Coevolutionary analysis of Pseudomonas syringae-phage interactions to help with rational design of phage treatments.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

Seed coating with phages for sustainable plant biocontrol of plant pathogens and influence of the seed coat mucilage.

Pathogens resistant to classical control strategies pose a significant threat to crop yield, with seeds being a major transmission route. Bacteriophages, viruses targeting bacteria, offer an environmentally sustainable biocontrol solution. In this study, we isolated and characterized two novel phages, Athelas and Alfirin, which infect Pseudomonas syringae and Agrobacterium fabrum, respectively, and included the recently published Pfeifenkraut phage infecting Xanthomonas translucens. Using a simple immersion method, phages coated onto seeds successfully lysed bacteria post air-drying. The seed coat mucilage (SCM), a polysaccharide-polymer matrix exuded by seeds, plays a critical role in phage binding. Seeds with removed mucilage formed five to 10 times less lysis zones compared to those with mucilage. The podovirus Athelas showed the highest mucilage dependency. Phages from the Autographiviridae family also depended on mucilage for seed adhesion. Comparative analysis of Arabidopsis SCM mutants suggested the diffusible cellulose as a key component for phage binding. Long-term activity tests demonstrated high phage stability on seed surfaces and significantly increasing seedling survival rates in the presence of pathogens. Using non-virulent host strains enhanced phage presence on seeds but also has potential limitations. These findings highlight phage-based interventions as promising, sustainable strategies for combating pathogen resistance and improving crop yield.

Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave.

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

Evaluating the Stability of Lytic and Lysogenic Bacteriophages in Various Protectants.

Phage therapy has regained value as a potential alternative and a complementary anti-infective approach to antibiotics in the fight against bacterial pathogens. Due to their host specificity, non-pathogenic nature for humans, and low production cost, phages offer an effective opportunity for utilization in healthcare, agriculture, and food preservation. Well-defined storage conditions are essential for commercialization and dissemination of phage usage. For this purpose, in our study, after the isolation and characterization of two different phages, one lytic and the other lysogenic; storage and shelf-life studies of phages were evaluated in a presence of various protectants (glycerol, sodium azide, DMSO with chloroform) and without any protectant during 8-month period at four different temperatures. The short-time stability of the lytic P. syringae phage and lysogenic MRSA phage, which were determined by STEM analysis to belong to the Straboviridae and Siphoviridae families, respectively were also examined for the different temperatures and the pH levels ranging from 1.0 to 14.0. This study revealed the storage-model of phages that exhibit distinct lifecycles, for the first time and provided a theoretical basis for development and application of phages, has yielded valuable findings contributing to understanding of phage biology.

Isolation of the Novel Phage PHB09 and Its Potential Use against the Plant Pathogen Pseudomonas syringae pv. actinidiae.

Bacteriophages are viruses that specifically infect target bacteria. Recently, bacteriophages have been considered potential biological control agents for bacterial pathogens due to their host specificity. Pseudomonas syringae pv. actinidiae (Psa) is a reemerging pathogen that causes bacterial canker of kiwifruit (Actinidia sp.). The economic impact of this pest and the development of resistance to antibiotics and copper sprays in Psa and other pathovars have led to investigation of alternative management strategies. Phage therapy may be a useful alternative to conventional treatments for controlling Psa infections. Although the efficacy of bacteriophage φ6 was evaluated for the control of Psa, the characteristics of other DNA bacteriophages infecting Psa remain unclear. In this study, the PHB09 lytic bacteriophage specific to Psa was isolated from kiwifruit orchard soil. Extensive host range testing using Psa isolated from kiwifruit orchards and other Pseudomonas strains showed PHB09 has a narrow host range. It remained stable over a wide range of temperatures (4-50 °C) and pH values (pH 3-11) and maintained stability for 50 min under ultraviolet irradiation. Complete genome sequence analysis indicated PHB09 might belong to a new myovirus genus in Caudoviricetes. Its genome contains a total of 94,844 bp and 186 predicted genes associated with phage structure, packaging, host lysis, DNA manipulation, transcription, and additional functions. The isolation and identification of PHB09 enrich the research on Pseudomonas phages and provide a promising biocontrol agent against kiwifruit bacterial canker.

Molecular Characterization and Taxonomic Assignment of Three Phage Isolates from a Collection Infecting Pseudomonas syringae pv. actinidiae and P. syringae pv. phaseolicola from Northern Italy.

Bacterial kiwifruit vine disease (Pseudomonas syringae pv. actinidiae, Psa) and halo blight of bean (P. syringae pv. phaseolicola, Pph) are routinely treated with copper, leading to environmental pollution and bacterial copper resistance. An alternative sustainable control method could be based on bacteriophages, as phage biocontrol offers high specificity and does not result in the spread of toxic residues into the environment or the food chain. In this research, specific phages suitable for phage-based biocontrol strategies effective against Psa and Pph were isolated and characterized. In total, sixteen lytic Pph phage isolates and seven lytic Psa phage isolates were isolated from soil in Piedmont and Veneto in northern Italy. Genome characterization of fifteen selected phages revealed that the isolated Pph phages were highly similar and could be considered as isolates of a novel species, whereas the isolated Psa phages grouped into four distinct clades, two of which represent putative novel species. No lysogeny-, virulence- or toxin-related genes were found in four phages, making them suitable for potential biocontrol purposes. A partial biological characterization including a host range analysis was performed on a representative subset of these isolates. This analysis was a prerequisite to assess their efficacy in greenhouse and in field trials, using different delivery strategies.

Higher Concentrations of Bacterial Enveloped Virus Phi6 Can Protect the Virus from Environmental Decay.

Phage Phi6 is an enveloped virus considered a possible nonpathogenic surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral pathogens in transmission studies. Larger input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phages are dried in plastic tubes and when they are stored in saline solution at 4°C. In contrast, when bacteriophage Phi6 is placed in LB (Luria-Bertani) growth medium (instead of saline) prior to placement on the plastic surface, the influence of the starting concentration on viral recovery is negligible. Protection is reflected in the phage half-lives at higher concentrations being longer than the half-lives at lower concentrations. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon the survival of infectious virus following inoculation onto various surfaces, large initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at higher concentrations but also because the half-lives themselves are extended at higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus and to consider the role of the carrier and matrix if the results are to be clinically relevant. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, much attention has been paid to the environmental decay of SARS-CoV-2 due to the proposed transmission of the virus via fomites. However, published experiments have commenced with inocula with very high virus titers, an experimental design not representative of real-life conditions. The study described here evaluated the impact of the initial virus titer on the environmental decay of an enveloped virus, using a nonpathogenic surrogate for the transmission of SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

The Bacteriophage Pf-10-A Component of the Biopesticide "Multiphage" Used to Control Agricultural Crop Diseases Caused by Pseudomonas syringae.

Phytopathogenic pseudomonads are widespread in the world and cause a wide range of plant diseases. In this work, we describe the Pseudomonas phage Pf-10, which is a part of the biopesticide "Multiphage" used for bacterial diseases of agricultural crops caused by Pseudomonas syringae. The Pf-10 chromosome is a dsDNA molecule with two direct terminal repeats (DTRs). The phage genomic DNA is 39,424 bp long with a GC-content of 56.5%. The Pf-10 phage uses a packaging mechanism based on T7-like short DTRs, and the length of each terminal repeat is 257 bp. Electron microscopic analysis has shown that phage Pf-10 has the podovirus morphotype. Phage Pf-10 is highly stable at pH values from 5 to 10 and temperatures from 4 to 60 °C and has a lytic activity against Pseudomonas strains. Phage Pf-10 is characterized by fast adsorption rate (80% of virions attach to the host cells in 10 min), but has a relatively small number of progeny (37 ± 8.5 phage particles per infected cell). According to the phylogenetic analysis, phage Pf-10 can be classified as a new phage species belonging to the genus Pifdecavirus, subfamily Studiervirinae, family Autographiviridae, order Caudovirales.

Preparing for the KIL: Receptor Analysis of Pseudomonas syringae pv. porri Phages and Their Impact on Bacterial Virulence.

The prevalence of Pseudomonas syringae pv. porri (Pspo) in Belgium continues to increase and sustainable treatments for this pathogen remain unavailable. A potentially attractive biocontrol strategy would be the application of bacteriophages. The ideal application strategy of phages in an agricultural setting remains unclear, especially in a field-based production such as for leek plants in Flanders. Therefore, more insight in bacteria-phage interaction is required, along with the evaluation of different application strategies. In this study, we further characterized the infection strategy of two Pspo phages, KIL3b and KIL5. We found that both phages recognize lipopolysaccharide (LPS) moieties on the surface of the bacterium. LPS is an important pathogenicity factor of Pspo. Our data also suggest that KIL5 requires an additional protein in the bacterial cytoplasmatic membrane to efficiently infect its host. Virulence tests showed that this protein also contributes to Pspo virulence. Furthermore, a cocktail of both phages was applied in a seed bioassay. A combination of KIL3b and KIL5 reduced the bacterial concentration 100-fold. However, in vitro Pspo resistance against phage infection developed quite rapidly. However, the impact of this phage resistance might be mitigated as is suggested by the fact that those resistance mutations preferably occur in genes involved in LPS metabolism, and that the virulence of those mutants is possibly reduced. Our data suggest that the phage cocktail has promising potential to lower the prevalence of Pspo and to be integrated in a pest management strategy. Targeted research is needed to further explore the applicability of the phages in combination with other disease control strategies.

Complete genome sequence of Phobos: a novel bacteriophage with unusual genomic features that infects Pseudomonas syringae.

The GenBank database contains over 2580 complete genome sequences from bacteriophages. However, limited reports are available concerning phages can that lyse members of Pseudomonas syringae, although this is a widespread bacterial species that can infect almost 200 plant species. In the present study, we isolated and characterized a new Siphoviridae phage, named "Pseudomonas phage vB_PsyS_Phobos" (for brevity, referred to here as Phobos). To our knowledge, this is one of the first genome sequences reported for a phage with lytic activity against P. syringae pv. syringae. The genome of Phobos is dsDNA of 56,734 bp with a GC content of 63.3%, containing 65 ORFs. Genome analysis revealed that Phobos is a novel lytic phage with unique genomic features and low similarity to other phages, suggesting that Phobos represents a new phage genus. Genome sequencing did not reveal sequences with significant similarity to known virulence factors, antibiotic resistance genes, potential immunoreactive allergens, or lysogeny-related proteins, suggesting suggests that phage Phobos is strictly lytic. Therefore, Phobos may be suitable for formulation as a biocontrol agent against P. syringae pv. syringae.

Isolation, Characterisation and Experimental Evolution of Phage that Infect the Horse Chestnut Tree Pathogen, Pseudomonas syringae pv. aesculi.

Bleeding canker of horse chestnut trees is a bacterial disease, caused by the bacterium Pseudomonas syringae pv. aesculi, estimated to be present in ~ 50% of UK horse chestnut trees. Currently, the disease has no cure and tree removal can be a common method of reducing inoculum and preventing spread. One potential method of control could be achieved using naturally occurring bacteriophages infective to the causative bacterium. Bacteriophages were isolated from symptomatic and asymptomatic horse chestnut trees in three locations in the South East of England. The phages were found to be belonging to both the Myoviridae and Podoviridae families by RAPD PCR and transmission electron microscopy. Experimental coevolution was carried out to understand the dynamics of bacterial resistance and phage infection and to determine whether new infective phage genotypes would emerge. The phages exhibited different coevolution patterns with their bacterial hosts across time. This approach could be used to generate novel phages for use in biocontrol cocktails in an effort to reduce the potential emergence of bacterial resistance.

Use of phage ϕ6 to inactivate Pseudomonas syringae pv. actinidiae in kiwifruit plants: in vitro and ex vivo experiments.

Over the last years, the global production and trade of kiwifruit has been severely impacted by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen that causes a disease in kiwifruit plants known as bacterial canker. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with disinfectants, copper-based bactericides and/or antibiotics. Moreover, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Phage therapy may be an alternative approach to inactivate Psa. The present study investigated the potential application of the already commercially available bacteriophage (or phage) ϕ6 to control Psa infections. The inactivation of Psa was assessed in vitro, using liquid culture medium, and ex vivo, using artificially contaminated kiwifruit leaves with two biovar 3 (a highly aggressive pathogen) strains (Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10). In the in vitro experiments, the phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). In the ex vivo tests, the decrease was lower (maximum reduction 1.1 log and 1.8 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). The results of this study suggest that the commercially available phage ϕ6 can be an effective alternative to control Psa infections in kiwifruit orchards.

Virus infection induces resistance to Pseudomonas syringae and to drought in both compatible and incompatible bacteria-host interactions, which are compromised under conditions of elevated temperature and CO2 levels.

Plants are simultaneously exposed to a variety of biotic and abiotic stresses, such as infections by viruses and bacteria, or drought. This study aimed to improve our understanding of interactions between viral and bacterial pathogens and the environment in the incompatible host Nicotiana benthamiana and the susceptible host Arabidopsis thaliana, and the contribution of viral virulence proteins to these responses. Infection by the Potato virus X (PVX)/Plum pox virus (PPV) pathosystem induced resistance to Pseudomonas syringae (Pst) and to drought in both compatible and incompatible bacteria-host interactions, once a threshold level of defence responses was triggered by the virulence proteins P25 of PVX and the helper component proteinase of PPV. Virus-induced resistance to Pst was compromised in salicylic acid and jasmonic acid signalling-deficient Arabidopsis but not in N. benthamiana lines. Elevated temperature and CO2 levels, parameters associated with climate change, negatively affected resistance to Pst and to drought induced by virus infection, and this correlated with diminished H2O2 production, decreased expression of defence genes and a drop in virus titres. Thus, diminished virulence should be considered as a potential factor limiting the outcome of beneficial trade-offs in the response of virus-infected plants to drought or bacterial pathogens under a climate change scenario.

Phage resistance evolution in vitro is not reflective of in vivo outcome in a plant-bacteria-phage system.

The evolution of resistance to parasites is fundamentally important to disease ecology, yet we remain unable to predict when and how resistance will evolve. This is largely due to the context-dependent nature of host-parasite interactions, as the benefit of resistance will depend on the abiotic and biotic environment. Through experimental evolution of the plant pathogenic bacterium Pseudomonas syringae and two lytic bacteriophages across two different environments (high-nutrient media and the tomato leaf apoplast), we demonstrate that de novo evolution of resistance is negligible in planta despite high levels of resistance evolution in vitro. We find no evidence supporting the evolution of phage-selected resistance in planta despite multiple passaging experiments, multiple assays for resistance, and high multiplicities of infection. Additionally, we find that phage-resistant mutants (evolved in vitro) did not realize a fitness benefit over phage-sensitive cells when grown in planta in the presence of phage, despite reduced growth of sensitive cells, evidence of phage replication in planta, and a large fitness benefit in the presence of phage observed in vitro. Thus, this context-dependent benefit of phage resistance led to different evolutionary outcomes across environments. These results underscore the importance of studying the evolution of parasite resistance in ecologically relevant environments.

Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6.

BACKGROUND: Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli. RESULTS: Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles. CONCLUSIONS: Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

Localized recombination drives diversification of killing spectra for phage-derived syringacins.

To better understand the potential for antagonistic interactions between members of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage-derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage-derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage-derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.

A bioassay-based protocol for chemical neutralization of human faecal wastes treated by physico-chemical disinfection processes: A case study on benzalkonium chloride.

In situ physico-chemical disinfection of high risk faecal waste is both effective and widely used as a sanitation management strategy for infection prevention and control. Systematic tests where the performance of alternative physico-chemical disinfection methods is systematically compared and optimized must be based on reliable protocols. These protocol are currently not adequately addressing the neutralization related issues: the neutralization of the tested disinfectant after specified conditions of concentration and contact time (CT) is necessary to prevent continued disinfection after the intended contact time; moreover such neutralization is often necessary in practice and on a large scale to prevent adverse health and ecological impacts from remaining disinfectant after the target CT is achieved. Few studies adequately assess the extent of neutralization of the chemical disinfectant and are intended to optimize on-site disinfection practices for waste matrices posing high microbial risks. Hence, there is a need for effective and reproducible neutralization protocols in chemical disinfection trials and practice. Furthermore, for most of chemical disinfectants used in healthcare settings there is no practical methodology to reliably and conveniently measure the residual disinfectant concentration after its neutralization and also determine the optimum concentration of the neutralizer. Because some neutralizing compounds can themselves be toxic to the test microorganisms, it is necessary to optimize neutralization procedures in disinfection experiments for the development of infection control practices using accepted positive control microbes. In the presented work, a stepwise bioassay-based protocol using representative faecal indicator microbes is described for optimizing chemical disinfection and subsequent disinfectant neutralization of any infectious faecal waste matrix. The example described is for the quaternary ammonium compound benzalkonium chloride and its recommended chemical neutralizer in a high strength human faecal waste matrix.

Existing Host Range Mutations Constrain Further Emergence of RNA Viruses.

RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the double-stranded RNA (dsRNA) bacteriophage φ6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type φ6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wild-type clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wild-type φ6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in φ6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.IMPORTANCE RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage φ6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type φ6. This research suggests that serial host-shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in φ6 host range expansion, expanding our knowledge of this important model system in experimental evolution.