Search Results (236)

Multidrug-resistant (MDR) strains of Klebsiella pneumoniae present an acute threat as they continue to disseminate globally. Phage therapy has shown promise as a powerful approach to combat MDR infections, but narrow phage host ranges make development of broad acting therapeutics more challenging. The goal of this effort was to use in vitro directed evolution (the “Appelmans protocol”) to isolate K. pneumoniae phages with broader host ranges for improved therapeutic cocktails. Five myophages in the genus Jiaodavirus (family Straboviridae) with complementary activity were mixed and passaged against a panel of 11 bacterial strains including a permissive host and phage-resistant clinical isolates. Following multiple rounds of training, we collected phage variants displaying altered specificity or expanded host ranges compared with parental phages when tested against a 100 strain diversity panel of K. pneumoniae. Some phage variants gained the ability to lyse previously phage-resistant strains but lost activity towards previously phage-susceptible strains, while several variants had expanded activity. Whole-genome sequencing identified mutations and recombination events impacting genes associated with host tropism including tail fiber genes that most likely underlie the observed changes in host ranges. Evolved phages with broader activity are promising candidates for improved K. pneumoniae therapeutic phage cocktails. Full article

A dearth of knowledge exists on identifying the begomoviruses and distributing cassava mosaic viruses across key cassava-growing regions of Sierra Leone. The study aimed to identify and map the distribution of cassava mosaic disease (CMD)-associated viruses in farmers’ fields in Sierra Leone. Cassava (Manihot esculenta Crantz) leaf samples were collected in 109 smallholder farms during a geo-referenced survey conducted from 10th May to 5th June 2024. Molecular diagnostics were carried out to identify the viral strains associated with CMD. Findings revealed that infection by stem cutting was more predominant in the south, east, north, and northwest regions than in the west region. In contrast, infection by whitefly was predominant in the west, north, and northwest regions. PCR screening of 426 samples coupled with sequence analysis revealed the presence of African cassava mosaic-like (ACMV-like) viruses, and East African cassava mosaic-like (EACMV-like) viruses as single infections at 78.1% and 1.3%, respectively. Co-infections of ACMV-like and EACMV-like viruses were detected in 20.6% of the tested samples. In addition, 70.6% of the samples positive for EACMV-like virus (single and mixed infections) were found to be positive for East African cassava mosaic Cameroon virus (EACMCMV). The ACMV and co-infection of ACMV and EACMV viruses were present in all regions, while EACMCV was detected in all regions except the western area. The results indicate more prevalence of the EACMCMV variant in Sierra Leone. This study suggests utilization of participatory surveillance and good agronomic practices to manage CMD in Sierra Leone. Full article

Background:Salmonella Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain—a double auxotroph for D-glutamate and D-alanine—by engineering the inducible expression of type 1 fimbriae. Methods: P_tetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. Results: IRTA ΔΔΔ P_tetA::fim showed robust fimA expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P_tetA::fim reduced infection burden in the cecum (−1.46/1.47-log), large intestine (−1.35/2.17-log), mLNs (−1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery. Full article

This review summarizes the current knowledge on the probiotic characteristics of dairy propionibacteria, represented by Propionibacterium freudenreichii and some Acidipropionibacterium species commonly consumed through raw milk cheese. For example, in Swiss-type cheeses, P. freudenreichii is added as a starter culture. Some strains of P. freudenreichii have been included in mixed probiotic commercial preparations or used to produce tablets from fermented culture media containing bioactive substances such as short-chain fatty acids (SCFAs), bifidogenic molecules, and vitamins. Acidipropionibacterium acidipropionici and A. jensenii strains have mainly been evaluated as health and productivity promoters in farm animals. For P. freudenreichii, the molecular mechanisms behind its probiotic action have been well elucidated, and recently, novel potential applications have been demonstrated in animal models. P. freudenreichii strains have been shown to mitigate inflammatory bowel diseases (IBDs) and mucositis and prevent necrotizing enterocolitis (NEC) in newborns. Their immunomodulation capacity has alleviated symptoms of food allergies, obesity, diabetes, colorectal cancer (CRC), and infections. Moreover, P. freudenreichii inhibited osteoclastogenesis in a rheumatoid arthritis model. Most observed effects are mediated by proteins on the cell surface or contained in extracellular vesicles (EVs) such as the surface layer (S-layer) protein SlpB, DlaT, and GroEL. No safety issues have been reported for these bacteria. However, investigations into transferable antibiotic resistance traits are still needed, and clinical trials are required to evaluate their effectiveness as probiotics for humans. Full article

Background: Group A Streptococcus (GAS) is a major human pathogen associated with serious diseases. Evaluating immune responses against GAS vaccines—immunogenicity, quality, and efficacy—is complicated by interference from co-infections, like Staphylococcus aureus (S. aureus). We aimed to evaluate peptide-based GAS vaccines in mice for antisera efficacy against standard and mutant GAS strains and to assess immunological methods under co-infection conditions. Methods: Female C57BL/6 mice were infected with S. aureus and immunized with various M-protein-derived peptide antigens: J8, J8i, J8i-J8i, and the native p145 sequence. Two novel, conserved M-protein-derived antigens (NTD and CTD2) were also evaluated. Enzyme-linked immunosorbent assays (ELISAs) were used to assess immunogenicity and GAS-specific antibody responses. Peptide antigens were either conjugated to or physically mixed with the PADRE T-helper epitope and tested for enhanced antisera immunogenicity and opsonic efficacy. Result: ELISA against the immunizing peptides as coating antigens reflected the immunogenicity, while p145-based ELISA correlated with GAS-specific antibody titres without S. aureus interference for J8-based vaccines. Immunogenicity ranked J8 > J8i ≈ J8i-J8i > p145. NTD and CTD2 antisera demonstrated opsonic activity, indicating protective potential. PADRE–J8 conjugates significantly enhanced antibody magnitude and quality, producing strong opsonic bactericidal responses against both standard and p145-mutant GAS strains. PADRE–J8i was effective only against standard strains. This is the first report to suggest at least two B-cell epitopes within the J8i peptide. Conclusion: These findings support the diagnostic utility of p145, NTD, and CTD2 under co-infection settings, and the vaccine potential of J8, NTD, and CTD2, particularly when conjugated to a T helper for enhanced antigen presentation. Full article

Diarrhea is a common disease in sika deer. The causes of diarrhea in sika deer are complex and involve a variety of pathogens. Additionally, new virulent pathogens are continuously emerging, which poses a serious threat to deer’s health and particularly affects fawns’ survival rate. In the present study, feces samples were collected from fawns with diarrhea in Jilin Province, in the northeast of China. The viral communities were investigated using the metagenomic method. Viral metagenome data revealed that the viruses in the fecal samples were mainly from 21 families in 14 orders. The major viruses in high abundance were astrovirus, rotavirus, coronavirus, and bovine viral diarrhea virus. In addition, a large number of phages, which mainly belonged to the family Siphoviridae, were identified. Then, the known causative virus species were investigated via RT-qPCR. The results showed that the infection rates of bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus were 59.44%, 58.89%, and 21.67%, respectively, and mixed infections were commonly seen in the samples. A bovine rotavirus strain was successfully isolated from the positive samples. Whole-genome sequencing revealed that the genotype of the strain was G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3, indicating the recombination of rotavirus. This study revealed the profiles and characteristics of viruses that cause sika deer diarrhea, which will be helpful for understanding diarrhea diseases in sika deer. Full article

Mixed infections of plant viruses are commonly found in natural patho-systems and present a valuable opportunity to understand how multiple viruses can co-infect the same host. Tomato spotted wilt orthotospovirus (TSWV) and impatiens necrotic spot orthotospovirus (INSV) are present in the same geographic areas and are closely related. More mixed infections of TSWV and INSV have been reported in recent years, and the INSV host range has been reported to be increasing. In a previous study, we isolated and characterized one strain of INSV and one of TSWV and found that they have an antagonistic relationship in their vectors. However, we were unable to determine whether this antagonism extends to the host plant or to uncover the underlying mechanisms and the host’s contribution. Here, we show that TSWV and INSV exhibit antagonistic interactions in the host plant, as evidenced by a lower viral titer in mixed infections compared to single infections. Using small RNA sequencing, we identified that the host plant contributes to this antagonism through differential small RNA processing, which appears to regulate viral replication and the success of infection. This research advances our understanding of virus–virus and virus-host interactions and presents opportunities for leveraging these dynamics in integrated pest management strategies. Full article

A biofilm is a group of bacterial cells in the polysaccharide matrix bonded to the surface (biotic or abiotic). Clinicians now realize that most infections are biofilm-related. Biofilm infections are often induced by more than one bacterial species. The aim of this study is to characterize a mixed biofilm composed of Pseudomonas aeruginosa and Proteus mirabilis strains. Forty-six isolates derived from chronic wound infections were cultivated to establish mature biofilms. The biofilm biomass and cell viability were measured by colorimetric assays. P. aeruginosa strains were tested for the presence of virulence and biofilm-related genes. The quorum sensing assay using the biosensor strain was also performed. A mixed biofilm of P. aeruginosa and P. mirabilis was visualized using fluorescence microscopy. Four groups of P. aeruginosa and P. mirabilis pairs, also visualized with fluorescence microscopy, were distinguished based on the biofilm biomass growth and metabolic activity loss. The exoY gene observed among P. aeruginosa isolates was connected to the metabolic activity loss of the biofilm. Generally, the interactions between P. aeruginosa and P. mirabilis species are not uniform. It is crucial to further research the interactions between microorganisms in biofilms. This may provide information on the mechanisms of biofilm formation in the complicated chronic wound environment. Full article

Canine circovirus (CanineCV) is an emerging virus of interest in both domestic and wild carnivores that is scarcely reported in southeastern Europe. This study examined the presence, genetic diversity, and evolutionary characteristics of CanineCV in red foxes (Vulpes vulpes) and golden jackals (Canis aureus) from northwestern Serbia, a region marked by expanding mesopredator populations overlapping with human habitats. Out of 98 sampled animals, circoviral DNA was detected in 31.6%. Jackals were mostly positive for CanineCV genotype 4, while genotype 5, associated with wild carnivores, was dominant in foxes. Mixed genotype 4/genotype 5 infections were only found in jackals. Phylogenetic and haplotype analyses indicated that most jackal-derived CanineCV strains clustered along sequences from Europe, Africa, and the Americas, while genotype 5 sequences grouped separately from other genotype representatives. A recombinant strain was identified as a divergent lineage, and several sequences showed evidence of recombination between Rep and Cap genes. Despite Cap protein amino acid differences, purifying selection dominated, suggesting functional constraints on viral evolution. The results indicate that jackals may act as recombination hotspots and bridging hosts between viral lineages. This study provides insight into the molecular epidemiology of CanineCV in the Balkans, highlighting the importance of ongoing surveillance. Full article

Background/Objectives: A 43-year-old male presented with neurological symptoms and asymptomatic cardiac dysfunction, left ventricular hypertrophy, and impaired global longitudinal strain with apical sparing, associated with elevated NT-proBNP. Methods: Multimodality imaging (bone scintigraphy and cardiac magnetic resonance) revealed cardiac amyloid deposition. Genetic testing confirmed variant transthyretin amyloidosis (ATTR) with mixed phenotype. Results: Treatment with tafamidis 20 mg for stage I polyneuropathy, available at that moment, was initiated with good neurological outcome. Three years later, cardiac function deteriorated, following a moderate COVID-19 infection, with heart failure symptoms and reduced ventricular and atrial functions. For progressive ATTR cardiomyopathy, we intensified therapy to tafamidis free acid 61 mg, associated with SGLT2 inhibitor, spironolactone, and furosemide with subsequent improvements of symptoms and stabilization of imaging findings. Conclusions: This case emphasizes the importance of multimodal imaging in early detection, monitoring, and guiding individualized management in ATTR cardiomyopathy. Full article

The Coronaviridae family has again demonstrated the potential for significant neurological complications in humans during the recent pandemic. In patients, these symptoms persist throughout the infection, often lasting for months. The consequences of most of these post-infection symptoms might be linked with abnormal cytokine production and reactive oxygen species (ROS) expression, resulting in neuron damage. We investigated the effect of infection with the Mouse Hepatitis Virus (MHV) JHM strain and Sialodacryoadenitis Virus (SDAV) on a primary microglia and astrocyte culture by analysing ROS production, cytokine and chemokine expression, and cell death during one month post infection. For this purpose, confocal microscopy, flow cytometry, and a high-throughput Luminex ProcartaPlex immunopanel for 48 cytokines and chemokines were utilised. The replication of MHV-JHM and SDAV in microglia and astrocytes has increased the production of pro-inflammatory cytokines and inhibited the production of anti-inflammatory cytokines. The cytokine expression induced by the two viruses differed, as did their detection after infection. SDAV infection resulted in a much broader cytokine response compared to that of MHV-JHM. Both viruses significantly increased ROS levels and induced apoptosis in a small percentage of the cells, but without necrosis. Full article

High-throughput sequencing (HTS) was employed for the first time to investigate plant viruses infecting solanaceous crops, including potato (Solanum tuberosum), tomato (Solanum lycopersicum), and pepper (Capsicum annuum), in Kosovo. Leaf samples showing virus-like symptoms were collected from various regions during the summer of 2023. Based on ribodepleted RNA sequencing and bioinformatics analysis, six viruses were identified: cucumber mosaic virus, broad bean wilt virus 2 (BBWV2), potato virus Y, pepper cryptic virus 2 (PCV2), bell pepper endornavirus (BPEV), and ranunculus white mottle virus. BBWV2, PCV2, and BPEV are reported for the first time in the Balkan region. Virus presence was validated using RT-PCR. Phylogenetic analyses revealed that the identified viral strains did not cluster according to their hosts and geographical origins. CMV and BBWV2 variants exhibited reassortment events, indicating possible local evolution or novel virus introductions. This research highlights the widespread occurrence of mixed infections in pepper plants and highlights the need for additional research into the virus transmission dynamics and potential reservoir hosts. These findings emphasize the need for continuous surveillance and integrated plant protection strategies to mitigate the impacts of viral infections on pepper and other economically important crops in Kosovo. Full article

Background Members of the Mycobacterium avium complex (MAC) have been documented to cause severe and disseminated infections in dogs, although such cases are sporadically reported. In this study, a comprehensive account of a rare case of generalised lymphadenomegaly caused by a mixed-strain infection with drug- and multidrug-resistant Mycobacterium avium subspecies hominissuis (Mah) in a Maremma sheepdog is presented. Methods Laboratory investigations, as well as the monitoring of the clinical signs displayed by the animal, were conducted throughout the course of a two-year drug therapy (based on rifampicin, azithromycin, and ciprofloxacin) and a two-year post-treatment follow-up period, until the death of the dog. Laboratory examinations included both solid and broth cultures from fine-needle aspiration samples of lymph nodes, molecular typing by 8-locus MIRUVNTR analysis and SNPs typing of five genetic regions (gyrB, rpsA, 3′hsp65, ITS and rpoB), and drug susceptibility testing towards seven antimycobacterial drugs. Results The results indicated the presence of two distinct genotypes of Mah, which exhibited different phenotypic characteristics, such as different drug susceptibility profiles and growth abilities in broth and solid media, suggesting a mixed-strain infection. Resistances to ethambutol alone, to ethambutol and clarithromycin, and to ethambutol, clarithromycin, rifampicin, and doxycycline were detected over the study. Conclusions Although the Mah strains isolated during the course of therapy showed sensitivity to the regiment, the complete eradication of the infection was never achieved. It has been hypothesised that the presence of drug-resistant and multidrug-resistant Mah strains in the animal may have been established at the onset of the infection or soon thereafter. The exposure to therapy has been suggested as a potential factor that could have favoured the growth of resistant strains, thereby rendering the therapy ineffective. The implications that the distinct phenotypic and genotypic profiles of Mah described here may have had for disease dynamics and control are discussed. Full article

Background: Helicobacter pylori is a Gram-negative bacterium responsible for various gastrointestinal diseases, including peptic ulcers and gastric cancer. Despite available antibiotic therapies, increasing resistance to clarithromycin—a key antibiotic in eradication regimens—poses a significant challenge. This resistance is primarily linked to point mutations in the 23S rRNA gene, particularly A2143G, A2142G, and A2142C, which hinder clarithromycin binding, reducing its bacteriostatic efficacy. This study aimed to assess the prevalence and variability of clarithromycin resistance mutations in pediatric patients from Bydgoszcz, Poland. Methods: A total of 45 gastric biopsy samples from pediatric patients were analyzed using the Bosphore^® Helicobacter pylori Genotyping Kit v1 to detect clarithromycin resistance-associated mutations. Results: Among the 45 tested samples, 30 were classified as wild-type, while 12 contained resistance-associated mutations. The most frequently detected mutation was A2143G (58.3%), followed by A2142G (33.3%). One sample exhibited both A2142G and A2143G mutations, and another contained a mixture of wild-type and mutant strains. The A2142C mutation was not detected in any sample. Conclusions: Our findings confirm the predominance of A2143G among clarithromycin-resistant H. pylori strains, consistent with global trends. The detection of both mutant and wild-type strains in a single patient highlights potential co-infections or subpopulations with varying resistance profiles. Continuous surveillance and improved diagnostic tools are crucial for optimizing treatment strategies. Tailored eradication protocols based on resistance profiling are necessary to enhance treatment efficacy and mitigate the spread of resistant strains. Further research is needed to understand the clinical implications of mixed infections and double mutations in H. pylori resistance development. Full article

Mycoviruses are viruses that infect fungi, including plant pathogens. The infection of these mycoviruses is sometimes associated with impaired phenotypes of their fungal hosts, a phenomenon known as hypovirulence. Thus, using mycoviruses as biological control agents has emerged as a promising tool to combat forest diseases. The invasive ascomycete fungus Fusarium circinatum, which causes pine pitch canker (PPC) disease in Pinus tree species and other coniferous trees, is infected by the mycovirus Fusarium circinatum mitovirus 1 (FcMV1), FcMV2-1, and FcMV2-2. However, its impact on pathogen fitness remains unclear. The most accurate method used to identify the effect of a mycovirus on its host is the generation of isogenic lines with and without the mycovirus. The present study aimed to cure F. circinatum isolates infected by FcMV1 using different approaches. For this purpose, three replicates of each isolate were exposed to thermal treatment (38 °C) and antibiotic treatment (ribavirin, cycloheximide, kanamycin, and rifampicin mixed with cAMP)(cyclic adenosine monophosphate) for five successive passages. The viral titer of FcMV1 was then assessed using qPCR (quantitative polymerase chain reaction) after the first week and after the fifth week of the treatment. The results revealed differences in treatment efficacy among F. circinatum isolates, with some showing very low virus titers at the end of the experiment. Both thermal and antibiotic treatment effectively reduced the viral load in all isolates. In addition, the antibiotic cycloheximide and rifampicin +cAMP reduced the viral titer more than ribavirin and kanamycin. The isolate Fc179 was found to be more prone to antibiotic treatment than the other two isolates (001 and Va221). This study demonstrated the possibility of using some isolates of F. circinatum for fine-tuning cures for mitovirus, in order to create virus-free strains for biological control in the future. Full article