Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

Pseudo-pac site sequences used by phage P22 in generalized transduction of Salmonella.

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains-LT2 and 14028S-for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage-host systems.

Phage-host co-evolution has led to distinct generalized transduction strategies.

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.

Versatile Platinum Nanoparticles-Decorated Phage Nanozyme Integrating Recognition, Bacteriolysis, and Catalysis Capabilities for On-Site Detection of Foodborne Pathogenic Strains Vitality Based on Bioluminescence/Pressure Dual-Mode Bioassay.

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 102-107 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

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.

Identification of a broad-spectrum lytic Myoviridae bacteriophage using multidrug resistant Salmonella isolates from pig slaughterhouses as the indicator and its application in combating Salmonella infections.

BACKGROUND: Salmonella is a leading foodborne and zoonotic pathogen, and is widely distributed in different nodes of the pork supply chain. In recent years, the increasing prevalence of antimicrobial resistant Salmonella poses a threat to global public health. The purpose of this study is to the prevalence of antimicrobial resistant Salmonella in pig slaughterhouses in Hubei Province in China, and explore the effect of using lytic bacteriophages fighting against antimicrobial resistant Salmonella. RESULTS: We collected a total of 1289 samples including anal swabs of pigs (862/1289), environmental swabs (204/1289), carcass surface swabs (36/1289) and environmental agar plates (187/1289) from eleven slaughterhouses in seven cities in Hubei Province and recovered 106 Salmonella isolates. Antimicrobial susceptibility testing revealed that these isolates showed a high rate of antimicrobial resistance; over 99.06% (105/106) of them were multidrug resistant. To combat these drug resistant Salmonella, we isolated 37 lytic phages using 106 isolates as indicator bacteria. One of them, designated ph 2-2, which belonged to the Myoviridae family, displayed good capacity to kill Salmonella under different adverse conditions (exposure to different temperatures, pHs, UV, and/or 75% ethanol) and had a wide lytic spectrum. Evaluation in mouse models showed that ph 2-2 was safe and saved 80% (administrated by gavage) and 100% (administrated through intraperitoneal injection) mice from infections caused by Salmonella Typhimurium. CONCLUSIONS: The data presented herein demonstrated that Salmonella contamination remains a problem in some pig slaughter houses in China and Salmonella isolates recovered in slaughter houses displayed a high rate of antimicrobial resistance. In addition, broad-spectrum lytic bacteriophages may represent a good candidate for the development of anti-antimicrobial resistant Salmonella agents.

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.

Evolution of a New Function by Fusion between Phage DNA and a Bacterial Gene.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene.

Bacteriophages benefit from generalized transduction.

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.

The Lytic Siphophage vB_StyS-LmqsSP1 Reduces the Number of Salmonella enterica Serovar Typhimurium Isolates on Chicken Skin.

Phage-based biocontrol of bacteria is considered a natural approach to combat foodborne pathogens. Salmonella spp. are notifiable and highly prevalent pathogens that cause foodborne diseases worldwide. In this study, six bacteriophages were isolated and further characterized that infect food-derived Salmonella isolates from different meat sources. The siphovirus VB_StyS-LmqsSP1, which was isolated from a cow's nasal swab, was further subjected to in-depth characterization. Phage-host interaction investigations in liquid medium showed that vB_StyS-LmqsSP1 can suppress the growth of Salmonella species isolates at 37°C for 10 h and significantly reduce the bacterial titer at 4°C. A reduction of 1.4 to 3 log units was observed in investigations with two food-derived Salmonella isolates and one reference strain under cooling conditions using multiplicities of infection (MOIs) of 104 and 105. Phage application on chicken skin resulted in a reduction of about 2 log units in the tested Salmonella isolates from the first 3 h throughout a 1-week experiment at cooling temperature and with an MOI of 105. The one-step growth curve analysis using vB_StyS-LmqsSP1 demonstrated a 60-min latent period and a burst size of 50 to 61 PFU/infected cell for all tested hosts. Furthermore, the genome of the phage was determined to be free from genes causing undesired effects. Based on the phenotypic and genotypic properties, LmqsSP1 was assigned as a promising candidate for biocontrol of Salmonella enterica serovar Typhimurium in food. IMPORTANCE Salmonella enterica is one of the major global causes of foodborne enteritis in humans. The use of chemical sanitizers for reducing bacterial pathogens in the food chain can result in the spread of bacterial resistance. Targeted and clean-label intervention strategies can reduce Salmonella contamination in food. The significance of our research demonstrates the suitability of a bacteriophage (vB_StyS-LmqsSP1) for biocontrol of Salmonella enterica serovar Typhimurium on poultry due to its lytic efficacy under conditions prevalent in food production environments.

Characterisation of IncI1 plasmids associated with change of phage type in isolates of Salmonella enterica serovar Typhimurium.

BACKGROUND: Acquisition of IncI1 plasmids by members of the Enterobacteriaceae sometimes leads to transfer of antimicrobial resistance and colicinogeny as well as change of phage type in Salmonella Typhimurium. Isolates of S. Typhimurium from a 2015 outbreak of food poisoning were found to contain an IncI1 plasmid implicated in change of phage type from PT135a to U307 not previously reported. The origin of the changes of phage type associated with this IncI1 plasmid was investigated. In addition, a comparison of its gene composition with that of IncI1 plasmids found in local isolates of S. Typhimurium typed as U307 from other times was undertaken. This comparison was extended to IncI1 plasmids in isolates of phage types PT6 and PT6 var. 1 which are thought to be associated with acquisition of IncI1 plasmids. RESULTS: Analysis of IncI1 plasmids from whole genome sequencing of isolates implicated a gene coding for a 1273 amino acid protein present only in U307 isolates as the likely source of change of phage type. The IncI1 plasmids from PT6 and PT6 var. 1 isolates all had the ibfA gene present in IncI1 plasmid R64. This gene inhibits growth of bacteriophage BF23 and was therefore the possible source of change of phage type. A fuller comparison of the genetic composition of IncI1 plasmids from U307 isolates and PT6 and PT6 var. 1 isolates along with two IncI1 plasmids from S. Typhimurium isolates not showing change of phage type was undertaken. Plasmids were classified as either 'Delta' or 'Col' IncI1 plasmids according to whether genes between repZ and the rfsF site showed high identity to genes in the same location in R64 or ColIb-P9 plasmids respectively. Comparison of the tra gene sets and the pil gene sets across the range of sequenced plasmids identified Delta and Col plasmids with almost identical sequences for both sets of genes. This indicated a genetic recombination event leading to a switch between Delta and Col gene sets at the rfsF site. Comparisons of other gene sets showing significant variation among the sequenced plasmids are reported. Searches of the NCBI GenBank database using DNA and protein sequences of interest from the sequenced plasmids identified global IncI1 plasmids with extensive regions showing 99 to 100% identity to some of the plasmids sequenced in this study indicating evidence for widespread distribution of these plasmids. CONCLUSION: Two genes possibly associated with change of phage type were identified in IncI1 plasmids. IncI1 plasmids were classified as either 'Delta' or 'Col' plasmids and other sequences of significant variation among these plasmids were identified. This study offers a new perspective on the understanding of the gene composition of IncI1 plasmids. The sequences of newly sequenced IncI1 plasmids could be compared against the regions of significant sequence variation identified in this study to understand better their overall gene composition and relatedness to other IncI1 plasmids in the databases.

Salmonella Typhimurium DT 104 response to Lytic bacteriophage and Lactobionic acid on raw chicken breast.

Bacterial food poisoning cases due to Salmonella have been linked with a variety of poultry products. This study evaluated the effects of a Salmonella-specific Lytic bacteriophage and Lactobionic acid (LBA) on Salmonella Typhimurium DT 104 growth on raw chicken breast meat. Each chicken breast was randomly assigned to a treatment group (Control, DI water, phage 1%, phage 5%, LBA 10 mg/mL, LBA 20 mg/mL, and phage 5% + LBA 20 mg/mL) with four chicken breasts per group. Samples were inoculated with 106 CFU/mL of Salmonella and stored at 4 °C for 30 min. The inoculated chicken breasts were randomly assigned to different storage time (0 h, 1 h, 24 h, or 48 h). Both time and treatment showed significance reduction (P < 0.0001) of microbial growth. The weight loss was significantly different (P < 0.0001) between treatments. The LBA treatments were not effective when compared to the control group, but Lytic bacteriophage significantly reduced the amount of microbial growth.

Endolysin LysSTG2: Characterization and application to control Salmonella Typhimurium biofilm alone and in combination with slightly acidic hypochlorous water.

The gene encoding LysSTG2, an endolysin from Salmonella-lytic bacteriophage STG2, was cloned, overexpressed, and characterized. LysSTG2 consists of a single domain belonging to the Peptidase_M15 superfamily. LysSTG2 showed strong lytic activity against chloroform-treated S. Typhimurium cells after incubation at 4-50 °C for 30 min, at pH ranging from 7.0 to 11.0, and in the presence of NaCl from 0 to 300 mmol/L. It also showed lytic activity against all the 14 tested Gram-negative strains treated with chloroform, including Salmonella, E. coli, and Pseudomonas aeruginosa, but not against the Gram-positive bacteria tested. In addition, LysSTG2 (100 µg/mL) reduced the viability of S. Typhimurium NBRC 12529 planktonic cells by 1.2 log and that of the biofilm cells after 1-h treatment. Sequential treatment of slightly acidic hypochlorous water (SAHW) containing 40 mg/L available chlorine and LysSTG2 (100 µg/mL) was effective on S. Typhimurium NBRC 12529 biofilm cells, removing more than 99% of biofilm cells. These results demonstrate that LysSTG2 alone can effectively kill S. Typhimurium cells after permeabilization treatment and successfully control S. Typhimurium in biofilms in combination with SAHW, suggesting that the combined use of LysSTG2 and SAHW might be a novel and promising method for combating S. Typhimurium in food industries.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

Global Proteomic Profiling of Salmonella Infection by a Giant Phage.

The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study, we sought to determine whether this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1,994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription, and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid protein, two head ejection proteins, and the functionally unassigned protein gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting that it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. We identified homologs to SPN3US gp22 in 45 related giant phages, including ϕKZ, whose counterpart is also abundant in infected bacteria but absent in the virion. We determined the ϕKZ counterpart to be cleaved in vitro by its prohead protease, an event that has been observed to promote head maturation of some other phages. Our findings are consistent with a scaffold protein assignment for SPN3US gp22, although direct evidence is required for its confirmation. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.IMPORTANCE "Giant" phages with genomes >200 kb are being isolated in increasing numbers from a range of environments. With hosts such as Salmonella enterica, Pseudomonas aeruginosa, and Erwinia amylovora, these phages are of interest for phage therapy of multidrug-resistant pathogens. However, our understanding of how these complex phages interact with their hosts is impeded by the proportion (∼80%) of their gene products that are functionally uncharacterized. To develop the repertoire of techniques for analysis of phages, we analyzed a liquid infection of Salmonella phage SPN3US (240-kb genome) using third-generation mass spectrometry. We observed the temporal production of phage proteins whose genes collectively represent 96% of the SPN3US genome. These findings demonstrate the sensitivity of mass spectrometry for global proteomic profiling of virus-infected cells, and the identification of a candidate for a major head morphogenesis protein will facilitate further studies into giant phage head assembly.

Associations between antibiotic resistance and bacteriophage resistance phenotypes in laboratory and clinical strains of Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages have received great attention as an alternative over antibiotics due to the host specificity. Therefore, this study was designed to evaluate the associations between bacteriophage-insensitive (BI) and antibiotic-resistant mutants of Salmonella Typhimurium strains. Bacteriophage-sensitive (BS) Salmonella enterica serovar Typhimurium ATCC 19585 (BSSTWT), ciprofloxacin-induced S. Typhimurium ATCC 19585 (BSSTCIP), S. Typhimurium KCCM 40253 (BSSTLAB), and clinically isolated multidrug-resistant S. Typhimurium CCARM 8009 (BSSTMDR) were used to induce the bacteriophage-insensitive mutants (BISTWT, BISTCIP, BISTLAB, and BISTMDR), which were characterized by measuring mutant frequency lysogenic induction, phage adsorption, antibiotic susceptibility, and differential gene expression. The numbers of BSSTWT, BSSTCIP, and BSSTLAB were reduced by P22 (>3 log), while the least lytic activity was observed for BSSTMDR, suggesting alteration in bacteriophage-binding receptors on the surface of multidrug-resistant strain. BSSTWT treated with P22 showed the large variation in the cell state (CV>40%) and highest mutant frequency (62%), followed by 25% for BSSTCIP. The least similarities between BSSTWT and BISTWT were observed for P22 and PBST-13 (<12%). The relative expression levels of bacteriophage-binding receptor-related genes (btuB, fhuA, fliK, fljB, ompC, ompF, rfaL, and tolC) were decreased in BISTCIP and BISTMDR. These results indicate that the bacteriophage resistance is highly associated with the antibiotic resistance. The findings in this study could pave the way for the application of bacteriophages as an alternative to control antibiotic-resistant bacteria.

Characterization of Salmonella bacteriophages and their potential use in dishwashing materials.

AIMS: The aims of this study were to isolate and characterize novel Salmonella phages and to evaluate the effectiveness of phage cocktails used as antibacterial agents in dishwashing materials. METHODS AND RESULTS: The effective phages, vB_STy-RN5i1 and vB_STy-RN29, were isolated from drain water samples collected from open markets using Salmonella Typhimurium as the host strain. These phages were identified as members of Podoviridae and Siphoviridae, respectively. Both phages infected at least six Salmonella serovars and rapidly lysed their host within one hour. They were stable at 4-45°C and at pH 6-9 for at least an hour while being evaluated in this study. The phage application results indicated that bacterial cells were reduced by 3⋅1 and 2⋅7 log CFU per ml at room temperature when they encountered the phage cocktail on scouring pads (SPs) and dishwashing sponges (DSs), respectively. CONCLUSIONS: The isolated Salmonella phages, vB_STy-RN5i1 and vB_STy-RN29, had potential against Salm. Typhimurium and could reduce the occurrence of bacterial-cross-contamination from dishwashing materials, which have been reported to be a source of bacteria, to other kitchen utensils and food. SIGNIFICANCE AND IMPACT OF THE STUDY: The successful reduction of bacterial contamination in dishwashing materials by the phage cocktail consisting of vB_STy-RN5i1 and vB_STy-RN29 reveals its potential to be an alternative antimicrobial agent for SPs and DSs.

Development of a broad-spectrum Salmonella phage cocktail containing Viunalike and Jerseylike viruses isolated from Thailand.

Salmonella is one of the most common agents of foodborne disease worldwide. As natural alternatives to traditional antimicrobial agents, bacteriophages (phages) are emerging as highly effective biocontrol agents against Salmonella and other foodborne bacteria. Due to the high diversity within the Salmonella genus and emergence of drug resistant strains, improved efforts are necessary to find broad range and strictly lytic Salmonella phages for use in food biocontrol. Here, we describe the isolation and characterization of two Salmonella phages: ST-W77 isolated on S. Typhimurium and SE-W109 isolated on S. Enteritidis with extraordinary Salmonella specificity. Whole genome sequencing identified ST-W77 as a Myovirus within the Viunalikevirus genus and SE-W109 as a Siphovirus within the Jerseylikevirus genus. Infectivity studies using a panel of S. Typhimurium cell wall mutants revealed both phages require the lipopolysaccharide O-antigen, with SE-W109 also recognizing the flagella, during infection of Salmonella. A combination of both phages was capable of prolonged (one-week) antibacterial activity when added to milk or chicken meat contaminated with Salmonella. Due to their broad host ranges, strictly lytic lifestyles and lack of lysogeny-related genes or virulence genes in their genomes, ST-W77 and SE-W109 are ideal phages for further development as Salmonella biocontrol agents for food production.

Assessment of the alteration in phage adsorption rates of antibiotic-resistant Salmonella typhimurium.

This study was designed to evaluate the phage-binding receptors on the surface of antibiotic-sensitive Salmonella typhimurium (ASST) and antibiotic-resistant S. typhimurium (ARST). The antibiotic susceptibilities of plasmid-cured ASST and ARST were evaluated against ampicillin, cephalothin, ciprofloxacin, kanamycin, penicillin, and tetracycline. The capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) were quantified using carbazole assay and HPLC, respectively. The amounts of CPSs and LPSs in ARST were decreased from 108 to 62 µg/ml and 284-111 ng/ml, respectively, after plasmid curing. The adsorption rates of P22, PBST10, and PBST13 to plasmid-uncured and plasmid-cured ASST and ARST were decreased after proteinase K and periodate treatments. The highest reduction in phage adsorption rate was observed for P22 to the plasmid-cured ARST treated with periodate (71%). The relative expression levels of btuB, fhuA, and rfaL were decreased by more than twofold in the plasmid-cured ASST, corresponding to the decrease in the adsorption rates of P22 and PBST10. The plasmid-cured ARST lost the ability to express the ß-lactamase gene, which was related to the loss of resistance to ampicillin, cephalothin, kanamycin, penicillin, and tetracycline. The results provide valuable insights into understanding the interaction between phage and antibiotic-resistant bacteria.