Evaluation of the Feasibility of Using Commercial Wound Coatings as a Carrier Matrix for Bacteriophages.

The aim of the investigation is to study the possibility of applying commercial wound coatings for treating infected wounds as a carrier matrix for bacteriophages. Materials and Methods: Twelve varieties of commercial wound coverings based on biopolymers of natural and synthetic origin, a biological preparation Staphylophag produced by scientific-industrial association Microgen (Russia), registration certificate P N001973/01, and the S. aureus 3196 test strain (GenBank JARQZO000000000) isolated from a patient with a burn wound have been used in our work. The ability of commercial biological wound coatings to absorb solutions was examined by immersing them in a physiological solution (pH 7.0-7.2) followed by weighing. The lytic activity of three bacteriophage series against the test strain was studied using the Appelman method and a spot test. The lytic activity of the bacteriophage in the wound samples was studied within 7 days after its absorption by the wound coatings. Results: The greatest volume of fluid was absorbed by the LycoSorb, NEOFIX FibroSorb Ag, Biatravm, and Chitocol-S wound coatings. All bacteriophage series have been found to have a high lytic activity against the test strain. It has also been shown that Chitocol-S, Collachit-FA, Algipran, and Aquacel Ag Extra possessed their own inherent antibacterial activity under in vitro conditions stable for 7 days; moreover, the lysis zones of the test strain increased after their saturation with bacteriophage. On day 0, a high level of bacteriophage lytic activity with the maximum size of the test strain lysis zones from 49 to 59 mm have been found to remain in all samples of the wound coverings. The bacteriophage activity persisted for 1 day in the samples of Hydrofilm, Polypran, and NEOFIX FibroCold Ag coatings, up to 4 days in Algipran, Nano-Aseptica, and Biatravm coatings; and for 7 days in the Chitocol-S, Collachit-FA, Opsite Post-Op Visible, NEOFIX FibroSorb Ag, Aquacel Ag Extra, and LycoSorb samples. Conclusion: Modern commercial wound dressings based on chitosan-collagen complex (Chitocol-S, Collachit-FA), polyurethane (Opsite Post-Op Visible, LycoSorb, NEOFIX FibroSorb Ag), and Hydrofiber (Aquacel Ag Extra) have a sufficient level of bacteriophage solution absorption, provide a stable preservation of the bacteriophage lytic activity under in vitro conditions up to 7 days. Thus, the in vitro studies prove the possibility of their use as a carrier matrix for bacteriophages.

Engineered Bacteriophage-Polymer Nanoassemblies for Treatment of Wound Biofilm Infections.

The antibacterial efficacy and specificity of lytic bacteriophages (phages) make them promising therapeutics for treatment of multidrug-resistant bacterial infections. Restricted penetration of phages through the protective matrix of biofilms, however, may limit their efficacy against biofilm infections. Here, engineered polymers were used to generate noncovalent phage-polymer nanoassemblies (PPNs) that penetrate bacterial biofilms and kill resident bacteria. Phage K, active against multiple strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), was assembled with cationic poly(oxanorbornene) polymers into PPNs. The PPNs retained phage infectivity, while demonstrating enhanced biofilm penetration and killing relative to free phages. PPNs achieved 3-log10 bacterial reduction (∼99.9%) against MRSA biofilms in vitro. PPNs were then incorporated into Poloxamer 407 (P407) hydrogels and applied onto in vivo wound biofilms, demonstrating controlled and sustained release. Hydrogel-incorporated PPNs were effective in a murine MRSA wound biofilm model, showing a 1.5-log10 reduction in bacterial load compared to a 0.5 log reduction with phage K in P407 hydrogel. Overall, this work showcases the therapeutic potential of phage K engineered with cationic polymers for treating wound biofilm infections.

Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus.

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

Bacteriophage therapy reduces Staphylococcus aureus in a porcine and human ex vivo burn wound infection model.

Burn wounds are a major burden, with high mortality rates due to infections. Staphylococcus aureus is a major causative agent of burn wound infections, which can be difficult to treat because of antibiotic resistance and biofilm formation. An alternative to antibiotics is the use of bacteriophages, viruses that infect and kill bacteria. We investigated the efficacy of bacteriophage therapy for burn wound infections, in both a porcine and a newly developed human ex vivo skin model. In both models, the efficacy of a reference antibiotic treatment (fusidic acid) and bacteriophage treatment was determined for a single treatment, successive treatment, and prophylaxis. Both models showed a reduction in bacterial load after a single bacteriophage treatment. Increasing the frequency of bacteriophage treatments increased bacteriophage efficacy in the human ex vivo skin model, but not in the porcine model. In both models, prophylaxis with bacteriophages increased treatment efficacy. In all cases, bacteriophage treatment outperformed fusidic acid treatment. Both models allowed investigation of bacteriophage-bacteria dynamics in burn wounds. Overall, bacteriophage treatment outperformed antibiotic control underlining the potential of bacteriophage therapy for the treatment of burn wound infections, especially when used prophylactically.

Isolation, characterization, and application of a lytic bacteriophage SSP49 to control Staphylococcus aureus contamination on baby spinach leaves.

Staphylococcus aureus, a major foodborne pathogen, is frequently detected in fresh produce. It often causes food poisoning accompanied by abdominal pain, diarrhea, and vomiting. Additionally, the abuse of antibiotics to control S. aureus has resulted in the emergence of antibiotics-resistant bacteria, such as methicillin resistant S. aureus. Therefore, bacteriophage, a natural antimicrobial agent, has been suggested as an alternative to antibiotics. In this study, a lytic phage SSP49 that specifically infects S. aureus was isolated from a sewage sample, and its morphological, biological, and genetic characteristics were determined. We found that phage SSP49 belongs to the Straboviridae family (Caudoviricetes class) and maintained host growth inhibition for 30 h in vitro. In addition, it showed high host specificity and a broad host range against various S. aureus strains. Receptor analysis revealed that phage SSP49 utilized cell wall teichoic acid as a host receptor. Whole genome sequencing revealed that the genome size of SSP49 was 137,283 bp and it contained 191 open reading frames. The genome of phage SSP49 did not contain genes related to lysogen formation, bacterial toxicity, and antibiotic resistance, suggesting its safety in food application. The activity of phage SSP49 was considerably stable under various high temperature and pH conditions. Furthermore, phage SSP49 effectively inhibited S. aureus growth on baby spinach leaves both at 4 °C and 25 °C while maintaining the numbers of active phage during treatments (reductions of 1.2 and 2.1 log CFU/cm2, respectively). Thus, this study demonstrated the potential of phage SSP49 as an alternative natural biocontrol agent against S. aureus contamination in fresh produce.

Exploring synergistic and antagonistic interactions in phage-antibiotic combinations against ESKAPE pathogens.

In the era of antimicrobial resistance, phage-antibiotic combinations offer a promising therapeutic option, yet research on their synergy and antagonism is limited. This study aims to assess these interactions, focusing on protein synthesis inhibitors and cell envelope-active agents against multidrug-resistant bacterial strains. We evaluated synergistic and antagonistic interactions in multidrug-resistant Staphylococcus aureus, Enterococcus faecium, and Pseudomonas aeruginosa strains. Phages were combined with protein synthesis inhibitors [linezolid (LZD), minocycline (MIN), gentamicin (GEN), and azithromycin (AZM)] or cell envelope-active agents [daptomycin (DAP), ceftaroline (CPT), and cefepime (FEP)]. Modified checkerboard minimum inhibitory concentration assays and 24-h time-kill analyses were conducted, alongside one-step growth curves to analyze phage growth kinetics. Statistical comparisons used one-way analysis of variance (ANOVA) and the Tukey test (P < 0.05). In the checkerboard and 24-h time-kill analyses (TKA) of S. aureus and E. faecium, phage-LZD and phage-MIN combinations were antagonistic (FIC > 4) while phage-DAP and phage-CPT were synergistic (FIC 0.5) (ANOVA range of mean differences 0.52-2.59 log10 CFU/mL; P < 0.001). For P. aeruginosa, phage-AZM was antagonistic (FIC > 4), phage-GEN was additive (FIC = 1), and phage-FEP was synergistic (ANOVA range of mean differences 1.04-1.95 log10 CFU/mL; P < 0.001). Phage growth kinetics were altered in the presence of LZD and MIN against S. aureus and in the presence of LZD against a single E. faecium strain (HOU503). Our findings indicate that select protein synthesis inhibitors may induce phage-antibiotic antagonism. However, this antagonism may not solely stem from changes in phage growth kinetics, warranting further investigation into the complex interplay among strains, phage attributes, and antibiotic mechanisms affecting bacterial inhibition.IMPORTANCEIn the face of escalating antimicrobial resistance, combining phages with antibiotics offers a promising avenue for treating infections unresponsive to traditional antibiotics. However, while studies have explored synergistic interactions, less attention has been given to potential antagonism and its impact on phage growth kinetics. This research evaluates the interplay between phages and antibiotics, revealing both synergistic and antagonistic patterns across various bacterial strains and shedding light on the complex dynamics that influence treatment efficacy. Understanding these interactions is crucial for optimizing combination therapies and advancing phage therapy as a viable solution for combating antimicrobial resistance.

High throughput platform technology for rapid target identification in personalized phage therapy.

As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.

Bacteriophage-driven emergence and expansion of Staphylococcus aureus in rodent populations.

Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species.

Distinct prophage gene profiles of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals.

Staphylococcus aureus strains exhibit varying associations with atopic dermatitis (AD), but the genetic determinants underpinning the pathogenicity are yet to be fully characterized. To reveal the genetic differences between S. aureus strains from AD patients and healthy individuals (HE), we developed and employed a random forest classifier to identify potential marker genes responsible for their phenotypic variations. The classifier was able to effectively distinguish strains from AD and HE. We also uncovered strong links between certain marker genes and phage functionalities, with phage holin emerging as the most pivotal differentiating factor. Further examination of S. aureus gene content highlighted the genetic diversity and functional implications of prophages in driving differentiation between strains from AD and HE. The HE group exhibited greater gene content diversity, largely influenced by their prophages. While strains from both AD and HE universally housed prophages, those in the HE group were distinctively higher at the strain level. Moreover, although prophages in the HE group exhibited variously higher enrichment of differential functions, the AD group displayed a notable enrichment of virulence factors within their prophages, underscoring the important contribution of prophages to the pathogenesis of AD-associated strains. Overall, prophages significantly shape the genetic and functional profiles of S. aureus strains, shedding light on their pathogenic potential and elucidating the mechanisms behind the phenotypic variations in AD and HE environments. IMPORTANCE: Through a nuanced exploration of Staphylococcus aureus strains obtained from atopic dermatitis (AD) patients and healthy controls (HE), our research unveils pivotal genetic determinants influencing their pathogenic associations. Utilizing a random forest classifier, we illuminate distinct marker genes, with phage holin emerging as a critical differential factor, revealing the profound impact of prophages on genetic and pathogenic profiles. HE strains exhibited a diverse gene content, notably shaped by unique, heightened prophages. Conversely, AD strains emphasized a pronounced enrichment of virulence factors within prophages, signifying their key role in AD pathogenesis. This work crucially highlights prophages as central architects of the genetic and functional attributes of S. aureus strains, providing vital insights into pathogenic mechanisms and phenotypic variations, thereby paving the way for targeted AD therapeutic approaches and management strategies by demystifying specific genetic and pathogenic mechanisms.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

OBJECTIVES: Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. METHODS: Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. RESULTS: At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. CONCLUSION: Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.

Bacteriophage ISP eliminates Staphylococcus aureus in planktonic phase, but not in the various stages of the biofilm cycle.

Metal-implant associated bacterial infections are a major clinical problem due to antibiotic treatment failure. As an alternative, we determined the effects of bacteriophage ISP on clinical isolates of Staphylococcus aureus in various stages of its life cycle in relation to biofilm formation and maturation. ISP effectively eliminated all planktonic phase bacteria, whereas its efficacy was reduced against bacteria attached to the metal implant and bacteria embedded within biofilms. The biofilm architecture hampered the bactericidal effects of ISP, as mechanical disruption of biofilms improved the efficacy of ISP against the bacteria. Phages penetrated the biofilm and interacted with the bacteria throughout the biofilm. However, most of the biofilm-embedded bacteria were phage-tolerant. In agreement, bacteria dispersed from mature biofilms of all clinical isolates, except for LUH15394, tolerated the lytic activity of ISP. Lastly, persisters within mature biofilms tolerated ISP and proliferated in its presence. Based on these findings, we conclude that ISP eliminates planktonic phase Staphylococcus aureus while its efficacy is limited against bacteria attached to the metal implant, embedded within (persister-enriched) biofilms, and dispersed from biofilms.

Evaluation of bacteriophage ϕ11 host recognition protein and its host-binding peptides for diagnosing/targeting Staphylococcus aureus infections.

BACKGROUND: Evaluating the potential of using both synthetic and biological products as targeting agents for the diagnosis, imaging, and treatment of infections due to particularly antibiotic-resistant pathogens is important for controlling infections. This study examined the interaction between Gp45, a receptor-binding protein of the ϕ11 lysogenic phage, and its host Staphylococcus aureus (S. aureus), a common cause of nosocomial infections. METHODS: Using molecular dynamics and docking simulations, this study identified the peptides that bind to S. aureus wall teichoic acids via Gp45. It compared the binding affinity of Gp45 and the two highest-scoring peptide sequences (P1 and P3) and their scrambled forms using microscopy, spectroscopy, and ELISA. RESULTS: It was found that rGp45 (recombinant Gp45) and chemically synthesised P1 had a higher binding affinity for S. aureus compared with all other peptides, except for Escherichia coli. Furthermore, rGp45 had a capture efficiency of > 86%; P1 had a capture efficiency of > 64%. CONCLUSION: These findings suggest that receptor-binding proteins such as rGp45, which provide a critical initiation of the phage life cycle for host adsorption, might play an important role in the diagnosis, imaging, and targeting of bacterial infections. Studying such proteins could accordingly enable the development of effective strategies for controlling infections.

Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis.

Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections. IMPORTANCE: Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

Isolation and characterization of two Staphylococcus aureus lytic bacteriophages "Huma" and "Simurgh".

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Investigating the effectiveness of liposome-bacteriophage nanocomplex in killing Staphylococcus aureus using epithelial cell coculture models.

Host cell invasion with strong antibiotics evading is a major feature of respiratory Staphylococcus aureus infections with severe recurrence. Bacteriophage (phage) therapy and design of liposomal phage to target intracellular pathogens have been described recently. The practicality for pulmonary delivery of liposomal phage, and how formulation compositions affecting the aerosolization and intracellular bacterial killing remain unexplored. In the present study, three commonly used phospholipids (SPC, EPC, and HSPC) were selected to investigate their ability for phage K nebulization and intracellular therapy in the form of liposome-phage nanocomplexes. The three lipid nanocarriers showed protection on phage K upon mesh nebulization and the pulmonary deposition efficiency was influenced by the lipid used. Moreover, the intracellular bacterial killing was strongly depended on the lipid types, where EPC-phage exhibited the best killing performance with no relapsing. Phage K with the aid of EPC liposomes was also observed to manage the tissue infection in a 3D spheroid model more effectively than other groups. Altogether, this novel EPC liposome-phage nanocomplex can be a promising formulation approach that enables inhalable phage to manage respiratory infections caused by bacteria strongly associated with human epithelial cells.

Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles.

Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned off during prophage induction, balancing immunity and autoimmunity. Our results show that the fine regulation of these processes is essential for the correct development of prophages' life cycle.

The CRISPR effector Cam1 mediates membrane depolarization for phage defence.

Prokaryotic type III CRISPR-Cas systems provide immunity against viruses and plasmids using CRISPR-associated Rossman fold (CARF) protein effectors1-5. Recognition of transcripts of these invaders with sequences that are complementary to CRISPR RNA guides leads to the production of cyclic oligoadenylate second messengers, which bind CARF domains and trigger the activity of an effector domain6,7. Whereas most effectors degrade host and invader nucleic acids, some are predicted to contain transmembrane helices without an enzymatic function. Whether and how these CARF-transmembrane helix fusion proteins facilitate the type III CRISPR-Cas immune response remains unknown. Here we investigate the role of cyclic oligoadenylate-activated membrane protein 1 (Cam1) during type III CRISPR immunity. Structural and biochemical analyses reveal that the CARF domains of a Cam1 dimer bind cyclic tetra-adenylate second messengers. In vivo, Cam1 localizes to the membrane, is predicted to form a tetrameric transmembrane pore, and provides defence against viral infection through the induction of membrane depolarization and growth arrest. These results reveal that CRISPR immunity does not always operate through the degradation of nucleic acids, but is instead mediated via a wider range of cellular responses.

Structure of the Portal Complex from Staphylococcus aureus Pathogenicity Island 1 Transducing Particles In Situ and In Isolation.

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

Aceite esencial de Arazá (Eugenia stipitata McVaugh) con actividad antibacteriana / Essential oil of Arazá (Eugenia stipitata McVaugh) with antibacterial activity

Introducción: El Perú es uno de los países con mayor biodiversidad en especies botánicas, algunas con propiedades medicinales conocidas. Objetivo: Determinar el efecto antibacteriano del aceite esencial de las hojas de Eugenia stipitata McVaugh frente a Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 y Salmonella enterica sv Enteritidis ATCC 13076. Métodos: Estudio de tipo básico con enfoque cuantitativo y experimental. Las plantas provienen del distrito de Belén, ciudad de Iquitos, Departamento de Loreto. La técnica para la extracción del aceite esencial fue la de arrastre de vapor y la técnica microbiológica para determinar el efecto antimicrobiano la de Kirby Bauer. Se trabajaron las muestras en 4 concentraciones 100, 75, 50 y un 25 por ciento; un control negativo solo con dimetilsulfóxido, se utilizaron 5 repeticiones por cada muestra. Resultados: La muestra a concentración al 100 por ciento tuvo actividad antibacteriana contra Staphylococcus aureus. La actividad del ensayo frente a Escherichia coli demostró ser efectiva en todas las muestras, sin embargo, se observó que los halos de inhibición de mayor diámetro se manifestaron en las muestras al 100 por ciento y 75 por ciento. Además, se evidenció actividad antibacteriana a concentraciones del 100 por ciento, 75 por ciento y un 50 por ciento frente a Salmonella enterica sv Enteritidis. Conclusiones: El aceite esencial de las hojas de Eugenia stipitata McVaugh presenta efecto antibacteriano frente a Staphylococcus aureus, Escherichia coli y Salmonella enterica sv Enteritidis(AU)

Introduction: Peru is one of the countries with the greatest biodiversity in botanical species, some with known medicinal properties. Objective: To determine the antibacterial effect of the essential oil of Eugenia stipitata McVaugh leaves against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella enterica sv Enteritidis ATCC 13076. Methods: Basic study with a quantitative and experimental approach. Plants came from the district of Belén, city of Iquitos, Department of Loreto. The technique for the extraction of the essential oil was steam dragging and the microbiological technique to determine the antimicrobial effect was Kirby Bauer's technique. The samples were worked in 4 concentrations 100, 75, 50 and 25 percent and a negative control only with dimethyl sulfoxide, using 5 replicates for each sample. Results: The sample at 100 percent concentration had antibacterial activity against Staphylococcus aureus. The activity of the assay against Escherichia coli proved to be effective in all the samples, however, it was observed that the inhibition halos of greater diameter were manifested in the samples at 100 percent and 75 percent. In addition, antibacterial activity was evidenced at concentrations of 100 percent, 75 percent and 50 percent against Salmonella enterica sv Enteritidis. Conclusions: The essential oil of Eugenia stipitata McVaugh leaves has an antibacterial effect against Staphylococcus aureus, Escherichia coli and Salmonella enterica sv Enteritidis(AU)