Due to the extensive use of antibiotics, the increase of infections caused by antibiotic-resistant bacteria is now a global health concern. Phages have proven useful for treating bacterial infections and represent a promising alternative or complement to antibiotic treatment. Yet, other alternatives exist, such as bacteria-produced non-replicative protein complexes that can kill their targeted bacteria by puncturing their membrane (Tailocins). To expand the repertoire of Tailocins available, we suggest a new approach that transforms phages into Tailocins. Here, we genetically engineered the virulent Ackermannviridae phage S117, as well as temperate phages Fels-1, -2 and Gifsy-1 and -2, targeting the food pathogen Salmonella, by deleting the portal vertex or major capsid gene using CRISPR-Cas9. We report the production of Tailocin particles from engineered virulent and temperate phages able to kill their native host. Our work represents a steppingstone that taps into the huge diversity of phages and transforms them into versatile puncturing new antimicrobials.

Food contamination by Salmonella can lead to serious foodborne diseases that constantly threaten public health. Innovative and effective strategies are needed to control foodborne pathogenic contamination since the incidence of foodborne diseases has increased gradually. In the present study, two broad-spectrum phages named Salmonella phage PSE-D1 and Salmonella phage PST-H1 were isolated from sewage in China. Phages PSE-D1 and PST-H1 were obtained by enrichment with Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) CVCC1806 and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) CVCC3384, respectively. They were able to lyse Salmonella, E. coli and K. pneumoniae and exhibited broad host range. Further study demonstrated that PSE-D1 and PST-H1 showed high pH and thermal tolerances. Phage PSE-D1 belongs to the Jiaodavirus genus, Tevenvirinae subfamily, while phage PST-H1 belongs to the Jerseyvirus genus, Guernseyvirinae subfamily according to morphology and phylogeny. The results of genome analysis showed that PSE-D1 and PST-H1 lack virulence and drug-resistance genes. The effects of PSE-D1 and PST-H1 on controlling S. Enteritidis CVCC1806 and S. Typhimurium CVCC3384 contamination in three kinds of foods (eggshells, sausages and milk) were further investigated, respectively. Our results showed that, compared to phage-free groups, PSE-D1 and PST-H1 inhibited the growth of their host strain significantly. A significant reduction of host bacteria titers (1.5 and 1.9 log10 CFU/sample, p < 0.001) on eggshells was observed under PSE-D1 and PST-H1 treatments, respectively. Furthermore, administration of PSE-D1 and PST-H1 decreased the counts of bacteria by 1.1 and 1.2 log10 CFU/cm2 (p < 0.001) in sausages as well as 1.5 and 1.8 log10 CFU/mL (p < 0.001) in milk, respectively. Interesting, the bacteriostasis efficacy of both phages exhibited more significantly at 4 °C than that at 28 °C in eggshells and milk and sausages. In sum, the purpose of our research was evaluating the counteracting effect of phage PSE-D1 and PST-H1 on the spread of Salmonella on contaminated foods products. Our results suggested that these two phage-based biocontrol treatments are promising strategies for controlling pathogenic Salmonella contaminated food.

A common cause of human food poisoning is through ingestion of pork products contaminated with Salmonella spp. Worryingly multi-drug resistant (MDR) Salmonella strains have been isolated from pigs, which motivates the need for alternative antimicrobials. In this study isolation and characterization of 21 lytic Salmonella phages is described. All 21 phages, labeled as SPFM phages were shown to efficiently infect MDR Salmonella strains isolated from United Kingdom pigs and phages SPFM1, SPFM3, SPFM10, SPFM14, SPFM15, SPFM17, and SPFM19 could lyse 100% of strains tested. The phage genome sizes range from 233 to 242 Kb, which qualifies them as jumbo phages. All SPFM phage genomes are approximately 95% similar to each other by average nucleotide identity, they encode between 258-307 coding sequences and share 188 core genes. Phylogenetic analysis shows these phages are most similar to phages of the genus Seoulvirus and to further characterize phages within the genus, genes under positive selection were identified. Several of the genes under evolutionary selection pressure were predicted to encode for proteins that interact with bacteria. We describe the phenotypic and genetic characterization of this novel Salmonella phage set. As the phages efficiently kill MDR Salmonella strains, they may offer a promising alternative to antibiotics.

Non-typhoidal Salmonella enterica infections are one of the leading causes of diarrhoeal diseases that spread to humans from animal sources such as poultry. Hence, keeping poultry farms free of Salmonella is essential for consumer safety and for a better yield of animal products. However, the emergence of antibiotic resistance due to over usage has sped up the search for alternative biocontrol methods such as the use of bacteriophages. Isolation and characterization of novel bacteriophages are key to adapt phage-based biocontrol applications. Here, we isolated and characterized Salmonella phages from samples collected at chicken farms and slaughterhouses in Kenya. The genomic characterization of these phage isolates revealed that they belong to four ICTV (International Committee on Taxonomy of Viruses) phage genera. All these phages are lytic and possibly suitable for biocontrol applications because no lysogenic genes or virulence factors were found in their genomes. Hence, we recommend further studies on these phages for their applications in Salmonella biocontrol.

Despite a wealth of knowledge on Salmonella phages worldwide, little is known about poultry-associated Salmonella phages from Thailand. Here, we isolated 108 phages from Thai poultry farms that infect Salmonellaenterica serovar Typhimurium. Phages STm101 and STm118 were identified as temperate Siphoviridae phages. Genome sequencing and analyses revealed these phages share approximately 96% nucleotide sequence similarity to phage SPN19, a member of the Chi-like virus genus. PCR amplification of the gene encoding capsid protein E of the Chi-like phage was positive for 50% of phage isolates, suggesting a predominance of this phage type among the sampled poultry farms. In addition to the flagella, two phages required the lipopolysaccharide to infect and lyse Salmonella. Furthermore, phylogenomic analysis demonstrated that phages STm101 and STm118 formed a monophyletic clade with phages isolated from Western countries, but not from closer isolated phages from Korea. However, further investigation and more phage isolates are required to investigate possible causes for this geographic distribution.

Escherichia coli and Salmonella Enteritidis are foodborne pathogens forming challenging biofilms that contribute to their virulence, antimicrobial resistance, and survival on surfaces. Interspecies interactions occur between species in mixed biofilms promoting different outcomes to each species. Here we describe the interactions between E. coli and S. Enteritidis strains, and their control using specific phages. Single-species biofilms presented more cells compared to dual-species biofilms. The spatial organization of strains, observed by confocal microscopy, revealed similar arrangements in both single- and dual-species biofilms. The EPS matrix composition, assessed by Fourier-transform infrared spectroscopy, disclosed that the spectra extracted from the different dual-species biofilms can either be a combination of both species EPS matrix components or that the EPS matrix of one species predominates. Phages damaged more the single-species biofilms than the mixed biofilms, showing also that the killing efficacy was greatly dependent on the phage growth characteristics, bacterial growth parameters, and bacterial spatial distribution in biofilms. This combination of methodologies provides new knowledge of species-species and phage-host interactions in biofilms of these two major foodborne pathogens.

Acute non-typhoidal salmonellosis (NTS) caused by a Gram-negative bacterium Salmonella enterica serovar Typhimurium (S. Tm) is one of the most common bacterial foodborne diseases worldwide. Bacteriophages (phages) can specifically target and lyse their host bacteria, including the multidrug-resistant strains, without collateral damage to other bacteria in the community. However, the therapeutic use of Salmonella phages in vivo is still poorly investigated. Salmonella phages ST-W77 and SE-W109 have previously been shown by our group to be useful for biocontrol properties. Here, we tested whether phages ST-W77 and SE-W109 can reduce Salmonella invasion into cultured human cells and confer a therapeutic benefit for acute NTS in a mammalian host. Human colonocytes, T84 cells, were treated with phages ST-W77, SE-W109, and its combination for 5 min before S. Tm infection. Gentamicin protection assays demonstrated that ST-W77 and SE-W109 significantly reduced S. Tm invasion and inflammatory response in human colonocytes. Next, streptomycin-pretreated mice were orally infected with S. Tm (108 CFU/mouse) and treated with a single or a combination of ST-W77 and SE-W109 (1010 PFU/mouse for 4 days) by oral feeding. Our data showed that phage-treated mice had lower S. Tm numbers and tissue inflammation compared to the untreated mice. Our study also revealed that ST-W77 and SE-W109 persist in the mouse gut lumen, but not in systemic sites. Together, these data suggested that Salmonella phages ST-W77 and SE-W109 could be further developed as an alternative approach for treating an acute NTS in mammalian hosts.

The Anderson phage typing scheme has been successfully used worldwide for epidemiological surveillance of Salmonella enterica serovar Typhimurium. Although the scheme is being replaced by whole genome sequence subtyping methods, it can provide a valuable model system for study of phage-host interaction. The phage typing scheme distinguishes more than 300 definitive types of Salmonella Typhimurium based on their patterns of lysis to a unique collection of 30 specific Salmonella phages. In this study, we sequenced the genomes of 28 Anderson typing phages of Salmonella Typhimurium to begin to characterize the genetic determinants that are responsible for the differences in these phage type profiles. Genomic analysis of typing phages reveals that Anderson phages can be classified into three different groups, the P22-like, ES18-like and SETP3-like clusters. Most Anderson phages are short tailed P22-like viruses (genus Lederbergvirus); but phages STMP8 and STMP18 are very closely related to the lambdoid long tailed phage ES18, and phages STMP12 and STMP13 are related to the long noncontractile tailed, virulent phage SETP3. Most of these typing phages have complex genome relationships, but interestingly, two phage pairs STMP5 and STMP16 as well as STMP12 and STMP13 differ by a single nucleotide. The former affects a P22-like protein involved in DNA passage through the periplasm during its injection, and the latter affects a gene whose function is unknown. Using the Anderson phage typing scheme would provide insights into phage biology and the development of phage therapy for the treatment of antibiotic resistant bacterial infections.

Effective phage cocktails consisting of multiple virus types are essential for successful phage therapy against pandrug-resistant pathogens, including Salmonella enterica serovar (S.) Typhimurium. Here we show that a Salmonella phage, F118P13, with non-productive infection and a lytic phage, PLL1, combined to inhibit pandrug-resistant S. Typhimurium growth and significantly limited resistance to phages in vitro. Further, intraperitoneal injection with this unique phage combination completely protected mice from Salmonella-induced death and inhibited bacterial proliferation rapidly in various organs. Furthermore, the phage combination treatment significantly attenuated the inflammatory response, restored the generation of CD4+ T cells repressed by Salmonella, and allowed macrophages and granulocytes to participate in immunophage synergy to promote bacterial clearance. Crucially, the non-productive phage F118P13 is less likely to be cleared by the immune system in vivo, thus providing an alternative to phage cocktail against bacterial infections.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

Bacterial inactivation using bacteriophages (or phages) has emerged as an effective solution for bacterial infections, but the screening methods used to evaluate the effectiveness of the phages to inactivate bacteria are not fast, reliable or precise enough. The efficiency of bacterial inactivation by phages has been evaluated by monitoring bacterial concentration either by counting colony-forming units (CFU), a laborious and time-consuming method, or by monitoring the optical density (OD), a less sensitive method. In this study, the resazurin cell viability assay was used to monitor the viability of bacteria from different genera during the inactivation by different phages, and the results were compared with the standard methods used to assess bacterial inactivation. The results showed that the resazurin colorimetric cell viability assay produces similar results to the standard method of colony-counting and giving, and also more sensitive results than the OD method. The resazurin assay can be used to quickly obtain the results of the cell viability effect profile using two different bacterial strains and several different phages at the same time, which is extremely valuable in screening studies. Moreover, this methodology is established as an effective, accurate and rapid method when compared to the ones widely used to monitor bacterial inactivation mediated by phages.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the common causes of human colitis. In the present study, two lytic phages vB_SenS-EnJE1 and vB_SenS-EnJE6 were isolated and the therapeutic effect of the combination of phages and faecal microbiota transplantation (FMT) on S. Typhimurium-induced mouse colitis was investigated. The characteristics and genome analysis indicated that they are suitable phages for phage therapy. Results showed that vB_SenS-EnJE1 lysis 41/54 Salmonella strains of serotype O4, and vB_SenS-EnJE6 lysis 46/54 Salmonella strains of serotypes O4 and O9. Severe inflammatory symptoms and disruption of the intestinal barrier were observed in S. Typhimurium -induced colitis. Interestingly, compared with a single phage cocktail (Pc) or single FMT, the combination of Pc and FMT (PcFMT) completely removed S. Typhimurium after 72 h of treatment, and significantly improved pathological damage and restored the intestinal barrier. Furthermore, PcFMT effectively restored the intestinal microbial diversity, especially for Firmicutes/Bacteroidetes [predominantly bacterial phyla responsible for the production of short-chain fatty acids (SCFA)]. Additionally, we found that PcFMT treatment significantly increased the levels of SCFA. All these data indicated that the combination of phages and FMT possesses excellent therapeutic effects on S. Typhimurium -induced intestinal microbiota disorder diseases. Pc and FMT played roles in "eliminating pathogens" and "strengthening vital qi," respectively. This study provides a new idea for the treatment of intestinal microbiota disorder diseases caused by specific bacterial infections.

Forty strains of Salmonella enterica (S. enterica) subspecies salamae (II), arizonae (IIIa), diarizonae (IIIb), and houtenae (IV) were isolated from human or environmental samples and tested for bacteriophage production. Production of bacteriophages was observed in 15 S. enterica strains (37.5%) belonging to either the subspecies salamae (8 strains) or diarizonae (7 strains). Activity of phages was tested against 52 pathogenic S. enterica subsp. enterica isolates and showed that phages produced by subsp. salamae had broader activity against pathogenic salmonellae compared to phages from the subsp. diarizonae. All 15 phages were analyzed using PCR amplification of phage-specific regions and 9 different amplification profiles were identified. Five phages (SEN1, SEN4, SEN5, SEN22, and SEN34) were completely sequenced and classified as temperate phages. Phages SEN4 and SEN5 were genetically identical, thus representing a single phage type (i.e. SEN4/5). SEN1 and SEN4/5 fit into the group of P2-like phages, while the SEN22 phage showed sequence relatedness to P22-like phages. Interestingly, while phage SEN34 was genetically distantly related to Lambda-like phages (Siphoviridae), it had the morphology of the Myoviridae family. Based on sequence analysis and electron microscopy, phages SEN1 and SEN4/5 were members of the Myoviridae family and phage SEN22 belonged to the Podoviridae family.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Salmonella infections continue to be one of the essential public health issues threatening millions of people. With the increasing occurrence of resistance against conventionally used antibiotics, the search for alternatives has become crucial. In this study, we aimed to isolate, characterize, and evaluate two lytic bacteriophages against clinically isolated multidrug-resistant (MDR) Salmonella serovars.

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

Bacteriophages display remarkable genetic diversity and host specificity. In this study, we explore phages infecting bacterial strains of the Enterobacteriaceae family because of their ability to infect related but distinct hosts. We isolated and characterized two novel virulent phages, SH6 and SH7, using a strain of Shigella flexneri as host bacterium. Morphological and genomic analyses revealed that phage SH6 belongs to the T1virus genus of the Siphoviridae family. Conversely, phage SH7 was classified in the T4virus genus of the Myoviridae family. Phage SH6 had a short latent period of 16 min and a burst size of 103 ± 16 PFU/infected cell while the phage SH7 latent period was 23 min with a much lower burst size of 26 ± 5 PFU/infected cell. Moreover, phage SH6 was sensitive to acidic conditions (pH < 5) while phage SH7 was stable from pH 3 to 11 for 1 hour. Of the 35 bacterial strains tested, SH6 infected its S. flexneri host strain and 8 strains of E. coli. Phage SH7 lysed additionally strains of E. coli O157:H7, Salmonella Paratyphi, and Shigella dysenteriae. The broader host ranges of these two phages as well as their microbiological properties suggest that they may be useful for controlling bacterial populations.

Citrobacter freundii causes opportunistic infections in humans and animals, which are becoming difficult to treat due to increased antibiotic resistance. The aim of this study was to explore phages as potential antimicrobial agents against this opportunistic pathogen. We isolated and characterized five new virulent phages, SH1, SH2, SH3, SH4, and SH5 from sewage samples in Tunisia. Morphological and genomic analyses revealed that the five C. freundii phages belong to the Caudovirales order, Podoviridae family, and Autographivirinae subfamily. Their linear double-stranded DNA genomes range from 39,158 to 39,832 bp and are terminally redundant with direct repeats between 183 and 242 bp. The five genomes share the same organization as coliphage T7. Based on genomic comparisons and on the phylogeny of the DNA polymerases, we assigned the five phages to the T7virus genus but separated them into two different groups. Phages SH1 and SH2 are very similar to previously characterized phages phiYeO3-12 and phiSG-JL2, infecting, respectively, Yersinia enterocolitica and Salmonella enterica, as well as sharing more than 80% identity with most genes of coliphage T7. Phages SH3, SH4, and SH5 are very similar to phages K1F and Dev2, infecting, respectively, Escherichia coli and Cronobacter turicensis. Several structural proteins of phages SH1, SH3, and SH4 were detected by mass spectrometry. The five phages were also stable from pH 5 to 10. No genes coding for known virulence factors or integrases were found, suggesting that the five isolated phages could be good candidates for therapeutic applications to prevent or treat C. freundii infections. In addition, this study increases our knowledge about the evolutionary relationships within the T7virus genus.

Coliphages T7M and T3, Yersinia phage ϕYeO3-12, and Salmonella phage ϕSG-JL2 share high homology in genomic sequences. Simple sequence repeats (SSRs) are found in their genomes and variations of SSRs among these phages are observed. Analyses on regions of sequences in T7M and T3 genomes that are likely derived from phage recombination, as well as the counterparts in ϕYeO3-12 and ϕSG-JL2, have been discussed by Lin in "Simple sequence repeat variations expedite phage divergence: mechanisms of indels and gene mutations" [1]. These regions are referred to as recombinant regions. The focus here is on SSRs in the whole genome and regions of sequences outside the recombinant regions, referred to as non-recombinant regions. This article provides SSR counts, relative abundance, relative density, and GC contents in the complete genome and non-recombinant regions of these phages. SSR period sizes and motifs in the non-recombinant regions of phage genomes are plotted. Genomic sequence changes between T7M and T3 due to insertions, deletions, and substitutions are also illustrated. SSRs and nearby sequences of T7M in the non-recombinant regions are compared to the sequences of ϕYeO3-12 and ϕSG-JL2 in the corresponding positions. The sequence variations of SSRs due to vertical evolution are classified into four categories and tabulated: (1) insertion/deletion of SSR units, (2) expansion/contraction of SSRs without alteration of genome length, (3) changes of repeat motifs, and (4) generation/loss of repeats.

Urinary tract infections (UTIs) are the second most frequent bacterial infections worldwide, with Escherichia coli being the main causative agent. The increase of antibiotic-resistance determinants among isolates from clinical samples, including UTIs, makes the development of novel therapeutic strategies a necessity. In this context, the use of bacteriophages as a therapeutic alternative has been proposed, due to their ability to efficiently kill bacteria. In this work, we isolated and characterized three novel bacteriophages, microbes laboratory phage 1 (MLP1), MLP2, and MLP3, belonging to the Chaseviridae, Myoviridae, and Podoviridae families, respectively. These phages efficiently infect and kill laboratory reference strains and multidrug-resistant clinical E. coli isolates from patients with diagnosed UTIs. Interestingly, these phages are also able to infect intestinal pathogenic Escherichia coli strains, such as enteroaggregative E. coli and diffusely adherent E. coli. Our data show that the MLP phages recognize different regions of the lipopolysaccharide (LPS) molecule, an important virulence factor in bacteria that is also highly variable among different E. coli strains. Altogether, our results suggest that these phages may represent an interesting alternative for the treatment of antibiotic-resistant E. coli. IMPORTANCE Urinary tract infections affect approximately 150 million people annually. The current antibiotic resistance crisis demands the development of novel therapeutic alternatives. Our results show that three novel phages, MLP1, MLP2, and MLP3 are able to infect both laboratory and multidrug-resistant clinical isolates of Escherichia coli. Since these phages (i) efficiently kill antibiotic-resistant clinical isolates of uropathogenic Escherichia coli (UPEC), (ii) recognize different portions of the LPS molecule, and (iii) are able to efficiently infect intestinal pathogenic Escherichia coli hosts, we believe that these novel phages are good candidates to be used as a therapeutic alternative to treat antibiotic-resistant E. coli strains generating urinary tract and/or intestinal infections.