Dual pathogenicity island transfer by piggybacking lateral transduction.

Lateral transduction (LT) is the process by which temperate phages mobilize large sections of bacterial genomes. Despite its importance, LT has only been observed during prophage induction. Here, we report that superantigen-carrying staphylococcal pathogenicity islands (SaPIs) employ a related but more versatile and complex mechanism of gene transfer to drive chromosomal hypermobility while self-transferring with additional virulence genes from the host. We found that after phage infection or prophage induction, activated SaPIs form concatamers in the bacterial chromosome by switching between parallel genomic tracks in replication bubbles. This dynamic life cycle enables SaPIbov1 to piggyback its LT of staphylococcal pathogenicity island vSaα, which encodes an array of genes involved in host-pathogen interactions, allowing both islands to be mobilized intact and transferred in a single infective particle. Our findings highlight previously unknown roles of pathogenicity islands in bacterial virulence and show that their evolutionary impact extends beyond the genes they carry.

Discovery of phage determinants that confer sensitivity to bacterial immune systems.

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.

Structural Basis for the Action of an All-Purpose Transcription Anti-termination Factor.

Bacteriophage λN protein, a model anti-termination factor, binds nascent RNA and host Nus factors, rendering RNA polymerase resistant to all pause and termination signals. A 3.7-Å-resolution cryo-electron microscopy structure and structure-informed functional analyses reveal a multi-pronged strategy by which the intrinsically unstructured λN directly modifies RNA polymerase interactions with the nucleic acids and subverts essential functions of NusA, NusE, and NusG to reprogram the transcriptional apparatus. λN repositions NusA and remodels the ß subunit flap tip, which likely precludes folding of pause or termination RNA hairpins in the exit tunnel and disrupts termination-supporting interactions of the α subunit C-terminal domains. λN invades and traverses the RNA polymerase hybrid cavity, likely stabilizing the hybrid and impeding pause- or termination-related conformational changes of polymerase. λN also lines upstream DNA, seemingly reinforcing anti-backtracking and anti-swiveling by NusG. Moreover, λN-repositioned NusA and NusE sequester the NusG C-terminal domain, counteracting ρ-dependent termination. Other anti-terminators likely utilize similar mechanisms to enable processive transcription.

Evaluation of the impact of repeated intravenous phage doses on mammalian host-phage interactions.

Phage therapy has shown great promise for the treatment of multidrug-resistant bacterial infections. However, the lack of a thorough and organized understanding of phage-body interactions has limited its clinical application. Here, we administered different purified phages (Salmonella phage SE_SZW1, Acinetobacter phage AB_SZ6, and Pseudomonas phage PA_LZ7) intravenously to healthy animals (rats and monkeys) to evaluate the phage-induced host responses and phage pharmacokinetics with different intravenous (IV) doses in healthy animals. The plasma and the organs were sampled after different IV doses to determine the phage biodistribution, phage-induced cytokines, and antibodies. The potential side effects of phages on animals were assessed. A non-compartment model revealed that the plasma phage titer gradually decreased over time following a single dose. Repeated doses resulted in a 2-3 Log10 decline of the plasma phage titer at 5 min compared to the first dose, regardless of the type of phage administered in rats. Host innate immune responses were activated including splenic enlargement following repeated doses. Phage-specific neutralization antibodies in animals receiving phages were detected. Similar results were obtained from monkeys. In conclusion, the mammalian bodies were well-tolerant to the administered phages. The animal responses to the phages and the phage biodistribution profiles could have a significant impact on the efficacy of phage therapy.IMPORTANCEPhage therapy has demonstrated potential in addressing multidrug-resistant bacterial infections. However, an insufficient understanding of phage-host interactions has impeded its broader clinical application. In our study, specific phages were administered intravenously (IV) to both rats and monkeys to elucidate phage-host interactions and evaluate phage pharmacokinetics (PK). Results revealed that with successive IV administrations, there was a decrease in plasma phage concentrations. Concurrently, these administrations elicited both innate and adaptive immune responses in the subjects. Notably, the observed immune responses and PK profiles exhibited variation contingent upon the phage type and the mammalian host. Despite these variations, the tested mammals exhibited a favorable tolerance to the IV-administered phages. This underscores the significance of comprehending these interactions for the optimization of phage therapy outcomes.

Acinetobacter baumannii satellite phage Aci01-2-Phanie depends on a helper myophage for its multiplication.

We report the discovery of a satellite-helper phage system with a novel type of dependence on a tail donor. The Acinetobacter baumannii satellite podovirus Aci01-2-Phanie (short name Phanie) uses a phage phi29-like DNA replication and packaging mode. Its linear 11,885 bp dsDNA genome bears 171 bp inverted terminal repeats (ITR). Phanie is related to phage DU-PP-III from Pectobacterium and to members of the Astrithrvirus from Salmonella enterica. Together, they form a new clade of phages with 27% to 30% identity over the whole genome. Detailed 3D protein structure prediction and mass spectrometry analyses demonstrate that Phanie encodes its capsid structural genes and genes necessary to form a short tail. However, our study reveals that Phanie virions are non-infectious unless they associate with the contractile tail of an unrelated phage, Aci01-1, to produce chimeric myoviruses. Following the coinfection of Phanie with myovirus Aci01-1, hybrid viral particles composed of Phanie capsids and Aci01-1 contractile tails are assembled together with Phanie and Aci01-1 particles.IMPORTANCEThere are few reported cases of satellite-helper phage interactions but many more may be yet undiscovered. Here we describe a new mode of satellite phage dependence on a helper phage. Phanie, like phage phi29, replicates its linear dsDNA by a protein primed-mechanism and protects it inside podovirus-like particles. However, these particles are defective, requiring the acquisition of the tail from a myovirus helper for production of infectious virions. The formation of chimeras between a phi29-like podovirus and a helper contractile tail reveals an unexpected association between very different bacterial viruses.

PhaTYP: predicting the lifestyle for bacteriophages using BERT.

Bacteriophages (or phages), which infect bacteria, have two distinct lifestyles: virulent and temperate. Predicting the lifestyle of phages helps decipher their interactions with their bacterial hosts, aiding phages' applications in fields such as phage therapy. Because experimental methods for annotating the lifestyle of phages cannot keep pace with the fast accumulation of sequenced phages, computational method for predicting phages' lifestyles has become an attractive alternative. Despite some promising results, computational lifestyle prediction remains difficult because of the limited known annotations and the sheer amount of sequenced phage contigs assembled from metagenomic data. In particular, most of the existing tools cannot precisely predict phages' lifestyles for short contigs. In this work, we develop PhaTYP (Phage TYPe prediction tool) to improve the accuracy of lifestyle prediction on short contigs. We design two different training tasks, self-supervised and fine-tuning tasks, to overcome lifestyle prediction difficulties. We rigorously tested and compared PhaTYP with four state-of-the-art methods: DeePhage, PHACTS, PhagePred and BACPHLIP. The experimental results show that PhaTYP outperforms all these methods and achieves more stable performance on short contigs. In addition, we demonstrated the utility of PhaTYP for analyzing the phage lifestyle on human neonates' gut data. This application shows that PhaTYP is a useful means for studying phages in metagenomic data and helps extend our understanding of microbial communities.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences.

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

My host's enemy is my enemy: plasmids carrying CRISPR-Cas as a defence against phages.

Bacteria are infected by mobile genetic elements like plasmids and virulent phages, and those infections significantly impact bacterial ecology and evolution. Recent discoveries reveal that some plasmids carry anti-phage immune systems like CRISPR-Cas, suggesting that plasmids may participate in the coevolutionary arms race between virulent phages and bacteria. Intuitively, this seems reasonable as virulent phages kill the plasmid's obligate host. However, the efficiency of CRISPR-Cas systems carried by plasmids can be expected to be lower than those carried by the chromosome due to continuous segregation loss, creating susceptible cells for phage amplification. To evaluate the anti-phage protection efficiency of CRISPR-Cas on plasmids, we develop a stochastic model describing the dynamics of a virulent phage infection against which a conjugative plasmid defends using CRISPR-Cas. We show that CRISPR-Cas on plasmids provides robust protection, except in limited parameter sets. In these cases, high segregation loss favours phage outbreaks by generating a population of defenceless cells on which the phage can evolve and escape CRISPR-Cas immunity. We show that the phage's ability to exploit segregation loss depends strongly on the evolvability of both CRISPR-Cas and the phage itself.

Diversity and phage sensitivity to phages of porcine enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diverse and poorly characterized E. coli pathotype that causes diarrhea in humans and animals. Phages have been proposed for the veterinary biocontrol of ETEC, but effective solutions require understanding of porcine ETEC diversity that affects phage infection. Here, we sequenced and analyzed the genomes of the PHAGEBio ETEC collection, gathering 79 diverse ETEC strains isolated from European pigs with post-weaning diarrhea (PWD). We identified the virulence factors characterizing the pathotype and several antibiotic resistance genes on plasmids, while phage resistance genes and other virulence factors were mostly chromosome encoded. We experienced that ETEC strains were highly resistant to Enterobacteriaceae phage infection. It was only by enrichment of numerous diverse samples with different media and conditions, using the 41 ETEC strains of our collection as hosts, that we could isolate two lytic phages that could infect a large part of our diverse ETEC collection: vB_EcoP_ETEP21B and vB_EcoS_ETEP102. Based on genome and host range analyses, we discussed the infection strategies of the two phages and identified components of lipopolysaccharides ( LPS) as receptors for the two phages. Our detailed computational structural analysis highlights several loops and pockets in the tail fibers that may allow recognition and binding of ETEC strains, also in the presence of O-antigens. Despite the importance of receptor recognition, the diversity of the ETEC strains remains a significant challenge for isolating ETEC phages and developing sustainable phage-based products to address ETEC-induced PWD.IMPORTANCEEnterotoxigenic Escherichia coli (ETEC)-induced post-weaning diarrhea is a severe disease in piglets that leads to weight loss and potentially death, with high economic and animal welfare costs worldwide. Phage-based approaches have been proposed, but available data are insufficient to ensure efficacy. Genome analysis of an extensive collection of ETEC strains revealed that phage defense mechanisms were mostly chromosome encoded, suggesting a lower chance of spread and selection by phage exposure. The difficulty in isolating lytic phages and the molecular and structural analyses of two ETEC phages point toward a multifactorial resistance of ETEC to phage infection and the importance of extensive phage screenings specifically against clinically relevant strains. The PHAGEBio ETEC collection and these two phages are valuable tools for the scientific community to expand our knowledge on the most studied, but still enigmatic, bacterial species-E. coli.

Phage therapy as a glimmer of hope in the fight against the recurrence or emergence of surgical site bacterial infections.

PURPOSE: Over the last decade, surgery rates have risen alarmingly, and surgical-site infections are expanding these concerns. In spite of advances in infection control practices, surgical infections continue to be a significant cause of death, prolonged hospitalization, and morbidity. As well as the presence of bacterial infections and their antibiotic resistance, biofilm formation is one of the challenges in the treatment of surgical wounds. METHODS: This review article was based on published studies on inpatients and laboratory animals receiving phage therapy for surgical wounds, phage therapy for tissue and bone infections treated with surgery to prevent recurrence, antibiotic-resistant wound infections treated with phage therapy, and biofilm-involved surgical wounds treated with phage therapy which were searched without date restrictions. RESULTS: It has been shown in this review article that phage therapy can be used to treat surgical-site infections in patients and animals, eliminate biofilms at the surgical site, prevent infection recurrence in wounds that have been operated on, and eradicate antibiotic-resistant infections in surgical wounds, including multi-drug resistance (MDR), extensively drug resistance (XDR), and pan-drug resistance (PDR). A cocktail of phages and antibiotics can also reduce surgical-site infections more effectively than phages alone. CONCLUSION: In light of these encouraging results, clinical trials and research with phages will continue in the near future to treat surgical-site infections, biofilm removal, and antibiotic-resistant wounds, all of which could be used to prescribe phages as an alternative to antibiotics.

Two novel phages, Klebsiella phage GADU21 and Escherichia phage GADU22, from the urine samples of patients with urinary tract infection.

Phages are found in a wide variety of places where bacteria exist including body fluids. The aim of the present study was to isolate phages from the urine samples of patients with urinary tract infection. The 10 urine samples were cultured to isolate bacteria and also used as phage sources against the isolated bacteria. From 10 urine samples with positive cultures, 3 phages were isolated (33%) and two of them were further studied. The Klebsiella phage GADU21 and Escherichia phage GADU22 phages infected Klebsiella pneumonia and Escherichia coli, respectively. Among the tested 14 species for host range analysis, the Klebsiella phage GADU21 was able to infect two species which are Klebsiella pneumonia and Proteus mirabilis, and Escherichia phage GADU22 was able to infect four species which are Shigella flexneri, Shigella sonnei and Escherichia coli. Among different isolates of the indicator bacteria for each phage, GADU21 infected half of the tested 20 Klebsiella pneumonia isolates while GADU22 infected 85% of the tested 20 E. coli isolates. The genome sizes and GC ratios were 75,968 bp and 44.4%, and 168,023 bp and 35.3% for GADU21 and GADU22, respectively. GADU21 and GADU22 were both lytic and had no antibiotic resistance and virulence genes. GADU21 was homologue with Klebsiella phage vB_KpP_FBKp27 but only 88% of the genome was covered by this phage. The non-covered parts of the GADU21 genome included genes for tail-fiber-proteins and HNH-endonuclease. GADU22 had 94.8% homology with Escherichia phage vB_Eco_OMNI12 and had genes for immunity proteins. Phylogenetic analysis showed GADU21 and GADU22 were members of Schitoviridae family and Efbeekayvirus genus and Straboviridae family and Tevenvirinae genus, respectively. VIRIDIC analysis classified these phages in new species clusters. Our study demonstrated the possibility to use infected body fluids as phage sources to isolate novel phages. GADU21 is the first reported Klebsiella phage isolated from human body fluid. The absence of virulence and antibiotic resistance genes in their genomes makes the phages a potential therapeutic tool against infections.

Viral Communities Contribute More to the Lysis of Antibiotic-Resistant Bacteria than the Transduction of Antibiotic Resistance Genes in Anaerobic Digestion Revealed by Metagenomics.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus-host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Bacteriophages as a potential substitute for antibiotics: A comprehensive review.

Over the years, the administration of antibiotics for the purpose of addressing bacterial infections has become increasingly challenging due to the increased prevalence of antimicrobial resistance exhibited by various strains of bacteria. Multidrug-resistant (MDR) bacterial species are rising due to the unavailability of novel antibiotics, leading to higher mortality rates. With these conditions, there is a need for alternatives in which phage therapy has made promising results. Phage-derived endolysins, phage cocktails, and bioengineered phages are effective and have antimicrobial properties against MDR and extensively drug-resistant strains. Despite these, it has been observed that phages can give antimicrobial activity to more than one bacterial species. Thus, phage cocktail against resistant strains provides broad spectrum treatment and magnitude of effectivity, which is many folds higher than antibiotics. Many commercially available endolysins such as Staphefekt SA.100, Exebacase (CF-301), and N-Rephasin®SAL200 are used in biofilm penetration and treating plant diseases. The role of CMP1 phage endolysin in transgenic tomato plants in preventing Clavibacter michiganensis infection and the effectiveness of phage in protecting Atlantic salmon from vibriosis have been reported. Furthermore, phage-derived endolysin therapy, such as TSPphg phage exogenous treatment, can aid in disrupting cell walls, leading to bacterial cell lysis. As animals in aquaculture and slaughterhouses are highly susceptible to bacterial infections, effective phage therapy instead of antibiotics can help treat poultry animals, preserve them, and facilitate disease-free trade. Using bioengineered phages and phage cocktails enhances the effectiveness by providing a broad spectrum of phages and target specificity. Research is currently being conducted on clinical trials to confirm the efficacy of engineered phages and phage cocktails in humans. Although obtaining commercial approval may be time-consuming, it will be beneficial in the postantibiotic era. This review provides an overview of the significance of phage therapy as a potential alternative to antibiotics in combating resistant bacterial strains and its application to various fields and emphasizes the importance of safeguarding and ensuring treatment efficacy.

Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Disarm The Bacteria: What Temperate Phages Can Do.

In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.

The analysis of the function, diversity, and evolution of the Bacillus phage genome.

BACKGROUND: Phages play a pivotal role in the evolution of microbial populations. The interactions between phages and their hosts are complex and may vary in response to host physiology and environmental conditions. Here, we have selected the genomes of some representative Bacillus prophages and lysosomes from the NCBI database for evolutionary analysis. We explored their evolutionary relationships and analyzed the protein information encoded by hundreds of Bacillus phages. RESULTS: We obtained the following conclusions: First, Bacillus phages carried some known functional gene fragments and a large number of unknown functional gene fragments, which might have an important impact on Bacillus populations, such as the formation of spores and biofilms and the transmission of virulence factors. Secondly, the Bacillus phage genome showed diversity, with a clear genome boundary between Bacillus prophages and Bacillus lytic phages. Furthermore, genetic mutations, sequence losses, duplications, and host-switching have occurred during the evolution of the Bacillus phage, resulting in low genome similarity between the Bacillus phages. Finally, the lysis module played an important influence on the process of Bacillus phage cross-species infestation. CONCLUSIONS: This study systematically described their protein function, diversity, and genome evolution, and the results of this study provide a basis for evolutionary diversity, horizontal gene transfer and co-evolution with the host in Bacillus phages.

Synergistic action of phages and lytic proteins with antibiotics: a combination strategy to target bacteria and biofilms.

BACKGROUND: Multidrug-resistant bacteria continue to emerge owing to the abuse of antibiotics and have a considerable negative impact on people and the environment. Bacteria can easily form biofilms to improve their survival, which reduces the efficacy of antibacterial drugs. Proteins such as endolysins and holins have been shown to have good antibacterial activity and effectively removal bacterial biofilms and reduce the production of drug-resistant bacteria. Recently, phages and their encoded lytic proteins have attracted attention as potential alternative antimicrobial agents. The aim of the present study was to investigate the sterilising efficacy of phages (SSE1, SGF2, and SGF3) and their encoded lytic proteins (lysozyme and holin), and to further explore their potential in combination with antibiotics. To the ultimate aim is to reduce or replace the use of antibiotics and provide more materials and options for sterilisation. RESULTS: Phages and their encoded lytic proteins were confirmed to have great advantages in sterilisation, and all exhibited significant potential for reducing bacterial resistance. Previous studies on the host spectrum demonstrated the bactericidal efficacy of three Shigella phages (SSE1, SGF2, and SGF3) and two lytic proteins (LysSSE1 and HolSSE1). In this study, we investigated the bactericidal effects on planktonic bacteria and bacterial biofilms. A combined sterilisation application of antibiotics, phages, and lytic proteins was performed. The results showed that phages and lytic proteins had better sterilisation effects than antibiotics with 1/2 minimum inhibitory concentrations (MIC) and their effect was further improved when used together with antibiotics. The best synergy was shown when combined with ß- lactam antibiotics, which might be related to their mechanism of sterilising action. This approach ensures a bactericidal effect at low antibiotic concentrations. CONCLUSIONS: This study strengthens the idea that phages and lytic proteins can significantly sterilise bacteria in vitro and achieve synergistic sterilisation effects with specific antibiotics. Therefore, a suitable combination strategy may decrease the risk of drug resistance.

The current status of phage therapy and its advancement towards establishing standard antimicrobials for combating multi drug-resistant bacterial pathogens.

Phage therapy; a revived antimicrobial weapon, has great therapeutic advantages with the main ones being its ability to eradicate multidrug-resistant pathogens as well as selective toxicity, which ensures that beneficial microbiota is not harmed, unlike antibiotics. These therapeutic properties make phage therapy a novel approach for combating resistant pathogens. Since millions of people across the globe succumb to multidrug-resistant infections, the implementation of phage therapy as a standard antimicrobial could transform global medicine as it offers greater therapeutic advantages than conventional antibiotics. Although phage therapy has incomplete clinical data, such as a lack of standard dosage and the ideal mode of administration, the conducted clinical studies report its safety and efficacy in some case studies, and therefore, this could lessen the concerns of its skeptics. Since its discovery, the development of phage therapeutics has been in a smooth progression. Concerns about phage resistance in populations of pathogenic bacteria are raised when bacteria are exposed to phages. Bacteria can use restriction-modification, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) defense, or mutations in the phage receptors to prevent phage invasion. Phage resistance, however, is often costly for the bacteria and may lead to a reduction in its virulence. The ongoing competition between bacteria and phage, on the other hand, ensures the emergence of phage strains that have evolved to infect resistant bacteria. A phage can quickly adapt by altering one or more aspects of its mode of infection, evading a resistance mechanism through genetic modifications, or directly thwarting the CRISPR-Cas defense. Using phage-bacterium coevolution as a technique could be crucial in the development of phage therapy as well. Through its recent advancement, gene-editing tools such as CRISPR-Cas allow the bioengineering of phages to produce phage cocktails that have broad spectrum activities, which could maximize the treatment's efficacy. This review presents the current state of phage therapy and its progression toward establishing standard medicine for combating antibiotic resistance. Recent clinical trials of phage therapy, some important case studies, and other ongoing clinical studies of phage therapy are all presented in this review. Furthermore, the recent advancement in the development of phage therapeutics, its application in various sectors, and concerns regarding its implementation are also highlighted here. Phage therapy has great potential and could help the fight against drug-resistant bacterial pathogens.

Complete genome sequence analysis, morphology and structural protein identification of two Bacillus subtilis phages, BSTP4 and BSTP6, which may form a new species in the genus Salasvirus.

In the present study, two new Bacillus subtilis phages, BSTP4 and BSTP6, were isolated and studied further. Morphologically, BSTP4 and BSTP6 are podoviruses with complete genome of 19,145 (39.9% G + C content) and 19,367 bp (39.8% G + C content), respectively, which became among the smallest Bacillus phages. Three most prominent structural proteins were separated and identified as pre-neck appendage, major head, and head fiber proteins using LC-MS/MS. Both phages encode putative terminal proteins (TP) and contain short inverted terminal repeats (ITRs) which may be important for their replication. In addition, non-coding RNA (pRNA) and parS sites were identified which may be required for DNA packaging and their maintenance inside the host, respectively. Furthermore, the phage genome sequences show significant similarity to B. subtilis group species genome sequences. Finally, phylogenomic and phylogenetic analyses suggest that BSTP4 and BSTP6 may form a new species in the genus Salasvirus, subfamily Picovirinae of family Salasmaviridae. Considering the small numbers of ICTV-accepted B. subtilis phages and the importance of the host in the food industry and biotechnology, the current study helps to improve our understanding of the diversity of B. subtilis phages and shed light on the phage-host relationships.