Search Results (29)

Photocatalysis and photodynamic therapy have been increasingly used in the management of diabetic foot ulcers (DFUs), and their integration into increasingly innovative treatment protocols enables effective infection control. Advanced techniques such as antibacterial photodynamic therapy (aPDT), liposomal photocatalytic carriers, nanoparticles, and nanomotors—used alone, in combination, or with the addition of antibiotics, lysozyme, or phage enzymes—offer promising solutions for wound treatment. These approaches are particularly effective even in the presence of comorbidities such as angiopathies, neuropathies, and immune system disorders, which are common among diabetic patients. Notably, the use of combination therapies holds great potential for addressing challenges within diabetic foot ulcers, including hypoxia, poor circulation, high glucose levels, increased oxidative stress, and rapid biofilm formation—factors that significantly hinder wound healing in diabetic patients. The integration of modern therapeutic strategies is essential for effective clinical practice, starting with halting infection progression, ensuring its effective eradication, and promoting proper tissue regeneration, especially considering that, according to the WHO, 830 million people worldwide suffer from diabetes. Full article

Background: Ocular predominant mucous membrane pemphigoid (oMMP) is a severe subtype of autoimmune blistering disease (AIBD), which can result in scarring and vision loss. The diagnosis of oMMP is challenging as patients often have undetectable levels of circulating autoantibodies by conventional assays. Likewise, the principal autoantigen in oMMP has been an area of debate. Methods: In this preliminary experiment, we performed Phage Immunoprecipitation Sequencing (PhIP-seq) on sera from patients with oMMP, as well as non-ocular MMP, bullous pemphigoid, and mucocutaneous-type pemphigus vulgaris. Results: We identified several autoantigens unique to oMMP relative to other AIBDs. We then cross-referenced these antigens against previously published single-nuclei datasets, as well as the International Mouse Phenotyping Consortium Database. Several protein hits identified in our study demonstrated enriched expression on the anterior surface epithelia, including TNKS1BP1, SEC16B, FNBP4, CASZ1, GOLGB1, DOT1L, PRDM 15, LARP4B, and RPL6. Likewise, a previous study of mouse knockout models of murine analogs CASZ1, HIP1, and ELOA2 reported that these mice showed abnormalities in terms of the ocular surface and development in the eyes. Notably, PhIP-seq failed to identify the canonical markers of AIBDs such as BP180, BP230, desmogleins 1 and 3, or integrin β4, indicating that the patient autoantibodies react with conformational epitopes rather than linear epitopes. Conclusions: oMMP patients demonstrate a unique autoantibody repertoire relative to the other AIBDs. Further validation of the identified autoantibodies will shed light on their potentially pathogenic role. Full article

Shiga toxin-producing Escherichia coli are zoonotic pathogens that cause food-borne human disease. Among these, the O157:H7 serotype has evolved from an enteropathogenic O55:H7 ancestor through the displacement of the somatic gene cluster and recurrent toxigenic conversion by Shiga toxin-converting bacteriophages. However, atypical strains that lack the Shiga toxin, the characteristic virulence hallmark, are circulating in this lineage. For this study, we analyzed the pathogenome and virulence inventories of the stx+ strain, TT12A, isolated from a patient with hemorrhagic colitis, and its respective co-isolated stx− strain, TT12B. Sequencing the genomes to closure proved critical to the cataloguing of subtle strain differentiating sequence and structural polymorphisms at a high-level of phylogenetic accuracy and resolution. Phylogenomic profiling revealed SNP and MLST profiles similar to the near clonal outbreak isolates. Their prophage inventories, however, were notably different. The attenuated atypical non-shigatoxigenic status of TT12B is explained by the absence of both the ΦStx_1a- and ΦStx_2a-prophages carried by TT12A, and we also recorded further alterations in the non-Stx prophage complement. Phenotypic characterization indicated that culture growth was directly impacted by the strains’ distinct lytic phage complement. Altogether, our phylogenomic and phenotypic analyses show that these intimately related isogenic strains are on divergent Stx(+/stx−) evolutionary paths. Full article

Phages of phytopathogenic bacteria are considered to be promising agents for the biological control of bacterial diseases in plants. This paper reports on the isolation and characterisation of a new Xanthomonas campestris pv. campestris phage, Murka. Phage morphology and basic kinetic characteristics of the infection were determined, and a phylogenomic analysis was performed. The phage was able to lyse a reasonably broad range (64%, 9 of the 14 of the Xanthomonas campestris pv. campestris strains used in the study) of circulating strains of the cabbage black rot pathogen. This lytic myovirus has a DNA genome of 44,044 bp and contains 83 predicted genes. Taxonomically, it belongs to the genus Foxunavirus. This bacteriophage is promising for use as a possible means of biological control of cabbage black rot. Full article

Nowadays there is a growing interest worldwide in using bacteriophages for therapeutic purposes to combat antibiotic-resistant bacterial strains, driven by the increasing ineffectiveness of drugs against bacterial infections. Despite this fact, no novel commercially available therapeutic phage products have been developed in the last two decades, as it is extremely difficult to register them under the current legal regulations. This paper presents a description of the interaction between a bacteriophage manufacturer and a clinical institution, the specificity of which is the selection of bacteriophages not for an individual patient, but for the entire spectrum of bacteria circulating in the intensive care unit with continuous clinical and microbiological monitoring of efficacy. The study presents the description of three clinical cases of patients who received bacteriophage complex via inhalation for 28 days according to the protocol without antibiotic use throughout the period. No adverse effects were observed and the elimination of multidrug-resistant microorganisms from the bronchoalveolar lavage contents was detected in all patients. A decrease in such inflammatory markers as C-reactive protein (CRP) and procalcitonin was also noted. The obtained results demonstrate the potential of an adaptive phage therapy protocol in intensive care units for reducing the amount of antibiotics used and preserving their efficacy. Full article

Antimicrobial resistance is rising as a major global public health threat and antibiotic resistance genes are widely spread among species, including human oral pathogens, e.g., Aggregatibacter actinomycetemcomitans. This Gram-negative, capnophilic, facultative anaerobe is well recognized as a causative agent leading to periodontal diseases, as well as seriously systemic infections including endocarditis. A. actinomycetemcomitans has also evolved mechanisms against complement-mediated phagocytosis and resiliently survives in serum-rich in vivo environments, i.e., inflamed periodontal pockets and blood circulations. This bacterium, however, demonstrated increasing sensitivity to human serum, when being infected by a pseudolysogenic bacteriophage S1249, which switched to the lytic state as a response to human serum. Concomitantly, the pyruvate dehydrogenase complex (PDHc), which is composed of multiple copies of three enzymes (E1, E2, and E3) and oxidatively decarboxylates pyruvate to acetyl-CoA available for tricarboxylic acid (TCA) cycle, was found up-regulated 10-fold in the bacterial lysogen after human serum exposure. The data clearly indicated that certain human serum components induced phage virion replication and egress, resulting in bacterial lysis. Phage manipulation of bacterial ATP production through regulation of PDHc, a gatekeeper linking glycolysis to TCA cycle through aerobic respiration, suggests that a more efficient energy production and delivery system is required for phage progeny replication and release in this in vivo environment. Insights into bacteriophage regulation of bacterial fitness in a mimic in vivo condition will provide alternative strategies to control bacterial infection, in addition to antibiotics. Full article

Gut bacterial/viral dysbiosis, changes in circulating metabolites, and plasma cytokines/chemokines have been previously associated with various liver diseases. Here, we analyzed the associations between fecal microbial composition, circulating metabolites, and plasma cytokines/chemokines in patients with liver cirrhosis (LC) and hepatocellular carcinoma (HCC). We recruited 10 HCC patients, 18 LC patients, and 17 healthy individuals. Their stool samples were used for gene sequencing of bacterial 16S rRNA and viral genomes, while plasma samples were utilized for the determination of endotoxin, zonulin, metabolite, and cytokine/chemokine levels. Dysbiosis was observed among gut bacteria and viruses, with significant changes in abundance at the genus and species levels, respectively. However, no differences were found between cohorts in the alpha and beta diversity. Plasma lipopolysaccharides and zonulin, but not trimethylamine N-oxide, were progressively increased in LC and HCC subjects. Profiling plasma metabolites and selected cytokines/chemokines revealed differential changes in the LC and HCC cohorts. Following joint correlation and correlation network analyses, regardless of etiology, common network signatures shared by LC and HCC patients were characterized by the gut virus Stenotrophomonas virus DLP5 and the uncultured Caudovirales phage, plasma metabolites pyruvic acid and acetic acid, and plasma cytokines/chemokines eotaxin and PDGF-AB/BB, respectively. Additionally, LC- and HCC-specific correlation networks were also identified. This study provides novel insights into altered gut microbial/viral composition that may contribute to pre-HCC disorders, metabolic reprogramming, or inflammatory microenvironments for hepatocarcinogenesis. Full article

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated K_D of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated K_D of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (K_D of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo. Full article

Neisseria meningitidis, a bacterium that colonizes in the human nasopharynx, occasionally causes invasive meningococcal disease leading to meningitis or septicemia. Different serogroups and lineages (clonal complexes) are related to the occurrence and epidemiology of N. meningitidis. Despite vaccines for most serogroups, N. meningitidis lineages causing unusual clinical manifestations and a higher fatality rate compared to other lineages have been reported in South America. The present study focused on exploring the diversity of N. meningitidis prophages from South America and their relationship with the epidemiological variables of these strains. We found a high diversity of prophages among the different clonal complexes. By comparing them with previously described N. meningitidis phages and prophages, we revealed groups of prophages sharing similar compositions, which could be useful for prophage comparison in N. meningitidis. Furthermore, we observed a high correlation between the prophage content and epidemiological features, e.g., pathogenicity or clonal complex. Additionally, a distinctive filamentous prophage named here as IMSAR-11 (Invasive Meningococci from South America Related to cc11) was identified. Interestingly, two versions of IMSAR-11, circular and chromosomally integrated, were found. Overall, this study reinforces the importance of the genomic characterization of circulating N. meningitidis lineages to generate new targets for lineage monitoring, diagnosis, or appropriateness of vaccine development. Further studies are necessary to understand the role of these prophages in the persistence, dispersal, and virulence of N. meningitidis in the world. Full article

With concern growing over antibiotics resistance, the use of bacteriophages to combat resistant bacteria has been suggested as an alternative strategy with which to enable the selective control of targeted pathogens. One major challenge that restrains the therapeutic application of bacteriophages as antibacterial agents is their short lifespan, which limits their antibacterial effect in vivo. Here, we developed a polylactic-co-glycolic acid (PLGA)/alginate-composite microsphere for increasing the lifespan of bacteriophages in vivo. The alginate matrix in PLGA microspheres encapsulated the bacteriophages and protected them against destabilization by an organic solvent. Encapsulated bacteriophages were detected in the tissue for 28 days post-administration, while the bacteriophages administered without advanced encapsulation survived in vivo for only 3–5 days. The bacteriophages with extended fate showed prophylaxis against the bacterial pathogens for 28 days post-administration. This enhanced prophylaxis is presumed to have originated from the diminished immune response against these encapsulated bacteriophages because of their controlled release. Collectively, composite encapsulation has prophylactic potential against bacterial pathogens that threaten food safety and public health. Full article

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 10¹¹ PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control. Full article

Shiga toxin (stx) is the principal virulence factor of the foodborne pathogen, Shiga toxin-producing Escherichia coli (STEC) O157:H7 and is associated with various lambdoid bacterio (phages). A comparative genomic analysis was performed on STEC O157 isolates from cattle (n = 125) and clinical (n = 127) samples to characterize virulence genes, stx-phage insertion sites and antimicrobial resistance genes that may segregate strains circulating in the same geographic region. In silico analyses revealed that O157 isolates harboured the toxin subtypes stx1a and stx2a. Most cattle (76.0%) and clinical (76.4%) isolates carried the virulence gene combination of stx1, stx2, eae and hlyA. Characterization of stx1 and stx2-carrying phages in assembled contigs revealed that they were associated with mlrA and wrbA insertion sites, respectively. In cattle isolates, mlrA and wrbA insertion sites were occupied more often (77% and 79% isolates respectively) than in clinical isolates (38% and 1.6% isolates, respectively). Profiling of antimicrobial resistance genes (ARGs) in the assembled contigs revealed that 8.8% of cattle (11/125) and 8.7% of clinical (11/127) isolates harboured ARGs. Eight antimicrobial resistance genes cassettes (ARCs) were identified in 14 isolates (cattle, n = 8 and clinical, n = 6) with streptomycin (aadA1, aadA2, ant(3’’)-Ia and aph(3’’)-Ib) being the most prevalent gene in ARCs. The profound disparity between the cattle and clinical strains in occupancy of the wrbA locus suggests that this trait may serve to differentiate cattle from human clinical STEC O157:H7. These findings are important for stx screening and stx-phage insertion site genotyping as well as monitoring ARGs in isolates from cattle and clinical samples. Full article

In this work, we analysed human isolates of nontyphoidal Salmonella enterica subsp. enterica (NTS), which were collected from salmonellosis cases in Armenia from 1996 to 2019. This disease became a leading food-borne bacterial infection in the region, with the younger age groups especially affected. The isolates were characterised by serotyping, Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) typing, and whole genome sequencing (WGS). The main serotypes were S. Typhimurium, S. Enteritidis, and S. Arizonae. ERIC-PCR indicated a high degree of clonality among S. Typhimurium strains, which were also multidrug-resistant and produced extended spectrum beta-lactamases. During the study period, the frequency of S. Typhimurium and S. Arizonae isolations decreased, but with the increase in S. Enteritidis and other NTS. A total of 42 NTS isolates were subjected to WGS and explored for virulence-related traits and the corresponding genetic elements. Some virulence and genetic factors were shared by all NTS serotypes, while the main differences were attributed to the serotype-specific diversity of virulence genes, SPIs, virulence plasmids, and phages. The results indicated the variability and dynamics in the epidemiology of salmonellosis and a high virulence potential of human NTS isolates circulating in the region. Full article

Plague pandemics and outbreaks have killed millions of people during the history of humankind. The disease, caused by the bacteria Yersinia pestis, is currently treated effectively with antibiotics. However, in the case of multidrug-resistant (MDR) bacteria, alternative treatments are required. Bacteriophage (phage) therapy has shown efficient antibacterial activity in various experimental animal models and in human patients infected with different MDR pathogens. Here, we evaluated the efficiency of фA1122 and PST phage therapy, alone or in combination with second-line antibiotics, using a well-established mouse model of pneumonic plague. Phage treatment significantly delayed mortality and limited bacterial proliferation in the lungs. However, the treatment did not prevent bacteremia, suggesting that phage efficiency may decrease in the circulation. Indeed, in vitro phage proliferation assays indicated that blood exerts inhibitory effects on lytic activity, which may be the major cause of treatment inefficiency. Combining phage therapy and second-line ceftriaxone treatment, which are individually insufficient, provided protection that led to the survival of all infected animals—a synergistic protective effect that represents a proof of concept for efficient combinatorial therapy in an emergency event of a plague outbreak involving MDR Y. pestis strains. Full article

Cytolysin-positive Enterococcus faecalis (E. faecalis) cause more severe alcohol-associated hepatitis, and phages might be used to specifically target these bacteria in a clinical trial. Using a humanized mouse model of ethanol-induced liver disease, the effect of cytolytic E. faecalis phage treatment on the intestinal and liver immune response was evaluated. The observed immune response was predominantly anti-inflammatory and tissue-restoring. Besides, live phages could be readily recovered from the serum, spleen, and liver following oral gavage in ethanol-fed mice. We also isolated 20 new phages from the sewage water; six of them exhibited a relatively broad host range. Taken together, the oral administration of cytolytic E. faecalis phages leads to the translocation of phages to the systemic circulation and appears to be safe, following chronic-binge ethanol administration. A cocktail of three phages covers the majority of tested cytolysin-positive E. faecalis strains and could be tested in a clinical trial. Full article