Trade-offs between receptor modification and fitness drive host-bacteriophage co-evolution leading to phage extinction or co-existence.

Parasite-host co-evolution results in population extinction or co-existence, yet the factors driving these distinct outcomes remain elusive. In this study, Salmonella strains were individually co-evolved with the lytic phage SF1 for 30 days, resulting in phage extinction or co-existence. We conducted a systematic investigation into the phenotypic and genetic dynamics of evolved host cells and phages to elucidate the evolutionary mechanisms. Throughout co-evolution, host cells displayed diverse phage resistance patterns: sensitivity, partial resistance, and complete resistance, to wild-type phage. Moreover, phage resistance strength showed a robust linear correlation with phage adsorption, suggesting that surface modification-mediated phage attachment predominates as the resistance mechanism in evolved bacterial populations. Additionally, bacterial isolates eliminating phages exhibited higher mutation rates and lower fitness costs in developing resistance compared to those leading to co-existence. Phage resistance genes were classified into two categories: key mutations, characterized by nonsense/frameshift mutations in rfaH-regulated rfb genes, leading to the removal of the receptor O-antigen; and secondary mutations, which involve less critical modifications, such as fimbrial synthesis and tRNA modification. The accumulation of secondary mutations resulted in partial and complete resistance, which could be overcome by evolved phages, whereas key mutations conferred undefeatable complete resistance by deleting receptors. In conclusion, higher key mutation frequencies with lower fitness costs promised strong resistance and eventual phage extinction, whereas deficiencies in fitness cost, mutation rate, and key mutation led to co-existence. Our findings reveal the distinct population dynamics and evolutionary trade-offs of phage resistance during co-evolution, thereby deepening our understanding of microbial interactions.

Biological and genomic characterization of a polyvalent phage PSH-1 against multidrug-resistant Salmonella Enteritidis.

BACKGROUND: Bacteriophage has been renewed attention as a new antibacterial agent due to the limitations of antibiotic treatment. Bacteriophages are generally thought to be highly host specific and even strain specific, but a small number of polyvalent bacteriophages have been found to infect bacteria of different genera. RESULTS: In this study, a virulent lytic bacteriophage (named Salmonella phage PSH-1) of Salmonella Enteritidis was isolated from the sewage samples of a large-scale pig farm, PSH-1 demonstrated lytic activity against four multidrug-resistant Salmonella Enteritidis isolates and Escherichia coli, and then its biological characteristics, genome and bacteriostatic ability were investigated. The results showed that the initial titer of PSH-1 was 1.15 × 1010 PFU/mL and the optimal multiplicity of infection (MOI) was 0.01, PSH-1 has stable activity in the range of pH 3.0-11.0. One-step growth curve showed that its latent period was 20 min, burst time was 80 min, and the burst was 495 particles. The whole-genome sequencing results revealed phage PSH-1 had a linear dsDNA with 48,466 bp length. The G/C content was 45.33%. Non-coding RNA genes and virulence factors were not found. Eighty- five open reading frames (ORFs) were identified after online annotation. By tests, the use of phage could succeed in controlling the artificial Salmonella contamination in milk at a range of temperatures. CONCLUSIONS: This study reports a novel Salmonella Enteritidis phage PSH-1, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of PSH-1 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling of salmonellosis.

Evaluation of lyophilized bacteriophage cocktail efficiency against multidrug-resistant Salmonella in broiler chickens.

Currently, phage biocontrol is increasingly used as a green and natural technology for treating Salmonella and other infections, but phages exhibit instability and activity loss during storage. Therefore, in this study, the effects of lyophilization on the activity and stability of phage cocktails for the control of multidrug-resistant Salmonella in broiler chickens were determined. Eight serotypes of Salmonella were isolated and identified from broiler chicken farms, and bacteriophages against multidrug-resistant Salmonella enterica subsp. enterica serovar Kentucky, Salmonella enterica subsp. enterica serovar Typhimrium and Salmonella enterica subsp. enterica serovar Enteritidis were isolated. The bacteriophage cocktail was prepared and lyophilized, and it was subjected to in vitro and in vivo examinations. A reconstituted lyophilized bacteriophage cocktail was used for the oral treatment of chicks before and after challenge with multidrug-resistant S. Kentucky. The colonization of cecum by S. Kentucky was detected by using real-time PCR, and the serum levels of IgM, IgA and IL-4 and pathological changes in the different groups were detected. Three Caudovirales phages families were identified including Autographiviridae, Straboviridae and Drexlerviridae against multidrug-resistant S. Kentucky, S. Typhimrium and S. Enteritidis. The groups treated with the bacteriophage cocktail showed no clinical signs, no postmortem lesions, and a mortality rate of 0%, which improved the growth performance parameters. Additionally, the estimated serum levels of IgM, IgA and IL-4 were significantly greater in the bacteriophage cocktail-treated groups. Lyophilization effectively preserves the long-term storage stability of phages. Therefore, lyophilized bacteriophage cocktail therapy is a valuable approach for controlling multidrug-resistant Salmonella infections in broiler chickens.

Isolation and Characterization of Salmonella Bacteriophages as Potential Agents for Phage Therapy of Antibiotic-Resistant Intestinal Infections.

Two bacteriophages specifically active against to pathogenic strains of the Salmonella genus were isolated. The morphology of phage colonies (size, transparency, and shape of the plaque edge, and halo) and the spectrum of their lytic activity and interaction with microbial cells (adsorption rate, duration of the latency, and reproductive efficiency) were examined. Using genome-wide sequencing, we determined the taxonomic position of bacteriophages and verified the absence of unwanted genes encoding toxins, adhesins, and invasins, as well as pathogenicity islands responsible for antibiotic resistance. In addition, phage stability under different physical conditions and their productivity were studied.

DNA Packaging Specificity in the λ-Like Phages: Gifsy-1.

DNA viruses recognize viral DNA and package it into virions. Specific recognition is needed to distinguish viral DNA from host cell DNA. The λ-like Escherichia coli phages are interesting and good models to examine genome packaging by large DNA viruses. Gifsy-1 is a λ-like Salmonella phage. Gifsy-1's DNA packaging specificity was compared with those of closely related phages λ, 21, and N15. In vivo packaging studies showed that a Gifsy-1-specific phage packaged λ DNA at ca. 50% efficiency and λ packages Gifsy-1-specific DNA at ~30% efficiency. The results indicate that Gifsy-1 and λ share the same DNA packaging specificity. N15 is also shown to package Gifsy-1 DNA. Phage 21 fails to package λ, N15, and Gifsy-1-specific DNAs; the efficiencies are 0.01%, 0.01%, and 1%, respectively. A known incompatibility between the 21 helix-turn-helix motif and cosBλ is proposed to account for the inability of 21 to package Gifsy-1 DNA. A model is proposed to explain the 100-fold difference in packaging efficiency between λ and Gifsy-1-specific DNAs by phage 21. Database sequences of enteric prophages indicate that phages with Gifsy-1's DNA packaging determinants are confined to Salmonella species. Similarly, prophages with λ DNA packaging specificity are rarely found in Salmonella. It is proposed that λ and Gifsy-1 have diverged from a common ancestor phage, and that the differences may reflect adaptation of their packaging systems to host cell differences.

Isolation and characterization of duck sewage source Salmonella phage P6 and antibacterial activity for recombinant endolysin LysP6.

Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.

Effect of Phage Spray on Hatchability and Chick Quality of Eggs Contaminated with Salmonella Typhimurium.

Salmonella Typhimurium (S. Typhimurium) contamination poses a significant challenge to breeder egg hatchability and chick health, necessitating the exploration of alternative disinfection methods. This study investigates the potential of phage vB_SPuM_SP02 (SP02) as a novel disinfectant for breeder eggs contaminated with S. Typhimurium SM022. Phage SP02 was isolated from poultry farm effluent and characterized for morphology, biological properties, and genome properties. Experimental groups of specific pathogen-free (SPF) eggs were treated with Salmonella and phage SP02, and efficacy was assessed through hatching rates, chick survival, weight, Salmonella load, immune organ indices, and intestinal flora. Phage treatment effectively eradicated Salmonella contamination on eggshells within 12 h, resulting in increased hatching and survival rates compared to controls. Furthermore, phage treatment mitigated weight loss and tissue Salmonella load in chicks without causing immune organ damage while reducing Salmonella spp. abundance in the intestinal tract. This study demonstrates the potential of phage SP02 as an eco-friendly and efficient disinfectant for S. Typhimurium-contaminated breeder eggs, offering promising prospects for practical application in poultry production.

Characterization and genome analysis of a broad host range lytic phage vB_SenS_TUMS_E19 against Salmonella enterica and its efficiency evaluation in the liquid egg.

Salmonella enterica serovars are zoonotic bacterial that cause foodborne enteritis. Due to bacteria's antibiotic resistance, using bacteriophages for biocontrol and treatment is a new therapeutic approach. In this study, we isolated, characterized, and analyzed the genome of vB_SenS_TUMS_E19 (E19), a broad host range Salmonella bacteriophage, and evaluated the influence of E19 on liquid eggs infected with Salmonella enterica serovar Enteritidis. Transmission electron microscopy showed that the isolated bacteriophage had a siphovirus morphotype. E19 showed rapid adsorption (92% in 5 min), a short latent period (18 min), a large burst size (156 PFU per cell), and a broad host range against different Salmonella enterica serovars. Whole-genome sequencing analysis indicated that the isolated phage had a 42 813 bp long genome with 49.8% G + C content. Neither tRNA genes nor those associated with antibiotic resistance, virulence factors, or lysogenic formation were detected in the genome. The efficacy of E19 was evaluated in liquid eggs inoculated with S. Enteritidis at 4 and 25 °C, and results showed that it could effectively eradicate S. Enteritidis in just 30 min and prevented its growth up to 72 h. Our findings indicate that E19 can be an alternative to a preservative to control Salmonella in food samples and help prevent and treat salmonellosis.

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.

Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

Bacteriophage Removal from Infected Salmonella Cultures.

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

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.

The effectiveness of endolysin ENDO-1252 from Salmonella bacteriophage-1252 against nontyphoidal Salmonella enterica.

Salmonella enterica (S. enterica) is the most common food and waterborne pathogen worldwide. The growing trend of antibiotic-resistant S. enterica poses severe healthcare threats. As an alternative antimicrobial agent, bacteriophage-encoded endolysins (endolysins) are a potential agent in controlling S. enterica infection. Endolysins are enzymes that particularly target the peptidoglycan layer of bacterial cells, leading to their rupture and destruction. However, the application of endolysins against Gram-negative bacteria is limited due to the presence of the outer membrane in the cell wall, which hinders the permeation of externally applied endolysins. This study aimed the prokaryotic expression system to produce the recombinant endolysin ENDO-1252, encoded by the Salmonella bacteriophage-1252 associated with S. Enteritidis. Subsequently, ENDO-1252 had strong lytic activity not only against S. Enteritidis but also against S. Typhimurium. In addition, ENDO-1252 showed optimal thermostability and lytic activity at 25°C with a pH of 7.0. In combination with 0.1 mM EDTA, the effect of 120 µg of ENDO-1252 for 6 hours exhibited the highest lytic activity, resulting in a reduction of 1.15 log or 92.87% on S. Enteritidis. These findings suggest that ENDO-1252 can be used as a potential and innovative antibacterial agent for controlling the growth of S. Enteritidis.

Tactical terminase: How a Salmonella prophage navigates oxidative stress.

Stress-induced prophages commonly "jump ship" by inducing lysis via the host SOS response. In a recent work, Uppalapati et al. reports an alternate, stress-selective strategy. Instead of promoting lysis, the Salmonella Gifsy-1 prophage arrests growth specifically when the SOS response coincides with oxidative stress.

Characterization of two virulent Salmonella phages and transient application in egg, meat and lettuce safety.

Salmonella, a prominent foodborne pathogen, has posed enduring challenges to the advancement of food safety and global public health. The escalating concern over antibiotic misuse, resulting in the excessive presence of drug residues in animal-derived food products, necessitates urgent exploration of alternative strategies for Salmonella control. Bacteriophages emerge as promising green biocontrol agents against pathogenic bacteria. This study delineates the identification of two novel virulent Salmonella phages, namely phage vB_SalS_ABTNLsp11241 (referred to as sp11241) and phage 8-19 (referred to as 8-19). Both phages exhibited efficient infectivity against Salmonella enterica serotype Enteritidis (SE). Furthermore, this study evaluated the effectiveness of two phages to control SE in three different foods (whole chicken eggs, raw chicken meat, and lettuce) at different MOIs (1, 100, and 10000) at 4°C. It's worth noting that sp11241 and 8-19 achieved complete elimination of SE on eggs after 3 h and 6 h at MOI = 100, and after 2 h and 5 h at MOI = 10000, respectively. After 12 h of treatment with sp11241, a maximum reduction of 3.17 log10 CFU/mL in SE was achieved on raw chicken meat, and a maximum reduction of 3.00 log10 CFU/mL was achieved on lettuce. Phage 8-19 has the same effect on lettuce as sp11241, but is slightly less effective than sp11241 on chicken meat (a maximum 2.69 log10 CFU/mL reduction). In conclusion, sp11241 and 8-19 exhibit considerable potential for controlling Salmonella contamination in food at a low temperature and represent viable candidates as green antibacterial agents for food applications.

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.

Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand-binding site of receptor-binding protein.

Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. A total of 97 bacteriophages, isolated from cattle farms (n = 48), poultry farms (n = 37), and wastewater (n = 5) samples in Türkiye, were subjected to host-range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. A total of 10 phages with the widest host range underwent further analysis, revealing seven unique genomes (32-243 kb), including a jumbophage (>200 kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining open reading frames (ORFs) of endolysin enzymes revealed surprising findings: five of seven unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor-binding protein sequences, with similar structures but distinct ligand-binding sites. These findings emphasize the importance of ligand-binding sites of receptor-binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.

Presence of phage-plasmids in multiple serovars of Salmonella enterica.

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a bla CTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.

Characterization of two novel Salmonella phages having biocontrol potential against Salmonella spp. in gastrointestinal conditions.

Salmonella is a primary enteric pathogen related to the contamination of poultry and other food products in numerous foodborne outbreaks. The continuous emergence of multidrug-resistant bacteria has become a serious issue due to the overuse of antibiotics. Hence, lytic phages are considered alternative biocontrol agents against these bacterial superbugs. Here, two Salmonella phages-S4lw and D5lw-were subjected to genomic and biological characterization and further encapsulated to improve the stability under acidic conditions mimicking gastrointestinal conditions. The two lytic phages, S4lw and D5lw, taxonomically belong to new species under the Guernseyvirinae and Ackermannviridae families, respectively. Each phage showed antimicrobial activities against diverse Salmonella spp., such as S. Enteritidis and S. Typhimurium, achieving 1.7-3.4 log reduction after 2-6 h of treatment. The phage cocktail at a multiplicity of infection (MOI) of 100 or 1000 completely inhibited these Salmonella strains for at least 14 h at 25 °C. Additionally, the bead-encapsulated phage cocktail could withstand low pH and different simulated gut environments for at least 1 h. Overall, the newly isolated phages can potentially mitigate Salmonella spp. under the gastrointestinal environments through encapsulation and may be further applied via oral administration to resolve common antimicrobial resistance issues in the poultry production chain.

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.