Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

Phage formulations and delivery strategies: Unleashing the potential against antibiotic-resistant bacteria.

Bacterial control promoted by bacteriophages (phages) is an attractive tool in the face of the antibiotic crisis triggered by the exacerbated use of these drugs. Despite the growing interest in using these viruses, some gaps still need answers, such as the protection and delivery of phages. Some limitation points involve the degradation of phage proteins by enzymes or inactivation in low-pH environments. In this review, a literature search using keywords related to the field of virus delivery formulations was done to understand the current scenario of using delivery techniques and phage formulations. A total of 2096 raw results were obtained, which resulted in 140 publications after refinement. These studies were analyzed for main application techniques and areas, keywords, and countries. Of the total, 57% of the publications occurred in the last five years, and the encapsulation technique was the most used among the articles analyzed. As excipient agents, lactose, trehalose, mannitol, PEG, and Leucine stand out. The development of phage formulations, protection approaches, their delivery routes, and the knowledge about the best application strategy enables the use of these organisms in several sectors. It can act as a powerful tool against antibiotic-resistant bacteria.

The Medicinal Phage-Regulatory Roadmap for Phage Therapy under EU Pharmaceutical Legislation.

Bacteriophage therapy is a promising approach to treating bacterial infections. Research and development of bacteriophage therapy is intensifying due to the increase in antibiotic resistance and the faltering development of new antibiotics. Bacteriophage therapy uses bacteriophages (phages), i.e., prokaryotic viruses, to specifically target and kill pathogenic bacteria. The legal handling of this type of therapy raises several questions. These include whether phage therapeutics belong to a specially regulated class of medicinal products, and which legal framework should be followed with regard to the various technical ways in which phage therapeutics can be manufactured and administered. The article shows to which class of medicinal products phage therapeutics from wild type phages and from genetically modified (designer) phages do or do not belong. Furthermore, the article explains which legal framework is relevant for the manufacture and administration of phage therapeutics, which are manufactured in advance in a uniform, patient-independent manner, and for tailor-made patient-specific phage therapeutics. For the systematically coherent, successful translation of phage therapy, the article considers pharmaceutical law and related legal areas, such as genetic engineering law. Finally, the article shows how the planned legislative revisions of Directive 2001/83/EC and Regulation (EC) No 726/2004 may affect the legal future of phage therapy.

Bacteriophage-Host Interactions and the Therapeutic Potential of Bacteriophages.

Healthcare faces a major problem with the increased emergence of antimicrobial resistance due to over-prescribing antibiotics. Bacteriophages may provide a solution to the treatment of bacterial infections given their specificity. Enzymes such as endolysins, exolysins, endopeptidases, endosialidases, and depolymerases produced by phages interact with bacterial surfaces, cell wall components, and exopolysaccharides, and may even destroy biofilms. Enzymatic cleavage of the host cell envelope components exposes specific receptors required for phage adhesion. Gram-positive bacteria are susceptible to phage infiltration through their peptidoglycan, cell wall teichoic acid (WTA), lipoteichoic acids (LTAs), and flagella. In Gram-negative bacteria, lipopolysaccharides (LPSs), pili, and capsules serve as targets. Defense mechanisms used by bacteria differ and include physical barriers (e.g., capsules) or endogenous mechanisms such as clustered regularly interspaced palindromic repeat (CRISPR)-associated protein (Cas) systems. Phage proteins stimulate immune responses against specific pathogens and improve antibiotic susceptibility. This review discusses the attachment of phages to bacterial cells, the penetration of bacterial cells, the use of phages in the treatment of bacterial infections, and the limitations of phage therapy. The therapeutic potential of phage-derived proteins and the impact that genomically engineered phages may have in the treatment of infections are summarized.

Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense.

CRISPR-Cas immune systems provide bacteria with adaptive immunity against bacteriophages, but they are often transcriptionally repressed to mitigate auto-immunity. In some cases, CRISPR-Cas expression increases in response to a phage infection, but the mechanisms of induction are largely unknown, and it is unclear whether induction occurs strongly and quickly enough to benefit the bacterial host. In S. pyogenes, Cas9 is both an immune effector and auto-repressor of CRISPR-Cas expression. Here, we show that phage-encoded anti-CRISPR proteins relieve Cas9 auto-repression and trigger a rapid increase in CRISPR-Cas levels during a single phage infective cycle. As a result, fewer cells succumb to lysis, leading to a striking survival benefit after multiple rounds of infection. CRISPR-Cas induction also reduces lysogeny, thereby limiting a route for horizontal gene transfer. Altogether, we show that Cas9 is not only a CRISPR-Cas effector and repressor but also a phage sensor that can mount an anti-anti-CRISPR transcriptional response.

Bacteriophage DNA induces an interrupted immune response during phage therapy in a chicken model.

One of the hopes for overcoming the antibiotic resistance crisis is the use of bacteriophages to combat bacterial infections, the so-called phage therapy. This therapeutic approach is generally believed to be safe for humans and animals as phages should infect only prokaryotic cells. Nevertheless, recent studies suggested that bacteriophages might be recognized by eukaryotic cells, inducing specific cellular responses. Here we show that in chickens infected with Salmonella enterica and treated with a phage cocktail, bacteriophages are initially recognized by animal cells as viruses, however, the cGAS-STING pathway (one of two major pathways of the innate antiviral response) is blocked at the stage of the IRF3 transcription factor phosphorylation. This inhibition is due to the inability of RNA polymerase III to recognize phage DNA and to produce dsRNA molecules which are necessary to stimulate a large protein complex indispensable for IRF3 phosphorylation, indicating the mechanism of the antiviral response impairment.

Sporadic regional re-emergent cholera: a 19th century problem in the 21st century.

Cholera, caused by Vibrio cholerae, is a severe diarrheal disease that necessitates prompt diagnosis and effective treatment. This review comprehensively examines various diagnostic methods, from traditional microscopy and culture to advanced nucleic acid testing like polymerase spiral reaction and rapid diagnostic tests, highlighting their advantages and limitations. Additionally, we explore evolving treatment strategies, with a focus on the challenges posed by antibiotic resistance due to the activation of the SOS response pathway in V. cholerae. We discuss promising alternative treatments, including low-pressure plasma sterilization, bacteriophages, and selenium nanoparticles. The paper emphasizes the importance of multidisciplinary approaches combining novel diagnostics and treatments in managing and preventing cholera, a persistent global health challenge. The current re-emergent 7th pandemic of cholera commenced in 1961 and shows no signs of abeyance. This is probably due to the changing genetic profile of V. cholerae concerning bacterial pathogenic toxins. Given this factor, we argue that the disease is effectively re-emergent, particularly in Eastern Mediterranean countries such as Lebanon, Syria, etc. This review considers the history of the current pandemic, the genetics of the causal agent, and current treatment regimes. In conclusion, cholera remains a significant global health challenge that requires prompt diagnosis and effective treatment. Understanding the history, genetics, and current treatments is crucial in effectively addressing this persistent and re-emergent disease.

Using a human colonoid-derived monolayer to study bacteriophage translocation.

Bacteriophages (phages) are estimated to be the most abundant microorganisms on Earth. Their presence in human blood suggests that they can translocate from non-sterile sites such as the gastrointestinal tract where they are concentrated. To examine phage translocation ex vivo, we adapted a primary colonoid monolayer model possessing cell diversity and architecture, and a thick layer of mucus akin to the colonic environment in vivo. We show that the colonoid monolayer is superior to the Caco-2 cell-line model, possessing intact and organized tight junctions and generating a physiologically relevant mucus layer. We showed, using two different phages, that translocation across the colonoid monolayer was largely absent in differentiated monolayers that express mucus, unlike Caco-2 cultures that expressed little to no mucus. By stimulating mucus production or removing mucus, we further demonstrated the importance of colonic mucus in preventing phage translocation. Finally, we used etiological drivers of gut permeability (alcohol, fat, and inflammatory cytokines) to measure their effects on phage translocation, demonstrating that all three stimuli have the capacity to amplify phage translocation. These findings suggest that phage translocation does occur in vivo but may be largely dependent on colonic mucus, an important insight to consider in future phage applications.

Feasibility of using bacteriophage therapy to treat Staphylococcal aureus fracture-related infections.

OBJECTIVE: Staphylococcus aureus fracture-related infections (FRIs) are associated with significant morbidity in part because conventional antibiotic therapies have limited ability to eradicate S. aureus in sessile states. Therefore, the objective of this study was to assess the feasibility of using Staphylococcal bacteriophages for FRI by testing the activity of a library of Staphylococcal bacteriophage therapeutics against historically preserved S. aureus FRI clinical isolates. METHODS: Current Procedural Terminology codes were used to identify patients with FRI from January 1, 2021 to December 31, 2021. Preserved S. aureus FRI isolates from the cases were then tested against a library of 51 Staphylococcal bacteriophages from an American company. This was conducted by assessing the ability of bacteriophages to reduce bacterial growth over time. Growth inhibition greater than 16 h was considered adequate for this study. RESULTS: All of the S. aureus preserved clinical isolates had at least one bacteriophage with robust lytic activity and six bacteriophages (11.8 %) had robust lytic activity to seven or more of the clinical isolates. However, 41 of the bacteriophages (80.4 %) had activity to less than three of the clinical isolates and no bacteriophage had activity to all the clinical isolates. CONCLUSION: Our findings show that Staphylococcal bacteriophage therapeutics are readily available for S. aureus FRI clinical isolates. However, when correlated with the current barriers to using bacteriophages to treat FRI, designated Staphylococcal bacteriophage cocktails with broad spectrum activity should be created.

Bacteriophage specificity is impacted by interactions between bacteria.

Predators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains of Escherichia coli and Salmonella enterica by competing a generalist T5-like phage able to infect both prey against P22vir, an S. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that the in vitro cost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest. IMPORTANCE: There is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and an in vitro system consisting of Escherichia coli, Salmonella enterica, a T5-like generalist phage, and the specialist phage P22vir to highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections.

Molecular characterization and environmental impact of newly isolated lytic phage SLAM_phiST1N3 in the Cornellvirus genus for biocontrol of a multidrug-resistant Salmonella Typhimurium in the swine industry chain.

Salmonella Typhimurium is a highly lethal pathogenic bacterium in weaned piglets, causing significant treatment costs and economic losses in the swine industry. Additionally, due to its ability to induce zoonotic diseases, resulting in harm to humans through the transmission of the pathogen from pork, it presents a serious public health issue. Bacteriophages (phages), viruses that infect specific bacterial strains, have been proposed as an alternative to antibiotics for controlling pathogenic bacteria. In this study, we isolated SLAM_phiST1N3, a phage infecting a multidrug-resistant (MDR) S. Typhimurium wild-type strain isolated from diseased pigs. First, comparative genomics and phylogenetic analysis revealed that SLAM_phiST1N3 belongs to the Cornellvirus genus. Moreover, utilizing a novel classification approach introduced in this study, SLAM_phiST1N3 was classified at the species level. Host range experiments demonstrated that SLAM_phiST1N3 did not infect other pathogenic bacteria or probiotics derived from pigs or other livestock. While complete eradication of Salmonella was not achievable in the liquid inhibition assay, surprisingly, we succeeded in largely eliminating Salmonella in the FIMM analysis, a gut simulation system using weaned piglet feces. Furthermore, using the C. elegans model, we showcased the potential of SLAM_phiST1N3 to prevent S. Typhimurium infection in living organisms. In addition, it was confirmed that bacterial control could be achieved when phage was applied to Salmonella-contaminated pork. pH and temperature stability experiments demonstrated that SLAM_phiST1N3 can endure swine industry processes and digestive conditions. In conclusion, SLAM_phiST1N3 demonstrates potential environmental impact as a substance for Salmonella prevention across various aspects of the swine industry chain.

Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections. IMPORTANCE: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Revisiting host identification of bacteriophage Enc34: from biochemical to molecular.

In our 2012 genome announcement (J Virol 86:11403-11404, 2012, https://doi.org/10.1128/JVI.01954-12), we initially identified the host bacterium of bacteriophage Enc34 as Enterobacter cancerogenus using biochemical tests. However, later in-house DNA sequencing revealed that the true host is a strain of Hafnia alvei. Capitalizing on our new DNA-sequencing capabilities, we also refined the genomic termini of Enc34, confirming a 60,496-bp genome with 12-nucleotide 5' cohesive ends. IMPORTANCE: Our correction reflects the evolving landscape of bacterial identification, where molecular methods have supplanted traditional biochemical tests. This case underscores the significance of revisiting past identifications, as seemingly known bacterial strains may yield unexpected discoveries, necessitating essential updates to the scientific record. Despite the host identity correction, our genome announcement retains importance as the first complete genome sequence of a Hafnia alvei bacteriophage.

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host's health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli, in the context of the murine gastrointestinal tract. The methods described here outline how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels. This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Bacteriophages-Dangerous Viruses Acting Incognito or Underestimated Saviors in the Fight against Bacteria?

The steadily increasing number of drug-resistant bacterial species has prompted the search for alternative treatments, resulting in a growing interest in bacteriophages. Although they are viruses infecting bacterial cells, bacteriophages are an extremely important part of the human microbiota. By interacting with eukaryotic cells, they are able to modulate the functioning of many systems, including the immune and nervous systems, affecting not only the homeostasis of the organism, but potentially also the regulation of pathological processes. Therefore, the aim of this review is to answer the questions of (i) how animal/human immune systems respond to bacteriophages under physiological conditions and under conditions of reduced immunity, especially during bacterial infection; (ii) whether bacteriophages can induce negative changes in brain functioning after crossing the blood-brain barrier, which could result in various disorders or in an increase in the risk of neurodegenerative diseases; and (iii) how bacteriophages can modify gut microbiota. The crucial dilemma is whether administration of bacteriophages is always beneficial or rather if it may involve any risks.

Thermal Inactivation of Escherichia Phage OSYSP and Host Strain Escherichia coli O157:H7 EDL933: A Comparative Kinetic Analysis.

Lytic bacteriophages are promising biocontrol agents against pathogenic bacteria for food and therapeutic applications. Investigating the feasibility of combining phage and physical lethal agents, such as heat, as an effective hurdle combination could lead to beneficial applications. The current research was initiated to compare the thermal inactivation kinetics of a lytic phage (Escherichia phage OSYSP) and its host (Shiga toxin-producing Escherichia coli O157:H7 EDL933), considering they have different critical thermal targets in their structures. To provide a basis for comparison, thermal inactivation kinetics were determined on suspensions of these agents in buffered peptone water using a thermally controlled circulating water bath. Results showed that the bacteriophage virions have a remarkable heat resistance (p < 0.05) compared to their host cells. The D-values of the populations of phage (PFU/mL) and EDL933 strain (CFU/mL) were 166.7 and 7.3 min at 55°C, compared to 44.4 and 0.3 min at 60°C, respectively. Additionally, D-values were significantly (p < 0.05) more influenced by temperature changes in the case of E. coli O157:H7 EDL933 (z-value 3.7°C) compared to that for phage OSYSP (z-value 7.7°C). When the phage suspension was heat-treated in a thermal cycler instead of a water bath, no significant differences between the two treatment procedures (p > 0.05) in estimating virus D- and z-values were observed. Based on these findings, it may be feasible to combine phage OSYSP with mild heat during processing of food to selectively inactivate E. coli O157:H7 EDL933 and subsequently maintain product safety during storage by the surviving phage population; however, the feasibility of this application needs to be investigated. Additionally, the relatively heat-resistant phage OSYSP could qualify as a biological indicator to validate thermal treatments of minimally processed foods in which E. coli O157:H7 EDL933 is the pathogen-of-concern.

Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

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.

Molecular mechanisms underlying the structural diversity of rhamnose-rich cell wall polysaccharides in lactococci.

In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.