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Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

Background Wheat dwarf virus (WDV) is a destructive cereal virus causing significant yield losses in wheat and barley. It is transmitted by the leafhopper Psammotettix alienus and can persist in wild grasses between growing seasons, making reliable detection and strain differentiation critical for disease management. Results We developed a comprehensive PCR toolkit for WDV by analysing 38 complete genome sequences, reviewing, validating, and upgrading existing primers and designing new primers spanning multiple viral genome regions. The primer toolkit achieved high diagnostic and analytical specificity as it consistently detected WDV in plants and insect vectors. This enabled the separation of WDV wheat- and barley-strains through a two-step workflow: screening with universal primers, and strain assignment with strain-specific primer pairs. Field testing across 13 Hungarian sites revealed barley strain dominance in the samples, infecting not only barley but also wheat and multiple grass species. Our surveys identified three previously undocumented reservoir grasses adding to the reviewed host range of 42 species. Complete genome sequencing of one wheat-strain and two barley-strain isolates confirmed > 99% intra-strain nucleotide identity but only ~ 85% between strains. Spatial mapping demonstrated virus concentration in grassy islands with declining titers toward cultivated areas, suggesting these serve as infection reservoirs. Conclusions This validated primer panel provides a robust framework for studying WDV epidemiology and developing targeted management strategies for this economically important pathogen. Understanding this model of virus-vector system and the improvement of the presented methods are key factors to combat other similarly operating plant-vector-pathogen systems.

Exploitation of engineered nanomaterials with unique properties has been dynamically growing in numerous fields, including the agricultural sector. Due to the increasing resistance of phytopathogenic microbes, human control over various plant pathogens in crop production is a big challenge and requires the development of novel antimicrobial materials. Photocatalytic active nanomaterials could offer an alternative solution to suppress the plant pathogens. In this work, titanium dioxide nanoparticles (TiO2 NPs) with high photocatalytic activity were synthesized by hydrothermal post-treatment of amorphous titania at different temperatures (250 °C or 310 °C) without using any additives or doping agents. The obtained samples were investigated through X-ray diffraction, N2-sorption measurements, diffuse reflectance UV-Vis spectroscopy, transmission electron microscopy, electron paramagnetic resonance spectroscopy, and X-ray photoelectron spectroscopy. The applied hydrothermal treatment led to the formation of TiO2 nanocrystallites with a predominant anatase crystal phase, with increasing crystallinity and crystallite size by prolonging treatment time. The photocatalytic activity of the TiO2 NPs was tested for the photo-degradation of phenol and applied for the inactivation of various plant pathogens such as Erwinia amylovora, Xanthomonas arboricola pv. juglandis, Pseudomonas syringae pv. tomato and Allorhizobium vitis. The studied bacteria showed different susceptibilities; their living cell numbers were quickly and remarkably reduced by UV-A-irradiated TiO2 NPs. The effectiveness of the most active sample prepared at 310 °C was much higher than that of commercial P25 TiO2. We found that fine-tuning of the structural properties by modulating the time and temperature of the hydrothermal treatment influenced the photocatalytic properties of the TiO2 NPs considerably. This work provides valuable information to the development of TiO2-based antimicrobial photocatalysts.

Contamination of meats and meat products with foodborne pathogenic bacteria raises serious safety issues in the food industry. The antibacterial activities of phosphorous-fluorine co-doped TiO2 nanoparticles (PF-TiO2) were investigated against seven foodborne pathogenic bacteria: Campylobacter jejuni, Salmonella Typhimurium, Enterohaemorrhagic E. coli, Yersinia enterocolitica, Shewanella putrefaciens, Listeria monocytogenes and Staphylococcus aureus. PF-TiO2 NPs were synthesized hydrothermally at 250 °C for 1, 3, 6 or 12 h, and then tested at three different concentrations (500 μg/mL, 100 μg/mL, 20 μg/mL) for the inactivation of foodborne bacteria under UVA irradiation, daylight exposure or dark conditions. The antibacterial efficacies were compared after 30 min of exposure to light. Distinct differences in the antibacterial activities of the PF-TiO2 NPs, and the susceptibilities of tested foodborne pathogenic bacterium species were found. PF-TiO2/3 h and PF-TiO2/6 h showed the highest antibacterial activity by decreasing the living bacterial cell number from ~106 by ~5 log (L. monocytogenes), ~4 log (EHEC), ~3 log (Y. enterolcolitca, S. putrefaciens) and ~2.5 log (S. aureus), along with complete eradication of C. jejuni and S. Typhimurium. Efficacy of PF-TiO2/1 h and PF-TiO2/12 h NPs was lower, typically causing a ~2–4 log decrease in colony forming units depending on the tested bacterium while the effect of PF-TiO2/0 h was comparable to P25 TiO2, a commercial TiO2 with high photocatalytic activity. Our results show that PF-co-doping of TiO2 NPs enhanced the antibacterial action against foodborne pathogenic bacteria and are potential candidates for use in the food industry as active surface components, potentially contributing to the production of meats that are safe for consumption.

Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to Caudovirales, Siphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45 kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.

Contamination of meats and meat products with foodborne pathogenic bacteria raises serious safety issues in the food industry. The antibacterial activities of phosphorous-fluorine co-doped TiO nanoparticles (PF-TiO ) were investigated against seven foodborne pathogenic bacteria: , Typhimurium, Enterohaemorrhagic , , , and . PF-TiO NPs were synthesized hydrothermally at 250 °C for 1, 3, 6 or 12 h, and then tested at three different concentrations (500 μg/mL, 100 μg/mL, 20 μg/mL) for the inactivation of foodborne bacteria under UVA irradiation, daylight exposure or dark conditions. The antibacterial efficacies were compared after 30 min of exposure to light. Distinct differences in the antibacterial activities of the PF-TiO NPs, and the susceptibilities of tested foodborne pathogenic bacterium species were found. PF-TiO /3 h and PF-TiO /6 h showed the highest antibacterial activity by decreasing the living bacterial cell number from ~10 by ~5 log ( ), ~4 log (EHEC), ~3 log ( , ) and ~2.5 log ( ), along with complete eradication of and Typhimurium. Efficacy of PF-TiO /1 h and PF-TiO /12 h NPs was lower, typically causing a ~2-4 log decrease in colony forming units depending on the tested bacterium while the effect of PF-TiO /0 h was comparable to P25 TiO , a commercial TiO with high photocatalytic activity. Our results show that PF-co-doping of TiO NPs enhanced the antibacterial action against foodborne pathogenic bacteria and are potential candidates for use in the food industry as active surface components, potentially contributing to the production of meats that are safe for consumption.

A dolgozat indokoltsága, tartalmaPhD munkám keretében lítikus bakteriofágok tulajdonságait és potenciális antibakteriális felhasználási lehetőségeit vizsgáltam. A munka jelentősége abban áll, hogy napjainkban a multidrog rezisztencia terjedése miatt a bakteriofágok terápiás célzatú felhasználását célzó ismeretek iránt fokozódik az érdeklődés.A dolgozat felépítéseMunkám két párhuzamosan futó részre bontható.Az első, nagyobbik és részletesebb rész egy K2 szerotípusú Klebsiella pneumoniae törzsön erős lítikus aktivitással rendelkező bakteriofág (B1) izolálására és jellemzésére fókuszál, valamint ezen fág tokbontó depolimerázának kifejezésére és karakterizálására. Ezen részben egy másik Klebsiella fágot (731) is jellemeztünk, azt az előbbivel összehasonlítva.A dolgozat másik része tartalmazza a Staphylococcus aureusfágokkal kapcsolatos munkák eredményeit, ahol szintén két fág (A1 és R4) kerül bemutatásra, egymással párhuzamosan.Jóllehet, az egyensúly néhol el-elbillent egyik vagy másik kórokozó fágjainak irányába (pl. Staphylococcus biofilm-degradálás, Klebsiella fág receptor-analízis), a koncepció a Klebsiella és Staphylococcus fág-párokkal kivitelezett kísérletek véghezvitelére vonatkozóan mégis hasonló vonalat követ, emiatt az irodalmi áttekintéshez hasonlatosan az „Anyagok és módszerek” fejezetet egyben, az összes fágra vonatkozólag közöljük, külön említve azon módszereket, melyek egyik vagy másik fág vizsgálatával függtek össze. Az „Eredmények I.” fejezetben a Klebsiella fágok, az „Eredmények II.” fejezetben a Staphylococcus fágok jellemzőit részletezzük. Az azonos jellegű eredmények prezentálási sorrendje a két részben nem megegyező: ez abból adódik, hogy a Klebsiella és a Staphylococcus fágok esetében más tulajdonságokra, vizsgálatokra fókuszáltunk (pl. más enzimekre, biofilmre stb.), így az egymásból következő, egymáshoz köthető eredményeket igyekeztünk folyamatában, egymás után közölni. A Diszkusszióban szintén külön, egymás után tekintjük át a két fágcsoport eredményeiből levont következtetéseket.