Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Prediction of key amino acids of Salmonella phage endolysin LysST-3 and detection of its mutants' activity.

Salmonella Typhimurium, a zoonotic pathogen, causes systemic and localized infection. The emergence of drug-resistant S. Typhimurium has increased; treating bacterial infections remains challenging. Phage endolysins derived from phages have a broader spectrum of bacteriolysis and better bacteriolytic activity than phages, and are less likely to induce drug resistance than antibiotics. LysST-3, the endolysin of Salmonella phage ST-3, was chosen in our study for its high lytic activity, broad cleavage spectrum, excellent bioactivity, and moderate safety profile. LysST-3 is a promising antimicrobial agent for inhibiting the development of drug resistance in Salmonella. The aim of this study is to investigate the molecular characteristics of LysST-3 through the prediction of key amino acid sites of LysST-3 and detection of its mutants' activity. We investigated its lytic effect on Salmonella and identified its key amino acid sites of interaction with substrate. LysST-3 may be a Ca2+, Mg2+ - dependent metalloenzyme. Its concave structure of the bottom "gripper" was found to be an important part of its amino acid active site. We identified its key sites (29P, 30T, 86D, 88 L, and 89 V) for substrate binding and activity using amino acid-targeted mutagenesis. Alterations in these sites did not affect protein secondary structure, but led to a significant reduction in the cleavage activity of the mutant proteins. Our study provides a basis for phage endolysin modification to target drug-resistant bacteria. Identifying the key amino acid site of the endolysin LysST-3 provides theoretical support for the functional modification of the endolysin and the development of subsequent effective therapeutic solutions.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Isolation and molecular characterization of the Salmonella Typhimurium orphan phage Arash.

The current threat of multidrug resistant strains necessitates development of alternatives to antibiotics such as bacteriophages. This study describes the isolation and characterization of a novel Salmonella Typhimurium phage 'Arash' from hospital wastewater in Leuven, Belgium. Arash has a myovirus morphology with a 95 nm capsid and a 140 nm tail. The host range of Arash is restricted to its isolation host. Approximately 86% of the phage particles are adsorbed to a host cell within 10 min. Arash has latent period of 65 min and burst size of 425 PFU/cell. Arash has a dsDNA genome of 180,819 bp with GC content of 53.02% with no similarities to any characterized phages, suggesting Arash as a novel species in the novel 'Arashvirus' genus. Arash carries no apparent lysogeny-, antibiotic resistance- nor virulence-related genes. Proteome analysis revealed 116 proteins as part of the mature phage particles of which 27 could be assigned a function. Therefore, the present findings shed light on the morphological, microbiological and genomic characteristics of Arash and suggest its potential application as therapeutic and/or biocontrol agent.

Antibiofilm activity of a lytic Salmonella phage on different Salmonella enterica serovars isolated from broiler farms.

Bacteriophages have been mainly used in treating infections caused by planktonic bacterial cells in the veterinary sector. However, their applications as antibiofilm agents have received little attention. Accordingly, a previously isolated Salmonella infecting Siphoviridae phage was investigated for host range against 15 Salmonella enterica isolates (S. Cape, S. Gallinarum, 4 S. Enteritidis, 3 S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, S. Agona, S. Daula, and S. Aba) recovered from the litters of commercial broiler farms. All S. enterica isolates were examined for their biofilm activity using a microtiter plate assay and for adrA, csgD, and gcpA genes using conventional PCR. The phage efficacy against established biofilms produced by the selected seven S. enterica isolates (S. Gallinarum, S. Enteritidis, S. Montevideo, S. Uno, S. Oritamerin, S. Belgdam, and S. Agona) was assessed using microtiter plate assay and reverse transcriptase real-time PCR over different incubation times of 5 and 24 h. All S. enterica isolates were strong biofilm formers. Moreover, the phage effectively reduced the biofilm activity of the established S. enterica biofilms in the microtiter plate assay using the independent sample t-test (P < 0.050). Furthermore, the relative expression levels of csgD, gcpA, and adrA genes in the biofilm cells of S. enterica isolate after phage treatment were significantly up-regulated to variable degrees using the independent sample t-test (P < 0.050). In conclusion, the present study revealed the potential use of Salmonella phage in reducing established biofilms produced by S. enterica serovars isolated from broiler farms.

A ToxIN homolog from Salmonella enterica serotype Enteritidis impairs bacteriophage infection.

AIMS: To determine if the bacteriophage abortive infection system ToxIN is present in foodborne Salmonella and if it protects against infection by bacteriophages specific to enteric bacteria. METHODS AND RESULTS: A set of foodborne Salmonella enteritidis isolates from a 2010 eggshell outbreak was identified via BLASTN (basic local alignment search tool nucleotide) queries as harboring a close homolog of ToxIN, carried on a plasmid with putative mobilization proteins. This homolog was cloned into a plasmid vector and transformed into the laboratory strain Salmonella typhimurium LT2 and tested against a set of Salmonella-specific phages (FelixO1, S16, Sp6, LPST153, and P22 HT105/1 int-201). ToxIN reduced infection by FelixO1, S16, and LPST153 by ∼1-4 log PFU ml-1 while reducing the plaque size of Sp6. When present in LT2 and Escherichia coli MG1655, ToxIN conferred cross-genus protection against phage isolates, which infect both bacteria. Finally, the putative ToxIN plasmid was found in whole-genome sequence contigs of several Salmonella serovars, pathogenic E. coli, and other pathogenic enterobacteria. CONCLUSIONS: Salmonella and E. coli can resist infection by several phages via ToxIN under laboratory conditions; ToxIN is present in foodborne pathogens including Salmonella and Shiga-toxigenic E. coli.

Newly Isolated Virulent Salmophages for Biocontrol of Multidrug-Resistant Salmonella in Ready-to-Eat Plant-Based Food.

Due to irrational antibiotic stewardship, an increase in the incidence of multidrug resistance of bacteria has been observed recently. Therefore, the search for new therapeutic methods for pathogen infection treatment seems to be necessary. One of the possibilities is the utilization of bacteriophages (phages)-the natural enemies of bacteria. Thus, this study is aimed at the genomic and functional characterization of two newly isolated phages targeting MDR Salmonella enterica strains and their efficacy in salmonellosis biocontrol in raw carrot-apple juice. The Salmonella phage vB_Sen-IAFB3829 (Salmonella phage strain KKP 3829) and Salmonella phage vB_Sen-IAFB3830 (Salmonella phage strain KKP 3830) were isolated against S. I (6,8:l,-:1,7) strain KKP 1762 and S. Typhimurium strain KKP 3080 host strains, respectively. Based on the transmission electron microscopy (TEM) and whole-genome sequencing (WGS) analyses, the viruses were identified as members of tailed bacteriophages from the Caudoviricetes class. Genome sequencing revealed that these phages have linear double-stranded DNA and sizes of 58,992 bp (vB_Sen-IAFB3829) and 50,514 bp (vB_Sen-IAFB3830). Phages retained their activity in a wide range of temperatures (from -20 °C to 60 °C) and active acidity values (pH from 3 to 11). The exposure of phages to UV radiation significantly decreased their activity in proportion to the exposure time. The application of phages to the food matrices significantly reduced the level of Salmonella contamination compared to the control. Genome analysis showed that both phages do not encode virulence or toxin genes and can be classified as virulent bacteriophages. Virulent characteristics and no possible pathogen factors make examined phages feasible to be potential candidates for food biocontrol.

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Modulation of Caecal Microbiota and Metabolome Profile in Salmonella-Infected Broilers by Phage Therapy.

Bacteriophage therapy is considered one of the most promising tools to control zoonotic bacteria, such as Salmonella, in broiler production. Phages exhibit high specificity for their targeted bacterial hosts, causing minimal disruption to the niche microbiota. However, data on the gut environment's response to phage therapy in poultry are limited. This study investigated the influence of Salmonella phage on host physiology through caecal microbiota and metabolome modulation using high-throughput 16S rRNA gene sequencing and an untargeted metabolomics approach. We employed 24 caecum content samples and 24 blood serum samples from 4-, 5- and 6-week-old broilers from a previous study where Salmonella phages were administered via feed in Salmonella-infected broilers, which were individually weighed weekly. Phage therapy did not affect the alpha or beta diversity of the microbiota. Specifically, we observed changes in the relative abundance of 14 out of the 110 genera using the PLS-DA and Bayes approaches. On the other hand, we noted changes in the caecal metabolites (63 up-accumulated and 37 down-accumulated out of the 1113 caecal metabolites). Nevertheless, the minimal changes in blood serum suggest a non-significant physiological response. The application of Salmonella phages under production conditions modulates the caecal microbiome and metabolome profiles in broilers without impacting the host physiology in terms of growth performance.

Expansion of the Plaquing Host Range and Improvement of the Absorption Rate of a T5-like Salmonella Phage by Altering the Long Tail Fibers.

The high host specificity of phages is a real challenge in the therapy applications of the individual phages. This study aimed to edit the long tail fiber proteins (pb1) of a T5-like phage to obtain the engineered phages with expanded plaquing host range. Two T5-like Salmonella phages with high genome sequence homology but different plaquing host ranges, narrow-host range phage vB STyj5-1 (STyj5-1) and wide-host range phage vB BD13 (BD13), were isolated and characterized. The pb1 parts of STyj5-1 were replaced by the corresponding part of BD13 using homologous recombination method to obtain the engineered phages. The alterations of the whole pb1 part or the N-terminal amino acids 1-400 of pb1 of STyj5-1 could expand their plaquing host ranges (from 20 strains to 30 strains) and improve their absorption rates (from 0.28-28.84% to 28.10-99.49%). Besides, the one-step growth curves of these engineered phages with modified pb1 parts were more similar to that of STyj5-1. The burst sizes of phages BD13, STyj5-1 and the engineered phages were 250, 236, 166, and 223 PFU per cell, respectively. The expanded plaquing host range and improved absorption rates of these engineered phages revealed that the pb1 part might be the primary determinant of the host specificities of some T5-like phages. IMPORTANCE Genetic editing can be used to change or expand the host range of phages and have been successfully applied in T2, T4 and other phages to obtain engineered phages. However, there are hardly any similar reports on T5-like phages due to that the determinant regions related to their host ranges have not been completely clarified and the editing of T5-like phages is more difficult compared to other phages. This study attempted and successfully expanded the host range of a narrow-host range T5-like phage (STyj5-1) by exchanging its whole pb1 part or the N-terminal 1-400aa of that part by a broad-host range phage (BD13). These demonstrated the pb1 part might be the primary determinant of the host specificities for some T5-like phages and provided an effective method of extension plaquing host range of these phages.

Expression and biological activity of lytic proteins HolST-3 and LysST-3 of Salmonella phage ST-3.

Salmonella infection is a major public health concern. Several strategies for Salmonella infection prevention and control are currently available including vaccines and antibiotics. However, vaccines are expensive and inefficient, and the use of antibiotics can lead to antibiotic resistance. Thus, alternative strategies for the treatment of Salmonella remain warrant. In this study, recombinant holin HolST-3 and lysin LysST-3 from Salmonella phage ST-3 were expressed and purified, and their bactericidal properties were analyzed. HolST-3 and LysST-3 possessed a wider lysis spectrum and more efficient bactericidal effect than phage ST-3, and a synergistic bactericidal effect was observed when combined in vitro. In addition, we explored the bactericidal properties of HolST-3 and LysST-3 in vivo using zebrafish as a model organism, and found that the bactericidal effects of both HolST-3 and LysST-3 in vivo were comparable to those of cefotaxime, an antibiotic. This study provides a basis for the development of HolST-3 and LysST-3 as novel bactericidal agents for the prevention and treatment of infectious diseases caused by Salmonella spp.

Salmonella phage akira, infecting selected Salmonella enterica Enteritidis and Typhimurium strains, represents a new lineage of bacteriophages.

Some serovars of Salmonella can cause life-threatening diarrhoeal diseases and bacteriemia. The emergence of multidrug-resistant strains has led to a need for alternative treatments such as phage therapy, which requires available, well-described, diverse, and suitable phages. Phage akira was found to lyse 19 out of 32 Salmonella enterica serovars and farm isolates tested, although plaque formation was observed with only two S. Enteritidis and one S. Typhimurium strain. Phage akira encodes anti-defence genes against type 1 R-M systems, is distinct (<65% nucleotide sequence identity) from related phages and has siphovirus morphology. We propose that akira represents a new genus in the class Caudoviricetes.

The Combination of Salmonella Phage ST-3 and Antibiotics to Prevent Salmonella Typhimurium In Vitro.

The novel phage ST-3, capable of infecting the foodborne pathogen Salmonella Typhimurium, was isolated from wastewater. The Biological characters and genome information of ST-3 were analyzed. In the in vitro assay, the phage ST-3 with a MOI of 10 effectively inhibited the growth of Salmonella Typhimurium CGMCC 1.1174 in 6 h. The inhibitory effect of combination phage ST-3 and antibiotics was also studied, the removal rate of planktonic host exposed to ST-3 and levofloxacin hydrochloride at the same time, or to ciprofloxacin followed by ST-3, is higher than that exposed to antibiotic dosing group alone and antibiotic + phage dosing group. The phage ST-3 combined with 0.5 µg/mL levofloxacin hydrochloride resulted in the largest decrease in biofilm biomass at 54%. The phage ST-3 could be a potential agent to control Salmonella Typhimurium growth and provide instruction for use it and antibiotics together.

Genetic Mining of Newly Isolated Salmophages for Phage Therapy.

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella.

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

Genomic and functional characterization of five novel Salmonella-targeting bacteriophages.

BACKGROUND: The host-unrestricted, non-typhoidal Salmonella enterica serovar Enteritidis (S. Enteritidis) and the serovar Typhimurium (S. Typhimurium) are major causative agents of food-borne gastroenteritis, and the host-restricted Salmonella enterica serovar Gallinarum (S. Gallinarum) is responsible for fowl typhoid. Increasing drug resistance in Salmonella contributes to the reduction of effective therapeutic and/or preventive options. Bacteriophages appear to be promising antibacterial tools, able to combat infectious diseases caused by a wide range of Salmonella strains belonging to both host-unrestricted and host-restricted Salmonella serovars. METHODS: In this study, five novel lytic Salmonella phages, named UPWr_S1-5, were isolated and characterized, including host range determination by plaque formation, morphology visualization with transmission electron microscopy, and establishment of physiological parameters. Moreover, phage genomes were sequenced, annotated and analyzed, and their genomes were compared with reference Salmonella phages by use of average nucleotide identity, phylogeny, dot plot, single nucleotide variation and protein function analysis. RESULTS: It was found that UPWr_S1-5 phages belong to the genus Jerseyvirus within the Siphoviridae family. All UPWr_S phages were found to efficiently infect various Salmonella serovars. Host range determination revealed differences in host infection profiles and exhibited ability to infect Salmonella enterica serovars such as Enteritidis, Gallinarum, Senftenberg, Stanley and Chester. The lytic life cycle of UPWr_S phages was confirmed using the mitomycin C test assay. Genomic analysis revealed that genomes of UPWr_S phages are composed of 51 core and 19 accessory genes, with 33 of all predicted genes having assigned functions. UPWr_S genome organization comparison revealed 3 kinds of genomes and mosaic structure. UPWr_S phages showed very high sequence similarity to each other, with more than 95% average nucleotide identity. CONCLUSIONS: Five novel UPWr_S1-5 bacteriophages were isolated and characterized. They exhibit host lysis range within 5 different serovars and are efficient in lysis of both host-unrestricted and host-restricted Salmonella serovars. Therefore, because of their ability to infect various Salmonella serovars and lytic life cycle, UPWr_S1-5 phages can be considered as useful tools in biological control of salmonellosis.

Genome analysis of Salmonella phage vB_SalM_8-19 (genus Rosemountvirus).

This work describes the characterization and genome annotation of Salmonella phage vB_SalM_8-19 (referred to as 8-19) isolated from sewage samples collected in a pig farm in Jilin, China. This phage was capable of infecting 60% Salmonella strains in our lab stock. The genome of phage 8-19 is composed of linear double-stranded DNA that is 52,648 bp in length with a G + C content of 46.02%; containing 74 ORFs and no tRNA genes. In October 2019, phylogenetic analyses indicated that phage 8-19 might belong to a novel cluster among the other similar phages which have not been specifically classified within some new genus in family Myoviridae. Recently, the International Committee on Taxonomy of Viruses (ICTV) defined phage 8-19 and its related phages as genus Rosemountvirus, family Myoviridae. This new genus, known as Rosemountvirus, is rarely reported in the literature.

Complete genome sequence of the extreme-pH-resistant Salmonella bacteriophage αα of the family Microviridae.

A novel Salmonella bacteriophage (phage), named αα, was the first reported member of the family Microviridae to exhibit tolerance to both extreme acidic and alkaline conditions (pH 2-12 for 1 h). Phage αα has a circular single-stranded DNA genome of 5,387 nt with a G+C content of 44.66%. A total of 11 putative gene products and no tRNA genes are encoded in the phage αα genome. Whole-genome sequence comparisons revealed that phage αα shares 95% identity with coliphage phiX174 and had a close evolutionary relationship to the phages NC1 and NC7. Phylogenetic analysis of the structural proteins of phage αα and 18 other phiX174-like phages showed that a phylogenetic tree based on protein B sequences had a topology similar to that obtained using whole genome sequences. In addition, variable sites in proteins F and G distributed on the surface of the mature capsid and the conserved protein J were probably involved in maintaining the structural integrity of the phage under extreme pH conditions. Our findings could open up new perspectives for identifying more extreme-pH-resistant phages and their structural proteins and understanding the mechanism of phage adaptation and evolution under extreme environmental stress.