genes in Pf4 and Pf5 (named xisF4 and xisF5, respectively). XisF4 and XisF5 represent two major subfamilies of functional excisionases and are commonly found in Pf prophages. While both of them can significantly promote prophage excision, only XisF5 is essential for Pf5 excision. XisF4 activates Pf4 phage replication by upregulating the phage initiator gene (PA0727). In addition, xisF4 and the neighboring phage repressor c gene pf4r are transcribed divergently and their 5'-untranslated regions overlap. XisF4 and Pf4r not only auto-activate their own expression but also repress each other. Furthermore, two H-NS family proteins, MvaT and MvaU, coordinately repress Pf4 production by directly repressing xisF4. Collectively, we reveal that Pf prophage excisionases cooperate in controlling lysogeny and phage production.

Immunogenicity and antimicrobial effectiveness of Pseudomonas aeruginosa specific bacteriophage in a human lung in vitro model.

The rise of antibiotic resistant bacteria is posing a serious threat to human health. For example, resistant strains of Pseudomonas aeruginosa have resulted in untreatable and potentially lethal infections in both cystic fibrosis and immunocompromised patients. Due to the growing need for alternative treatment options, bacteriophage, or phage, therapy is gaining considerable attention. While previous studies have demonstrated the effectiveness of phage in combating persistent bacterial infections, there is currently a lack of knowledge regarding the host immunological response following phage exposure. In the present study, the bioresponses of an enhanced in vitro model were characterized following exposure to either DMS3 or PEV2, P. aeruginosa targeting phages. Results demonstrated a PEV2-dependent increase in IL-6 and TNF-α production, but no changes associated with DMS3 exposure. Additionally, following the establishment of an in vitro infection model, DMS3 was found to successfully protect mammalian lung cells from P. aeruginosa. Taken together, the biocompatibility and antibacterial effectiveness distinguish DMS3 bacteriophage as a strong candidate for phage therapy. However, as DMS3 is pilin dependent and bacterial receptor expression varies significantly, this work highlights the necessity of generating phage cocktails.

Effect of acute predation with bacteriophage on intermicrobial aggression by Pseudomonas aeruginosa.

In persons with structural lung disease, particularly those with cystic fibrosis (CF), chronic airway infections cause progressive loss of lung function. CF airways can be colonized by a variety of microorganisms; the most frequently encountered bacterial and fungal pathogens are Pseudomonas aeruginosa and Aspergillus fumigatus, respectively. Co-infection with P. aeruginosa and A. fumigatus often results in a more rapid loss of lung function, indicating that interactions between these pathogens affect infection pathogenesis. There has been renewed interest in the use of viruses (bacteriophage, mycoviruses) as alternatives to antibiotics to treat these infections. In previous work, we found that filamentous Pf bacteriophage produced by P. aeruginosa directly inhibited the metabolic activity of A. fumigatus by binding to and sequestering iron. In the current study, we further examined how filamentous Pf bacteriophage affected interactions between P. aeruginosa and A. fumigatus. Here, we report that the antifungal properties of supernatants collected from P. aeruginosa cultures infected with Pf bacteriophage were substantially less inhibitory towards A. fumigatus biofilms. In particular, we found that acute infection of P. aeruginosa by Pf bacteriophage inhibited the production of the virulence factor pyoverdine. Our results raise the possibility that the reduced production of antimicrobials by P. aeruginosa infected by Pf bacteriophage may promote conditions in CF airways that allow co-infection with A. fumigatus to occur, exacerbating disease severity. Our results also highlight the importance of considering how the use of bacteriophage as therapeutic agents could affect the behavior and composition of polymicrobial communities colonizing sites of chronic infection.

Pseudomonas predators: understanding and exploiting phage-host interactions.

Species in the genus Pseudomonas thrive in a diverse set of ecological niches and include crucial pathogens, such as the human pathogen Pseudomonas aeruginosa and the plant pathogen Pseudomonas syringae. The bacteriophages that infect Pseudomonas spp. mirror the widespread and diverse nature of their hosts. Therefore, Pseudomonas spp. and their phages are an ideal system to study the molecular mechanisms that govern virus-host interactions. Furthermore, phages are principal catalysts of host evolution and diversity, which directly affects the ecological roles of environmental and pathogenic Pseudomonas spp. Understanding these interactions not only provides novel insights into phage biology but also advances the development of phage therapy, phage-derived antimicrobial strategies and innovative biotechnological tools that may be derived from phage-bacteria interactions.

Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an efficient biofilm-dwelling microbial pathogen, associated with nosocomial infections. These biofilm-associated infections are resistant to antibiotics and immune defenses, therefore pose major problem against their treatment. This scenario demands alternative therapeutic regimens, and bacteriophage therapy is one among potential strategies for clinical management of multiple drug resistance. In this investigation, the efficacy of a bacteriophage, JHP, is evaluated to eradicate P. aeruginosa biofilms. Growth kinetics of P. aeruginosa biofilm revealed that the highest cell density biofilm (1.5 × 1016 CFU/mL) was established within the polystyrene microtiter plate at 72 h post inoculation. Pseudomonas aeruginosa biofilms of different ages, treated with JHP (0.6 MOI) for different post-infection durations, reduced biomass from 2 to 4.5 logs (60-90%). JHP treatment before biofilm development reduced the bacterial load up to 9 logs (>95% bacterial load reduction) as compared with untreated control, which highlights its potential to prevent biofilm formation in indwelling medical devices. Combinations of JHP with other phages or antibiotics could be an efficient alternative for P. aeruginosa biofilm removal in clinical and industrial settings.

Isolation and characterization of wetland VSW-3, a novel lytic cold-active bacteriophage of Pseudomonas fluorescens.

Wetlands are often called the "kidneys of the Earth" and contribute substantially to environmental improvement. Pseudomonas fluorescens is a major contaminant of milk products and causes the spoilage of refrigerated foods and fresh poultry. In this study, we isolated and characterized a lytic cold-active bacteriophage named VSW-3 together with P. fluorescens SW-3 cells from the Napahai wetland in China. Electron microscopy showed that VSW-3 had an icosahedral head (56 nm) and a tapering tail (20 nm × 12 nm) and a genome size of approximate 40 kb. On the basis of the top-scoring hits in the BLASTP analysis, VSW-3 showed a high degree of module similarity to the Pseudomonas phages Andromeda and Bf7. The latent and burst periods were 45 and 20 min, respectively, with an average burst size of 90 phage particles per infected cell. The pH and thermal stability of VSW-3 were also explored. The optimal pH was found to be 7.0 and the activity decreased rapidly when the temperature exceeded 60 °C. VSW-3 is a cold-active bacteriophage, hence, it is important to research its ability to prevent product contamination caused by P. fluorescens and to characterize its relationship with its host P. fluorescens in the future.

Quorum sensing influences phage infection efficiency via affecting cell population and physiological state.

Bacterial growth phase has been reported affecting phage infection. To underpin the related mechanism, infection efficiency of Pseudomonas aeruginosa phage K5 is characterized. When infecting the logarithmic cells, phage K5 produced significantly more infection centers than the stationary cells, well concordant with the viable cell ratio in the different growth phases. Additionally, the burst size decreased dramatically in the stationary cells, implying that the physiological state of the viable cells contributed to the productivity of phage K5, and it was consistent with the expression variation of the phage RNA polymerase. Quorum sensing inhibitor penicillic acid was applied and could significantly improve the viable cell proportion and the infection center numbers, but had less effect on the corresponding burst sizes. Moreover, the effect of penicillic acid and the quorum sensing regulator mutants on the production of phage C11 was also analyzed. Taken together, our data suggest that quorum sensing is involved in the defense of phage K5 infection by influencing the viable cell population and their physiological state, and it is an efficient and intrinsic pathway allowing bacteria to resist phage attacks in natural environment.

[Effects of bacteriophages on biofilm formation by strains of Pseudomonas aeruginosa].

The effects of two Pseudomonas aeruginosa bacteriophages, vB-Pa 4 and vB-Pa 5, on the formation and development of biofilms of six polyresistant hospital strains of P. aeruginosa have been investigated. Pretreatment of bacteriophages prevented the formation or almost completely prevented the growth of adequate biofilms. The biofilms that had already formed were partially or completely destroyed after phage treatment. The results demonstrate the prospects of using isolated bacteriophages of P. aeruginosa to destroy biofilms and prevent their formation.

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model.

The Galleria mellonella infection model was used to assess the in vivo efficacy of phage therapy against laboratory and clinical strains of Pseudomonas aeruginosa. In a first series of experiments, Galleria were infected with the laboratory strain P. aeruginosa PAO1 and were treated with varying multiplicity of infection (MOI) of phages either 2h post-infection (treatment) or 2h pre-infection (prevention) via injection into the haemolymph. To address the kinetics of infection, larvae were bled over a period of 24h for quantification of bacteria and phages. Survival rates at 24h when infected with 10 cells/larvae were greater in the prevention versus treatment model (47% vs. 40%, MOI=10; 47% vs. 20%, MOI=1; and 33% vs. 7%, MOI=0.1). This pattern held true when 100 cells/larvae were used (87% vs. 20%, MOI=10; 53% vs. 13%, MOI=1; 67% vs. 7%, MOI=0.1). By 24h post-infection, phages kept bacterial cell numbers in the haemolymph 1000-fold lower than in the non-treated group. In a second series of experiments using clinical strains to further validate the prevention model, phages protected Galleria when infected with both a bacteraemia (0% vs. 85%) and a cystic fibrosis (80% vs. 100%) isolate. Therefore, this study validates the use of G. mellonella as a simple, robust and cost-effective model for initial in vivo examination of P. aeruginosa-targeted phage therapy, which may be applied to other pathogens with similarly low infective doses.

In vitro and in vivo antibacterial activity of environmental bacteriophages against Pseudomonas aeruginosa strains from cystic fibrosis patients.

The goal of the study was to determine the relationship between in vitro/in vivo efficacy of environmental Pseudomonas phages and certain phenotypical properties of Pseudomonas aeruginosa (PA) strains. We studied the diversity between particular isolates and determined phage sensitivity in vitro and in vivo in the Galleria mellonella insect model. Twenty-eight lytic bacteriophages specific for PA were tested against 121 CF PA isolates including 29 mucoid PA strains. Most strains from cystic fibrosis (CF) patients were lysed by at least three phages (93.6 %), but completely insensitive strains were also present (6.4 %). Two phages PA5oct and KT28 exhibited high rates of lytic potency on 55-68 % of PA strains (72-86 % of mucoid isolates). We further explored phage activity against six PA strains (CF and non-CF) in vitro, comparing clonal differences in phage susceptibility with bacterial properties such as the ability to form biofilms, mucosity, twitching motility, and biochemical profiles. We observed the relationship between variation in phage susceptibility and Fourier transform infrared spectroscopy (FTIR) analysis in the spectra window of carbohydrates. The protective efficacy of two selected phages against PA PAO1 and 0038 infection was confirmed in vivo in G. mellonella larvae. Generally, the wax moth model results confirmed the data from in vitro assays, but in massive infection of CF isolates, the application of lytic phages probably led to the release of toxic compound causing an increase in larvae mortality. We assumed that apart of in vitro phage activity testing, a simple and convenient wax moth larvae model should be applied for the evaluation of in vivo effectiveness of particular phage preparations.

Isolation and characterization of T7-like lytic bacteriophages infecting multidrug resistant Pseudomonas aeruginosa isolated from Egypt.

In this study, two lytic phages designated as ϕPSZ1 and ϕPSZ2 infecting multidrug resistant Pseudomonas aeruginosa were isolated from sewage samples collected in Zagazig, Egypt. Morphological analysis by transmission electron microscopy revealed that both phages belong to the podoviridae family and resembles typical T7-like phages. ϕPSZ1 has a head of about 60 ± 5 nm in diameter with a short tail of 19 ± 2 nm in length, while ϕPSZ2 has a head of about 57 ± 5 nm in diameter with a short tail of 14 ± 2 nm in length. Both phages were shown to be able to infect 13 different P. aeruginosa strains and has no effect on other tested bacteria. In spite of morphological similarity, these phages showed diverged genomic sequences revealed by restriction enzyme digestion analysis. One-step growth curves of bacteriophages revealed eclipse and latent periods of 12 min for ϕPSZ1 and 15 min for ϕPSZ2, respectively, with burst sizes of about 100 per infected cell. Phage treatment prevented the growth of P. aeruginosa for up to 18 h with multiplicity of infection ratios of 1. These results suggest that both phages have a high potential for phage application to control P. aeruginosa.

Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa.

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets.

Development of giant bacteriophage ϕKZ is independent of the host transcription apparatus.

UNLABELLED: Pseudomonas aeruginosa bacteriophage ϕKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the ∼ 280-kb ϕKZ genome to single-nucleotide resolution, we combine 369 ϕKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the ϕKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to ϕKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the ϕKZ genome is performed by the consecutive action of two ϕKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage ϕKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial ß and ß'-like RNAP subunits.

Effectiveness of bacteriophages in the sputum of cystic fibrosis patients.

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections.