Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4 °C and 2.38 log units for the storage at 25 °C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Impact of different hand-drying methods on surrounding environment: aerosolization of virus and bacteria, and transfer to surfaces.

BACKGROUND: In recent years, hand drying has been highlighted as a key step in appropriate hand hygiene, as moisture on hands can increase the transfer of micro-organisms from hands to surfaces and vice versa. AIM: To understand bacterial and viral aerosolization following hand drying, and study the transfer of micro-organisms from hands to surfaces after drying using different methods. METHODS: Groups of five volunteers had their hands pre-washed with soap, rinsed and dried, then inoculated with a concentrated mixture of Pseudomonas fluorescens and MS2 bacteriophage. Volunteers entered an empty washroom, one at a time, and rinsed their hands with water or washed their hands with soap prior to drying with a jet dryer or paper towels. Each volunteer applied one hand successively to various surfaces, while their other hand was sampled using the glove juice method. Both residual bacteria and viruses were quantified from the washroom air, surface swabs and hand samples. FINDINGS: P. fluorescens and MS2 bacteriophages were rarely aerosolized while drying hands for any of the drying methods studied. Results also showed limited, and similar, transfer of both micro-organisms studied on to surfaces for all drying methods. CONCLUSION: The use of jet dryers or paper towels produces low levels of aerosolization when drying hands in a washroom. Similarly, all drying methods result in low transfer to surfaces. While the coronavirus disease 2019 pandemic raised concerns regarding public washrooms, this study shows that all methods tested are hygienic solutions for dry washed hands.

Lytic bacteriophages UFJF_PfDIW6 and UFJF_PfSW6 prevent Pseudomonas fluorescens growth in vitro and the proteolytic-caused spoilage of raw milk during chilled storage.

In this study, P. fluorescens-infecting phages were isolated, characterized, and evaluated to their potential to control the bacterial counts and, consequently, the proteolytic spoilage of raw milk during cold storage. The UFJF_PfDIW6 and UFJF_PfSW6 phages showed titers of 9.7 and 7.6 log PFU/ml; latent period of 115 and 25 min, and burst size of 145 and 25 PFU/infected cell, respectively. They also were highly specific to the host bacterium, morphologically classified as the Podoviridae family, stable at pH 5 to 11 and were not inactivated at 63 °C or 72 °C for 30 min. These phages found to be effective against P. fluorescens, reducing bacterial count throughout the entire exponential growth phase in broth formulated with milk at both 4 °C and 10 °C. This effect on bacteria growth led to inhibition by at least 2 days in proteases production, delaying the degradation of milk proteins. When applied together in raw milk stored at 4 °C, they reduced the total bacteria, psychrotrophic, and Pseudomonas by 3 log CFU/ml. This study's findings indicate that these phages have a great potential to prevent the growth of Pseudomonas and, consequently, to retard proteolytic spoilage of raw milk during chilled storage.

Temperature-dependent changes to host-parasite interactions alter the thermal performance of a bacterial host.

Thermal performance curves (TPCs) are used to predict changes in species interactions, and hence, range shifts, disease dynamics and community composition, under forecasted climate change. Species interactions might in turn affect TPCs. Here, we investigate how temperature-dependent changes in a microbial host-parasite interaction (the bacterium Pseudomonas fluorescens, and its lytic bacteriophage, SBW[Formula: see text]) changes the host TPC and the ecological and evolutionary mechanisms underlying these changes. The bacteriophage had a narrower thermal tolerance for infection, with their critical thermal maximum ~6 °C lower than those at which the bacteria still had high growth. Consequently, in the presence of phage, the host TPC changed, resulting in a lower maximum growth rate. These changes were not just driven by differences in thermal tolerance, with temperature-dependent costs of evolved resistance also playing a major role: the largest cost of resistance occurred at the temperature at which bacteria grew best in the absence of phage. Our work highlights how ecological and evolutionary mechanisms can alter the effect of a parasite on host thermal performance, even over very short timescales.

Isolation of virulent phages infecting dominant mesophilic aerobic bacteria in cucumber pickle fermentation.

Pickle is a type of mildly lactic acid fermented vegetable and is a traditional dish favored in China, Japan, and Korea. Corruption of spoilage bacteria and accumulation of nitrite during vegetable fermentation are common problems that affect the pickle industry and consumer health. In this work, cucumber juice was used as a vegetable model to study the dominant mesophilic aerobic bacteria (MAB) producing nitrite during pickle fermentation. Virulent phages infecting the dominant MABs combined with Lactobacillus plantarum M6 were used to control these bacteria. Enterobacter cloacae and Pseudomonas fluorescens are the dominant MABs in the fermentation of cucumber juice containing 4% or 8% NaCl, with isolation percentages reaching 30.6% and 23.1%, respectively. Virulent phages PspYZU5415 and EcpYZU01 were isolated using P. fluorescens J5415 and E. cloacae J01 as the host bacteria, respectively. These two phages show a broad host range and strong lytic activity, and their genomes contain no toxins and antibiotic resistance genes. PspYZU5415 and EcpYZU01 were combined into a cocktail (designated as Phage MIX) that effectively inhibits the growth of E. cloacae and P. fluorescens in cucumber juice with different salt concentrations. PhageMIX combined with L. plantarum M6 decreased the counts of P. mendocina and E. cloacae to undetectable levels at 48 h during the fermentation of cucumber juice artificially contaminated with P. mendocina and E. cloacae. In addition, nitrite content increased to 11.3 mg/L at 20 h and then degraded completely at 36 h. By contrast, P. mendocina and E. cloacae remained in the groups without PhageMIX during fermentation (0-48 h). Nitrite content rapidly increased to 65.7 mg/L at 12 h and then decreased to 21.6 mg/L at 48 h in the control group. This study suggests that PhageMIX combined with lactic acid bacterial strains can be used as an ecological starter for controlling the dominant MABs P. mendocina and E. cloacae and for reducing nitrate production during the early stage of pickle fermentation.

Coevolutionary dynamics shape the structure of bacteria-phage infection networks.

Coevolution-reciprocal evolutionary change among interacting species driven by natural selection-is thought to be an important force in shaping biodiversity. This ongoing process takes place within tangled networks of species interactions. In microbial communities, evolutionary change between hosts and parasites occurs at the same time scale as ecological change. Yet, we still lack experimental evidence of the role of coevolution in driving changes in the structure of such species interaction networks. Filling this gap is important because network structure influences community persistence through indirect effects. Here, we quantified experimentally to what extent coevolutionary dynamics lead to contrasting patterns in the architecture of bacteria-phage infection networks. Specifically, we look at the tendency of these networks to be organized in a nested pattern by which the more specialist phages tend to infect only a proper subset of those bacteria infected by the most generalist phages. We found that interactions between coevolving bacteria and phages become less nested over time under fluctuating dynamics, and more nested under arms race dynamics. Moreover, when coevolution results in high average infectivity, phages and bacteria differ more from each other over time under arms race dynamics than under fluctuating dynamics. The tradeoff between the fitness benefits of evolving resistance/infectivity traits and the costs of maintaining them might explain these differences in network structure. Our study shows that the interaction pattern between bacteria and phages at the community level depends on the way coevolution unfolds.

Bacteriophage ϕIBB-PF7A loaded on sodium alginate-based films to prevent microbial meat spoilage.

Despite the recent advances achieved in food industries to fulfil the growing consumer demand for high quality and food safety, microbial contamination remains a serious issue. This study aimed to incorporate ϕIBB-PF7A bacteriophage (phage) onto sodium alginate-based films crosslinked with calcium chloride, to prevent poultry spoilage caused by Pseudomonas fluorescens. Films were prepared by casting and characterized in terms of phage loading, distribution, stability, release profile and antimicrobial performance. Results showed that phages were successfully incorporated as evidenced by their viability and homogeneous distribution within the films as assessed by microscopy. A decrease in phage viability was only detected after 8 weeks when stored under refrigerated conditions. Antimicrobial activity demonstrated that incorporated phages significantly impaired P. fluorescens growth. Films' antimicrobial efficacy was further demonstrated on chicken breast fillets artificially inoculated, decreasing 2Log P. fluorescens viable cell counts in the first two days and reductions were maintained up to 5 days of exposure (1 Log). These results highlight that phage incorporation onto sodium-alginate-based films constitutes a simple approach of preserving the antimicrobial activity of phages in a dried and insoluble format, that can further be applied in food industry for the prevention of microbial spoilage.

Complete genome sequence of PFP1, a novel T7-like Pseudomonas fluorescens bacteriophage.

The complete genomic sequence of Pseudomonas fluorescens bacteriophage PFP1, isolated from sewage samples collected in Liaoning Province, China, were sequenced in this study and found to be 40,914 bp long. The PFP1 genome is composed of linear double-stranded DNA with 55.81% G+C content and 45 putative protein-coding genes, and no rRNA and tRNA genes. Comparative genomics and phylogenetic analysis revealed that the Pseudomonas fluorescens phage PFP1 is a new member of the genus T7virus. This information can be used to develop novel phage-based control strategies against Pseudomonas fluorescens.

Genome sequence of the novel virulent bacteriophage PMBT14 with lytic activity against Pseudomonas fluorescens DSM 50090R.

Psychrotrophic gram-negative Pseudomonas spp. represent a serious problem in the dairy industry as they can cause spoilage of milk and dairy products. Bacteriophages have moved into focus as promising biocontrol agents for such food spoilage bacteria. The virulent Siphoviridae phage PMBT14 was isolated on a mutant variant of P. fluorescens DSM 50090 challenged with an unrelated virulent P. fluorescens DSM 50090 Podoviridae phage (i.e., mutant strain DSM 50090R). PMBT14 has a 47,820-bp dsDNA genome with 76 predicted open reading frames (ORFs). Its genome shows no significant sequence similarity to that of known phages, suggesting that PMBT14 represents a novel phage. Phage PMBT14 could be a promising biocontrol agent for P. fluorescens in milk or dairy foods.

Isolation and characterization of two lytic cold-active bacteriophages infecting Pseudomonas fluorescens from the Napahai plateau wetland.

As the "kidneys of the Earth", wetlands play important roles as biodiversity reservoirs, in water purification, and in flood control. In this study, 2 lytic cold-active bacteriophages, named VW-6S and VW-6B, infecting Pseudomonas fluorescens W-6 cells from the Napahai plateau wetland in China were isolated and characterized. Electron microscopy showed that both VW-6S and VW-6B had an icosahedral head (66.7 and 61.1 nm, respectively) and a long tail (8.3 nm width × 233.3 nm length and 11.1 nm width × 166.7 nm length, respectively). The bacteriophages VW-6S and VW-6B were classified as Siphoviridae and had an approximate genome size of 30-40 kb. The latent and burst periods of VW-6S were 60 and 30 min, whereas those of VW-6B were 30 and 30 min, respectively. The optimal pH values for the bacteriophages VW-6S and VW-6B were 8.0 and 10.0, respectively, and their activity decreased rapidly at temperatures higher than 60 °C. These cold-active bacteriophages provide good materials for further study of cold-adaptation mechanisms and interaction with the host P. fluorescens.

Evolutionary contribution to coexistence of competitors in microbial food webs.

The theory of species coexistence is a key concept in ecology that has received much attention. The role of rapid evolution for determining species coexistence is still poorly understood although evolutionary change on ecological time-scales has the potential to change almost any ecological process. The influence of evolution on coexistence can be especially pronounced in microbial communities where organisms often have large population sizes and short generation times. Previous work on coexistence has assumed that traits involved in resource use and species interactions are constant or change very slowly in terms of ecological time-scales. However, recent work suggests that these traits can evolve rapidly. Nevertheless, the importance of rapid evolution to coexistence has not been tested experimentally. Here, we show how rapid evolution alters the frequency of two bacterial competitors over time when grown together with specialist consumers (bacteriophages), a generalist consumer (protozoan) and all in combination. We find that consumers facilitate coexistence in a manner consistent with classic ecological theory. However, through disentangling the relative contributions of ecology (changes in consumer abundance) and evolution (changes in traits mediating species interactions) on the frequency of the two competitors over time, we find differences between the consumer types and combinations. Overall, our results indicate that the influence of evolution on species coexistence strongly depends on the traits and species interactions considered.

Complete genome sequences of two novel autographiviruses infecting a bacterium from the Pseudomonas fluorescens group.

In this paper, we describe two independent isolates of a new member of the subfamily Autographivirinae, Pseudomonas phage KNP. The type strain (KNP) has a linear, 40,491-bp-long genome with GC content of 57.3%, and 50 coding DNA sequences (CDSs). The genome of the second strain (WRT) contains one CDS less, encodes a significantly different tail fiber protein and is shorter (40,214 bp; GC content, 57.4%). Phylogenetic analysis indicates that both KNP and WRT belong to the genus T7virus. Together with genetically similar Pseudomonas phages (gh-1, phiPSA2, phiPsa17, PPPL-1, shl2, phi15, PPpW-4, UNO-SLW4, phiIBB-PF7A, Pf-10, and Phi-S1), they form a divergent yet coherent group that stands apart from the T7-like viruses (sensu lato). Analysis of the diversity of this group and its relatedness to other members of the subfamily Autographivirinae led us to the conclusion that this group might be considered as a candidate for a new genus.

Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria-plasmid-phage interaction.

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities, bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here, we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage, SBW25φ2, and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions.

Stability of A Coevolving Host-parasite System Peaks at Intermediate Productivity.

Habitat productivity may affect the stability of consumer-resource systems, through both ecological and evolutionary mechanisms. We hypothesize that coevolving consumer-resource systems show more stable dynamics at intermediate resource availability, while very low-level resource supply cannot support sufficiently large populations of resource and consumer species to avoid stochastic extinction, and extremely resource-rich environments may promote escalatory arms-race-like coevolution that can cause strong fluctuations in species abundance and even extinction of one or both trophic levels. We tested these ideas by carrying out an experimental evolution study with a model bacterium-phage system (Pseudomonas fluorescens SBW25 and its phage SBW25Φ2). Consistent with our hypothesis, this system was most stable at intermediate resource supply (fewer extinction events and smaller magnitude of population fluctuation). In our experiment, the rate of coevolution between bacterial resistance and phage infectivity was correlated with the magnitude of population fluctuation, which may explain the different in stability between levels of resource supply. Crucially, our results are consistent with a suggestion that, among the two major modes of antagonistic coevolution, arms race is more likely than fluctuation selection dynamics to cause extinction events in consumer-resource systems. This study suggests an important role of environment-dependent coevolutionary dynamics for the stability of consumer-resource species systems, therefore highlights the importance to consider contemporaneous evolutionary dynamics when studying the stability of ecosystems, particularly those under environmental changes.

Genomic evolution of bacterial populations under coselection by antibiotics and phage.

Bacteria live in dynamic systems where selection pressures can alter rapidly, forcing adaptation to the prevailing conditions. In particular, bacteriophages and antibiotics of anthropogenic origin are major bacterial stressors in many environments. We previously observed that populations of the bacterium Pseudomonas fluorescens SBW25 exposed to the lytic bacteriophage SBW25Φ2 and a noninhibitive concentration of the antibiotic streptomycin (coselection) achieved higher levels of phage resistance compared to populations exposed to the phage alone. In addition, the phage became extinct under coselection while remaining present in the phage alone environment. Further, phenotypic tests indicated that these observations might be associated with increased mutation rate under coselection. In this study, we examined the genetic causes behind these phenotypes by whole-genome sequencing clones isolated from the end of the experiments. We were able to identify genetic factors likely responsible for streptomycin resistance, phage resistance and hypermutable (mutator) phenotypes. This constitutes genomic evidence in support of the observation that while the presence of phage did not affect antibiotic resistance, the presence of antibiotic affected phage resistance. We had previously hypothesized an association between mutators and elevated levels of phage resistance under coselection. However, our evidence regarding the mechanism was inconclusive, as although with phage mutators were only found under coselection, additional genomic evidence was lacking and phage resistance was also observed in nonmutators under coselection. More generally, our study provides novel insights into evolution between univariate and multivariate selection (here two stressors), as well as the potential role of hypermutability in natural communities.

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.

Sublethal streptomycin concentrations and lytic bacteriophage together promote resistance evolution.

Sub-minimum inhibiting concentrations (sub-MICs) of antibiotics frequently occur in natural environments owing to wide-spread antibiotic leakage by human action. Even though the concentrations are very low, these sub-MICs have recently been shown to alter bacterial populations by selecting for antibiotic resistance and increasing the rate of adaptive evolution. However, studies are lacking on how these effects reverberate into key ecological interactions, such as bacteria-phage interactions. Previously, co-selection of bacteria by phages and antibiotic concentrations exceeding MICs has been hypothesized to decrease the rate of resistance evolution because of fitness costs associated with resistance mutations. By contrast, here we show that sub-MICs of the antibiotic streptomycin (Sm) increased the rate of phage resistance evolution, as well as causing extinction of the phage. Notably, Sm and the phage in combination also enhanced the evolution of Sm resistance compared with Sm alone. These observations demonstrate the potential of sub-MICs of antibiotics to impact key ecological interactions in microbial communities with evolutionary outcomes that can radically differ from those associated with high concentrations. Our findings also contribute to the understanding of ecological and evolutionary factors essential for the management of the antibiotic resistance problem.This article is part of the themed issue 'Human influences on evolution, and the ecological and societal consequences'.

Complete genome sequence of the lytic cold-active Pseudomonas fluorescens bacteriophage VSW-3 from Napahai plateau wetland.

The lytic cold-active bacteriophage VSW-3, belonging to the Podoviridae family and infecting the host Pseudomonas fluorescens SW-3, was isolated from the Napahai plateau wetland in China. With the development of sequencing technology, the study of Pseudomonas genomic diversity has increased; however, knowledge of cold-active phages infecting Pseudomonas is limited. The newly sequenced phage VSW-3 was classified based on virion morphology by transmission electron microscope. Sequence analysis revealed that the genome size was 40,556 bp with an overall GC content of 57.54 % and 46 open reading frames. The genome was organized into several modules containing genes for packaging, structural proteins, replication/transcription, and phage lysis. The sequence contained 45 potential promoters, 3 transcription terminators, and yet no tRNAs. This is the first report of cold-active Pseudomonas fluorescens bacteriophage genome sequencing.

Parasite genetic distance and local adaptation in co-evolving bacteria-bacteriophage populations.

Antagonistic co-evolution between hosts and parasites can lead to local adaptation (LA) such that parasite fitness is greatest in sympatric hosts (or vice versa). The magnitude of LA typically increases with geographical distance, which is assumed to be because genetic (and hence phenotypic) distance increases with geographical distance. Here, we explicitly test the relationships between parasite genetic and phenotypic distance and LA using isolates of co-evolved viral parasites (lytic bacteriophage ϕ2) and the host bacterium Pseudomonas fluorescens SBW25. We find positive relationships between parasite genotype and infectivity phenotype, but the strength of the relationship was greater when infectivity was defined by the identity of hosts that could be infected rather than the actual number of hosts infected (host range), and when measurements were compared within rather than among populations. Crucially, we find a monotonic relationship between LA and genetic distance across phage isolates from different populations, although in contrast to many geographical studies, parasite LA decreased with genetic distance. These results can be explained by the fact that bacteria can rapidly adapt to phage infectivity mutations, but that evolved resistance has a degree of specificity to the local phage population. Our results show that antagonistic co-evolution alone can result in predictable links between genetic distance and host-parasite local adaptation.

Migration highways and migration barriers created by host-parasite interactions.

Co-evolving parasites may play a key role in host migration and population structure. Using co-evolving bacteria and viruses, we test general hypotheses as to how co-evolving parasites affect the success of passive host migration between habitats that can support different intensities of host-parasite interactions. First, we show that parasites aid migration from areas of intense to weak co-evolutionary interactions and impede migration in the opposite direction, as a result of intraspecific apparent competition mediated via parasites. Second, when habitats show qualitative difference such that some environments support parasite persistence while others do not, different population regulation forces (either parasitism or competitive exclusion) will reduce the success of migration in both directions. Our study shows that co-evolution with parasites can predictably homogenises or isolates host populations, depending on heterogeneity of abiotic conditions, with the second scenario constituting a novel type of 'isolation by adaptation'.