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

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.

Closely Related Vibrio alginolyticus Strains Encode an Identical Repertoire of Caudovirales-Like Regions and Filamentous Phages

Many filamentous vibriophages encode virulence genes that lead to the emergence of pathogenic bacteria. Most genomes of filamentous vibriophages characterized up until today were isolated from human pathogens. Despite genome-based predictions that environmental Vibrios also contain filamentous phages that contribute to bacterial virulence, empirical evidence is scarce. This study aimed to characterize the bacteriophages of a marine pathogen, Vibrio alginolyticus (Kiel-alginolyticus ecotype) and to determine their role in bacterial virulence. To do so, we sequenced the phage-containing supernatant of eight different V. alginolyticus strains, characterized the phages therein and performed infection experiments on juvenile pipefish to assess their contribution to bacterial virulence. We were able to identify two actively replicating filamentous phages. Unique to this study was that all eight bacteria of the Kiel-alginolyticus ecotype have identical bacteriophages, supporting our previously established theory of a clonal expansion of the Kiel-alginolyticus ecotype. We further found that in one of the two filamentous phages, two phage-morphogenesis proteins (Zot and Ace) share high sequence similarity with putative toxins encoded on the Vibrio cholerae phage CTXΦ. The coverage of this filamentous phage correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phage contributes to bacterial virulence.

First Report of Filamentous Phages Isolated from Tunisian Orchards to Control Erwinia amylovora

Newly discovered Erwinia amylovora phages PEar1, PEar2, PEar4 and PEar6 were isolated from three different orchards in North Tunisia to study their potential as biocontrol agents. Illumina sequencing revealed that the PEar viruses carry a single-strand DNA genome between 6608 and 6801 nucleotides and belong to the Inoviridae, making them the first described filamentous phages of E. amylovora. Interestingly, phage-infected cells show a decreased swimming and swarming motility and a cocktail of the four phages can significantly reduce infection of E. amylovora in a pear bioassay, potentially making them suitable candidates for phage biocontrol.

The Impact of Pf Bacteriophages on the Fitness of Pseudomonas aeruginosa; A Mathematical Modeling Approach

Pseudomonas aeruginosa (Pa) is a major bacterial pathogen responsible for chronic lung infections in cystic fibrosis patients. Recent work by ourselves and others has implicated Pf bacteriophages, non-lytic 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+ 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- 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 predicts that Pf+ strains of Pa can only outcompete Pf- strains if the benefits of phage production falls solely onto Pf+ strains and not onto the overall bacterial community in the lung. Further, phage production only leads to a net positive gain in fitness at antibiotic concentrations slightly above the minimum inhibitory concentration (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 predicts that frequent administration of intermediate doses of antibiotics with low decay 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.ImportanceFilamentous 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 and may facilitate improved treatment strategies for combating antibiotic tolerance.

UPΦ phages, a new group of filamentous phages found in several members of Enterobacteriales

Filamentous phages establish chronic infections in their bacterial hosts, and new phages are secreted by infected bacteria for multiple generations, typically without causing host death. Often, these viruses integrate in their host’s genome by co-opting the host’s XerCD recombinase system. In several cases, these viruses also encode genes that increase bacterial virulence in plants and animals. Here, we describe a new filamentous phage, UPϕ901, which we originally found integrated in a clinical isolate of Escherichia coli from urine. UPϕ901 and closely related phages can be found in published genomes of over 200 other bacteria, including strains of Citrobacter koseri, Salmonella enterica, Yersinia enterocolitica, and Klebsiella pneumoniae. Its closest relatives are consistently found in urine or in the blood and feces of patients with urinary tract infections. More distant relatives can be found in isolates from other environments, including sewage, water, soil, and contaminated food. Each of these phages, which we collectively call ‘UPϕ viruses’, also harbors two or more novel genes of unknown function.

UPϕ phages, a new group of filamentous phages found in several members of Enterobacteriales

Filamentous phages establish chronic infections in their bacterial hosts, and new phages are secreted by infected bacteria for multiple generations, typically without causing host death. Often, these viruses integrate in their host’s genome by co-opting the host’s XerCD recombinase system. In several cases, these viruses also encode genes that increase bacterial virulence in plants and animals. Here, we describe a new filamentous phage, UPϕ901, which we originally found integrated in a clinical isolate of uropathogenic Escherichia coli. UPϕ901 and closely related phages can be found in published genomes of over 200 other bacteria, including strains of Citrobacter koseri, Salmonella enterica, Yersinia enterocolitica, and Klebsiella pneumoniae. Its closest relatives are consistently found in urine or in the blood and feces of patients with urinary tract infections. More distant relatives can be found in isolates from other environments, including sewage, water, soil, and contaminated food. Each of these phages, which we collectively call “UPϕ viruses,” also harbors two novel genes of unknown function.

Filamentous phages reduce bacterial growth in low salinities

Being non-lytic, filamentous phages can replicate at high frequencies and often carry virulence factors, which are important in the evolution and emergence of novel pathogens. However, their net effect on bacterial fitness remains unknown. To understand the ecology and evolution between filamentous phages and their hosts, it is important to assess (i) fitness effects of filamentous phages on their hosts and (ii) how these effects depend on the environment. To determine how the net effect on bacterial fitness by filamentous phages changes across environments, we constructed phage–bacteria infection networks at ambient 15 practical salinity units (PSU) and stressful salinities (11 and 7 PSU) using the marine bacterium, Vibrio alginolyticus and its derived filamentous phages as model system. We observed no significant difference in network structure at 15 and 11 PSU. However, at 7 PSU phages significantly reduced bacterial growth changing network structure. This pattern was mainly driven by a significant increase in bacterial susceptibility. Our findings suggest that filamentous phages decrease bacterial growth, an indirect measure of fitness in stressful environmental conditions, which might impact bacterial communities, alter horizontal gene transfer events and possibly favour the emergence of novel pathogens in environmental Vibrios .

Closely related Vibrio alginolyticus strains encode an identical repertoire of prophages and filamentous phages

Filamentous vibriophages represent a massive repertoire of virulence factors which can be transferred across species boundaries, leading to the emergence of deadly pathogens. All filamentous vibriophages that were characterized until today were isolated from human pathogens. Considering frequent horizontal gene transfer among vibrios, we predict that other environmental isolates, including non-human pathogens also carry filamentous phages, of which some may encode virulence factors.The aim of this study was to characterize the phage repertoire, consisting of prophages and filamentous phages, of a marine pathogen, Vibrio alginolyticus. To do so, we sequenced eight different V. alginolyticus strains, isolated from different pipefish and characterised their phage repertoire using a combination of morphological analyses and comparative genomics.We were able to identify a total of five novel phage regions (three different Caudovirales and two different Inoviridae), whereby only those two loci predicted to correspond to filamentous phages (family Inoviridae) represent actively replicating phages. Unique for this study was that all eight host strains, which were isolated from different eukaryotic hosts have identical bacteriophages, suggesting a clonal expansion of this strain after the phages had been acquired by a common ancestor. We further found that co-occurrence of two different filamentous phages leads to within-host competition resulting in reduced phage replication by one of the two phages. One of the two filamentous phages encoded two virulence genes (Ace and Zot), homologous to those encoded on the V. cholerae phage CTXΦ. The coverage of these zot-encoding phages correlated positively with virulence (measured in controlled infection experiments on the eukaryotic host), suggesting that this phages is an important virulence determinant.Impact statementMany bacteria of the genus Vibrio, such as V. cholerae or V. parahaemolyticus impose a strong threat to human health. Often, small viruses, known as filamentous phages encode virulence genes. Upon infecting a bacterial cell, these phages can transform a previously harmless bacterium into a deadly pathogen. While filamentous phages and their virulence factors are well-characterized for human pathogenic vibrios, filamentous phages of marine vibrios, pathogenic for a wide range of marine organisms, are predicted to carry virulence factors, but have so far not been characterized in depth. Using whole genome sequencing and comparative genomics of phages isolated from a marine fish pathogen V. alginolyticus, we show that also environmental strains harbour filamentous phages that carry virulence genes. These phages were most likely acquired from other vibrios by a process known as horizontal gene transfer. We found that these phages are identical across eight different pathogenic V. alginolyticus strains, suggesting that they have been acquired by a common ancestor before a clonal expansion of this ecotype took place. The phages characterized in this study have not been described before and are unique for the Kiel V. alginolyticus ecotype.Data SummaryThe GenBank accession numbers for all genomic sequence data analysed in the present study can be found in Table S1.All phage regions identified by PHASTER analysis of each chromosome and the respective coverage of active phage loci are listed in Table S2.GenBank files were deposited at NCBI for the two actively replicating filamentous phages VALGΦ6 (Accession number: MN719123) and VALGΦ8 (Accession number: MN690600)The virulence data from the infection experiments have been deposited at PANGAEA: Accession number will be provided upon acceptance of the manuscript.Data statementAll supporting data have been provided within the article or through supplementary data files. Four supplementary tables and six supplementary figures are available with the online version of this article.