Isolation and characterization of bacteriophages specific to Acidovorax citrulli

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon worldwide. Biological approach in the disease control might be a potential solution and substitute for available bactericides of poor efficacy. Therefore, we isolated twelve bacteriophage strains specific to A. citrulli from rhizosphere of watermelon plants showing symptoms of the disease. The strains were characterized based on host range, plaque and virion morphology, thermal point of inactivation, adsorption rate, one step growth curve, and RFLP analysis. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related species. They produced clear plaques on bacterial lawn of different A. citrulli strains after 24 h of incubation. Examination by transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. The thermal inactivation point of phages was 66 or 67 °C. They were sensitive to chloroform, stable in pH 5-9, but inactivated after 5-10 min exposure to UV. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages

Unveiling therapeutic potential of bacteriophage treatment in acinetobacter baumannii-infected zebrafish embryo model

Introduction: There is an urgent demand for the development of new therapeutic approachesto combat multidrug-resistant Acinetobacter baumannii, and bacteriophages appear to be a highly promising solution. Phages are suitable to precisely target the infection-causing bacteria without disrupting the beneficial microbiota. The zebrafish (Danio rerio) embryo model represents an insightful animal model for preclinical studying of various infectious diseases and for discovery of novel safe and effective antimicrobial drugs. Methods: Systemic bacterial infection was established by microinjection of 2000 cells of nosocomial carbapenem-resistant A. baumannii strain 6077/12 into the bloodstream of 48 hour old zebrafish embryos. Infected embryos were treated by parenteral administration of 4 different doses (10, 50, 100, 500 PFU) of bacteriophage vB_AbaM_ISTD at 6 hours after infection (hpi). Efficacy of treatment was evaluated according to embryo survival, morphological malformations and bacterial burden (CFU) over a 3- day period. Results: A. baumannii-infected embryos treated with bacteriophage resulted with 100% survival rate, while 70% of untreated embryos survived to 24 hpi and none to the end of the experiment. Viable bacterial cell count and embryo morphology observations indicated that the administered phage effectively reduced A. baumanii infection in vivo. The most effective dose was 500 PFU, decreasing the bacterial load by 3.09 log units during 24 hpi, while lower bacteriophage doses(10, 50 and 100 PFU) produced less prominent, but also significant bacterial reduction of 2.10, 2.19 and 2.67 log units, respectively. Conclusion: Parentheral administration of phage ISTD demonstrated potent therapeutic activity against A. baumannii infection in every investigated dose

Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5–9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days

EMPOWERING ANTIFUNGAL DRUGS DISCOVERY THROUGH THE ZEBRAFISH-INFECTIOUS DISEASES MODELLING

Fungal infections, once considered a rare disease, have become an everyday problem in modern societies, posing major challenges to global health. It is estimated that more than one billion people are affected by fungal infections and 1.6 million people succumb to these diseases every year. Of the 600 species of fungi capable of causing infections in humans, species of the genus Candida cause more than 85% of infections, especially C. albicans, which has become a serious threat to human health in immunocompromised and immunosuppressed individuals. Unfortunately, the current arsenal of clinical drugs relies on only four classes of approved drugs (polyenes, azoles, echinocandins and allylamines), which are only partially effective, resulting in incomplete eradication of the fungal infection. In addition, the serious side effects, ranging from systemic or organ-specific toxicity to poor bioavailability and low activity, significantly hamper the clinical use of antifungals. These problems call for new effective and safe antifungal agents,but also for appropriate preclinical models to accurately study potential adverse effects on the human population and test their efficacy against fungal infections. In this sense, zebrafish (Danio rerio) embryos have become one of the most powerful preclinical animal models in infection biology and drug discovery, offering the unique opportunity to simultaneously monitor the safety and efficacy of the applied molecule in real time. With the aim of providing a preclinical platform for the identification of new safe antifungal drugs to effectively control C. albicans infection, we comprehensively tested the toxicity of 13 clinical antifungal drugs in the zebrafish embryo model. The 21 toxicity endpoints, including survival, teratogenicity, cardiotoxicity and hepatotoxicity, were evaluated and compared with adverse effects described in rats and humans. Of the clinical drugs, the efficacy of fluconazole and voriconazole was evaluated in the zebrafish - C. albicans model of systemic and wound biofilm infection.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-ResistantAcinetobacter baumannii

Acinetobacter baumanniiis a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistantA. baumanniistrain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates ofA. baumanniifrom a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combatingA. baumanniiinfections

The importance of tests applied to evaluate the effectiveness of antiplatelet therapy in patients with recurrent coronary stent thrombosis

Background. Stent thrombosis is potentially lethal complication with huge economic burden. The role of insufficient response to antiplatelet therapy is still unclear reason for its occurrence. Case report. We presented 54-year-old man with recurrent stent thrombosis on the 4th, 9th and 12th day after the primary percutaneous coronary intervention in spite of double antiaggregation therapy (aspirin+ clopidogrel). All possible procedural causes were excluded and reimplantation of intracoronary stent was insufficient to resolve the problem, so four platelet tests were performed: flow cytometry, Platelet Function Analyzer-100 test, aggregometry, and determination of gene polymorphism for P2Y12 receptor (directly involved in the mechanism of thienopyridine), and GPIIbIIIa receptor (final receptor in aggregation). The patient was the carrier of the major haplotype H1H1 for P2Y12 receptor and minor A1A2 for GPIIbIIIa receptor. The results of all the performed tests showed insufficient antiplatelet effect of aspirin and sufficient response to thienopyridin (not to clopidogrel, but to ticlopidine). Conclusion. Performance of platelet function tests is necessary in the case of major adverse cardiac events especially stent thrombosis, after implantation of intracoronary stent

MEDICINAL MUSHROOM EXTRACTS ATTENUATE PSEUDOMONAS AERUGINOSA QUORUM SENSING AND VIRULENCE

Pseudomonas aeruginosa has been recognized as a priority pathogen by World Health Organization, due to the emergence of multidrug-resistant (MDR) strains. Thus, new treatment options such as antivirulence strategy is urgently needed. This strategy is based on the disruption of quorum sensing (QS) activity of this pathogen. The focus of this research was to explore the anti-QS activity of four selected medicinal mushrooms (Lentinula edodes, Cantharellus cibarius, Trametes versicolor and Pleurotus ostreatus) extracts on MDR clinical isolate P. aeruginosa MMA83. Another aim was to check their cytotoxicity on Caenorhabditis elegans AU37 (glp-4(bn2) I; sec-1(km4). Among three types of mushroom extracts - hot water polysaccharide extracts (WPE), hot alkali polysaccharide extracts (APE) and methanol extracts (Met), APE extracts downregulated all tested QS and virulence factors genes of P. aeruginosa MMA83. The most prominent effect was observed for C. cibarius APE extract, lowering expression from 2-fold (for lasI gene) to 20-fold for lasB gene. Extracts didn’t show cytotoxic effect on C. elegans. The efficacy of APE extracts in lowering the expression of QS and virulence factors genes of P. aeruginosa MMA83 indicate that these extracts can reduce pathogenicity of P. aeruginosa. Also, they possess one of the desirable biotechnology features – the absence of cytotoxicity. Anti-QS and antivirulence effect of APE extracts on P. aeruginosa envisages these extracts as the promising therapeutic candidates for the development of next-generation antivirulence agents.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

NOVEL BACTERIOPHAGE ISOLATION FROM BELGRADE WASTEWATERS

Anti-microbial drug resistance (AMR) is one of the global health threats caused by the misuse of drugs typically used to treat microbial infections in humans, animals and plants. AMR in nosocomial infections not only significantly hinders treatment and endangers the patients’ lives, but also elevates the costs of healthcare. Multiple research approaches have been initiated to combat AMR, and one promising method is bacteriophage therapy. Bacteriophages (phages) are viruses that naturally exploit bacteria as their hosts for replication and can cause cell lysis, which makes them promising candidates for treating the infections that do not respond to conventional antibiotic therapies. In this study, we screened wastewater samples from four different collectors in Belgrade urban area for bacteriophages active against clinically isolated strains of two biofilm-producing bacteria that readily persist in hospital environment - Klebsiella pneumoniae (6 strains) and Pseudomonas aeruginosa (2 strains). Wastewaters were screened for phage presence through phage enrichment process, in which bacteria were grown in a mixture of water samples and nutrient-rich broth. Obtained cultures were screened for antimicrobial activity against the respective host strains, and candidates were subjected to a first-round plaque assay to detect the phages. Finally, the activity of all the candidates was tested against all strains of the same species to gain the first insight into their host range. We discovered 20 potentially distinct bacteriophages active against K. pneumoniae strains and two potentially different candidates targeting P. aeruginosa. Notably, one phage exhibited activity against all tested K. pneumoniae strains, and four were active against 5 out of 6 tested strains. Among 22 candidates in total, five showed depolymerizing activity, indicating promise in combating biofilm formation. Currently, isolation of new phages, as well as purification and host range analysis is underway for several candidates targeting K. pneumoniae and two targeting P. aeruginosa strains.Book of abstract: From biotechnology to human and planetary health XIII congress of microbiologists of Serbia with international participation Mikromed regio 5, ums series 24: 4th – 6th april 2024, Mona Plaza hotel, Belgrade, Serbi

Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome

Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-termeffect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated.Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region(mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library.Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not leadto potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positivechanges, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatmenthad a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatmentcannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainlyreflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey.Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees

Short-term effect of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome

Introduction: Brevibacillus laterosporus is a promising microbiological agent that can be used to prevent and control destructive diseases affecting honey bee colonies. In the presentstudy, the short-term effect of the B. laterosporus BGSP11 bee diet on microbiota and mycobiota was investigated. Methods: The honey bee diet was supplemented with spores of B. laterosporus BGSP11 at a concentration of 1×108 CFU/mL in sucrose solution. Metabarcoding analysis of the bee microbial community profile was performed based on 16S RNA (bacteriobiota) and Internally Transcribes Spacer (ITS) region (mycobiota) obtained using MiSeq Illumina sequencing. The QIIME2 v2021.4 pipeline was used to analyze the obtained amplicon data library. Results: The results show that the BGSP11 bee diet slightly altered the bee microbiota and did not lead to potentially harmful changes in the bacterial microbiota. Moreover, it can potentially induce positive changes, mainly reflected in the reduction of opportunistic bacteria. On the other hand, the treatment had a greater effect on mycobiota. However, the changesin the bee mycobiome caused by the treatment cannot be considered a priori as beneficial or harmful,since the interaction between the bee and its mycobiome is not sufficiently studied. The observed positive changes in the bee mycobiome are mainly reflected in the reduction of phytopathogenic fungi that may affect the organoleptic and techno-functional properties of honey. Conclusion: This pilot study suggests that the introduction of BGSP11 in beekeeping practice as a biological agent could be considered due to no harmful effects observed on the microbiota of bees