Relative Virulence Research Articles

A Novel and Robust Method for Investigating Fungal Biofilm.

Candida auris, labeled an urgent threat by the CDC, shows significant resilience to treatments and disinfectants via biofilm formation, complicating treatment/disease management. The inconsistencies in biofilm architecture observed across studies hinder the understanding of its role in pathogenesis. Our novel in vitro technique cultivates C. auris biofilms on gelatin-coated coverslips, reliably producing multilayer biofilms with extracellular polymeric substances (EPS). This method, applicable to other Candida species like C. glabrata and C. albicans, is cost-effective and mimics the niche of biofilm formation. It is suitable for high-throughput drug screening and repurposing efforts, aiding in the development of new therapeutics. Our technique represents a significant advancement in Candida biofilm research, addressing the need for consistent, reproducible biofilm models. We detail a step-by-step procedure for creating a substratum for biofilm growth and measuring biofilm thickness using confocal laser scanning microscopy (CLSM) and ultrastructure by scanning electron microscopy (SEM). This method provides consistent outcomes across various Candida species. Key features • The biofilm formed on gelatin surfaces mimics host conditions, replicating the multilayered structure and EPS, offering a more accurate model for studying C. auris biofilms. • This method is highly reproducible and suitable for drug screening and biofilm analysis through three-dimensional (3D) reconstruction. • This in vitro technique aids in studying biofilm formation, related virulence properties, and drug tolerance of C. auris and other Candida species. • The simple, cost-effective technique is ideal for screening novel inhibitors and repurposed drug libraries, facilitating the design/identification of new therapeutics against Candida species.

The magnitude of Ascochyta blight of chickpea and its relationship with prevailing environmental conditions in the Thal region of Punjab, Pakistan

This study assessed Ascochyta blight disease in chickpea across five districts in the ‘Thal’ region of Punjab, Pakistan: Bhakkar, Jhang, Layyah, Muzaffargarh, and Mianwali. The disease prevalence, incidence, and severity were recorded over two consecutive years, 2020-2021 and 2021-2022, during the chickpea-growing season. Disease prevalence ranged from 76% in Layyah to 40% in Muzaffargarh. Layyah also exhibited the highest disease incidence (84.2%) and severity (59.41%), while Muzaffargarh recorded the lowest disease incidence (20.55%) and severity (8.55%). All thirty-seven fungal isolates obtained from diseased samples were morphologically identified as Ascochyta rabiei. Twenty-five isolates, including five representatives from each district, were characterized based on their pathogenicity. The isolate ARL1 from Layyah exhibited the highest pathogenicity, with disease rating scores of 8.5 in detached leaf assays and 7.3 in attached leaf assays. Pathogen virulence showed a positive relationship with the disease intensity observed in the respective districts. Disease incidence and severity were further correlated with various prevailing environmental factors in these districts. Disease incidence was positively correlated with relative humidity and wind speed, while disease severity showed a positive correlation with relative humidity and rainfall but a negative correlation with maximum temperature. Other epidemiological factors did not exhibit significant relationships with the disease. This study concludes that the ‘Thal’ region is highly threatened by Ascochyta blight disease due to relatively higher relative humidity, rainfall, wind speed, and fungal virulence. Therefore, proper and timely management practices are essential to mitigate the impact of Ascochyta blight disease and address these influencing factors effectively.

Metagenome mining divulges virulent and multidrug resistant Pseudomonas aeruginosa ST242 and Klebsiella michiganensis ST∗1b23 coinfecting an 8-month-old meningitis infant under ICU in Kampala, Uganda, East Africa

Pediatric meningitis is a global health problem, with insufficiently known pathogens and antibiotic resistance (AMR) especially in low-resource settings. Here, we sought to uncover the virulence and AMR of pathogens associated with infant meningitis, treated with ceftriaxone, in Kampala, Uganda. In a bid to isolate Klebsiella oxytoca, we coincidentally recovered a co-culture and challenged it with antibiotic susceptibility testing (AST) on a panel of 14 antibiotics. We then combined metagenome binning with antiSMASH/PRISM genome mining to unveil the pathogens, their virulence and molecular targets in relation to meningitis. From AST, the co-culture exhibited resistance to multiple aminoglycosides, fluroquinolones, and β-lactams, including ceftriaxone, the inherently used drug. From metagenome annotation, the first bin was identified as Pseudomonas aeruginosa ST242 and the second as Klebsiella michiganensis ST∗1b23. Among others, P. aeruginosa ST242 virulence factors include type 3 and type 6 secretion systems, biofilm, and nonribosomal peptides (NRPs) of the pyoverdine synthase operon, targeting claudin-5, a component of the tight junctions of the blood-brain barrier (BBB). The P. aeruginosa ST242 genome portrays intrinsic resistance to beta-lactamases (blaOXA-50 and blaPAO), aminoglycosides [aph(3′)-IIb)], fluoroquinolones (crpP), tetracycline (tmexD2) and fosfomycin (fosA), among others. From K. michiganensis ST∗1b23 genome mining we elucidated a yersiniabactin-related metabolite, targeting the ligand-binding domain of the human polymeric immunoglobulin receptor (pIgR) and other components of the BBB. The K. michiganensis ST∗1b23 chromosome encodes the genes blaOXY-1 and OqxA/B, conferring resistance to β-lactams, fluoroquinolones, and trimethoprim respectively. Notably, we found one frameshift and nine substitution mutations conferring carbapenem and cephalosporin resistance mechanisms. Overall, our findings strongly suggest coinfection and a mechanistic crosstalk between P. aeruginosa ST242 and K. michiganensis ST∗1b23 in the pathogenesis of meningitis in this case. Importantly, ceftriaxone could be an inappropriate treatment choice for these pathogens. Hence, serious surveillance and experimental studies will improve the management of pediatric meningitis.

Antimicrobial potential of carvacrol against Edwardsiella piscicida in vitro

With the alarming rise of antibiotic-resistant bacteria, novel antibacterial substances are urgently needed for controlling and treating multidrug-resistant bacterial infections. Edwardsiella piscicida is an important zoonotic enteric pathogen, that can cause systemic hemorrhagic septicemia in fish. Carvacrol, a major terpene of oregano essential oil, has a wide range of antibacterial activities. This study aimed to analyze the effect of carvacrol on the growth and virulence of E. piscicida in vitro. The minimum inhibitory concentration (MIC) of carvacrol against E. piscicida was 125 μg/mL. The sub-inhibitory concentrations of carvacrol significantly decreased the biofilm formation of E. piscicida in a dose dependent manner, whereas increased the hemolytic activity with a negative correlation. The quantitative real-time PCR results showed that carvacrol at sub-MICs downregulated the expression of related virulence genes, including flagellum (fimA, fliC, flgN), hemolysins (ethA, ethB), quorum sensing systems (luxR, qseB), T3SS (esrB, esrC) and T6SS (evpB, evpC). Moreover, carvacrol (≤1/8 MIC) reduced the cytotoxicity, adherence and internalization activities of E. piscicida to the EPC cells. In vivo trial, the diet mixed with carvacrol increased the survival of zebrafish infected with E. piscicida. Overall, these findings suggested that carvacrol might be a promising therapeutic agent against E. piscicida infection in aquaculture.

Complete genome sequence provides information on quorum sensing related spoilage and virulence of Aeromonas salmonicida GMT3 isolated from spoiled sturgeon

Foodborne bacteria can pose a threat to the public health due to their spoilage and virulence potential, which can be regulated by quorum sensing (QS) system. In the study, we isolated a spoilage bacteria strain Aeromonas salmonicida GMT3 from refrigerated sturgeon. The complete genome of A. salmonicida GMT3 was sequenced, and the QS related genes were assigned. QS signal molecules N-acyl-homoserine lactones (AHLs) and AI-2 were detected. Genes regulating the spoilage-related metabolic pathways, including protease and lipase secretion, amines metabolism, sulfur metabolism, motility and biofilm formation were analyzed. Furthermore, genes encoding for several virulence factors, e.g. hemolysin, aerolysin, type II secretion system (T2SS), type VI secretion system (T6SS), antibiotic and multidrug resistance were also identified. In addition, the spoilage and virulence phenotypes associated with QS including protease, swimming and swarming activity, biofilm and hemolytic activity were detected. This study provided new insights into spoilage and virulence mechanisms correlated with QS of A. salmonicida GMT3, which might promote development of new approaches for spoilage and virulence control based on QS target.

Evaluation of laboratory-prepared water dispersible granules of Metarhizium rileyi (Farlow) Kepler, S.A. Rehner, and Humber against major soil pupating insect pests

Entomopathogenic fungi (EPF) exhibit formidable effectiveness that, under specific conditions, can offset the shortcomings of chemical insecticides within pest control programs. Among these EPFs, Metarhizium rileyi stands as one of the most extensively studied agents for global pest control. The study delves into the assessment of the relative virulence of M. rileyi water-dispersible granules (WDG) when deployed against distinct soil-pupating stages of lepidopteran and dipteran insects. M. rileyi WDG formulations were subjected to evaluation against various soil-pupating stages of insects, including lepidopteran and dipteran species. The results, based on dose-response relationships and LC50 values, revealed distinct levels of virulence exhibited by M. rileyi. Among the lepidopteran insects tested, M. rileyi demonstrated the highest level of virulence against H. armigera, followed by A. albistriga, A. flava, S. frugiperda, A. ipsilon, S. litura, S. obliqua, and A. janata. The LC50 values of these treatments, arranged in ascending order, were 2.01 x 107, 7.17 x 107, 1.13 x 108, 1.41 x 108, 1.54 x 108, 3.35 x 108, 3.62 x 108, and 1.00 x 109, respectively. In contrast, M. rileyi exhibited the least virulence against S. exigua, with an LC50 value of 1.35 x 109. Notably, the efficacy of M. rileyi WDG formulations was significantly lower when targeting dipteran pupae compared to lepidopteran pupae. For B. cucurbitae and B. dorsalis, the LC50 values were 7.26 x 109 and 1.29 x 1010, respectively. M. rileyi WDG demonstrated remarkable virulence when tested against the pupae of H. armigera, marking the highest level of efficacy observed. In contrast, these WDG exhibited limited efficacy when targeting dipteran pupae, such as those of B. dorsalis and B. Cucurbitae.

First report of Streptococcus agalactiae isolated from a healthy captive sichuan golden snub-nosed monkey (Rhinopithecus roxellana) in China

Streptococcus agalactiae (S. agalactiae) is an opportunistic pathogen, and to date, studies have mainly focused on S. agalactiae strains isolated from humans, dairy cows, and fish. We reported one S. agalactiae strain, named CFFB, which was isolated from a healthy Sichuan golden snub-nosed monkey. Classical bacteriological approaches, as well as, next-generation sequencing, comparative genomics, and mice challenge test were used to characterize this strain. CFFB was identified as serotype III, ST19 combination which is a common type found in human strains. Phylogenetic analysis showed that the genome of CFFB was closely related to human clinical isolates, rather far away from animal strains. In total, CFFB contained fewer virulence-associated genes and antibiotic resistance genes than human isolates that were close to CFFB in evolutionary relationships. In the mice challenge test, CFFB had a relative weak virulence that just caused death in 33 % of ICR mice at a dose of 108 CFU by intraperitoneal injection, and CFFB was reisolated from the cardiac blood of the dead mice. Meanwhile, two intact prophages (prophage 1 and 2) were identified in the CFFB genome and shared high similarities with phage Javan52 and Javan29 which from human S. agalactiae isolate Gottschalk 1002A and RBH03, respectively. Moreover, the type II-A CRISPR-Cas system was detected in the CFFB genome, and the spacers from CFFB were the same to the streptococci isolates from human. These results suggest that CFFB isolated from healthy Sichuan golden snub-nosed monkeys may have its origin in human S. agalactiae. Our results suggested some genomic similarities between the S. agalactiae colonized in Sichuan golden snub-nosed monkey and those in infected humans.

Study on molecular epidemiology of carbapenem resistant Pseudomonas aeruginosa and related genes of quorum sensing signal system

This study aims to investigate the drug resistance, regulation mechanism of quorum sensing system, expression of related virulence genes, and epidemiological characteristics of carbapenem-resistant Pseudomonas aeruginosa (CRPA).In this study, Polymerase chain reaction amplification was performed to evaluate carbapenemase genes, OprD2 gene, quorum sensing system, and related virulence genes. Bacterial genotypes were analyzed using multilocus sequence typing and evolutionary analysis was conducted based on the goeBURST algorithm. The results demonstrated that a total of 47 CRPA strains were collected in this study, primarily from respiratory specimens in the ICU. Drug sensitivity results showed that the resistance rates of the 47 CRPA strains were highest for imipenem (97.87 %). The loss of OprD2 may be the main factor contributing to carbapenem resistance in our hospital's CRPA strains.All isolates tested positive for the quorum sensing system genes lasI and rhlI/R, and the virulence gene lasB was detected in all isolates, while the algD gene was detected in 19.15 % of the isolates. Among the 47 strains, 6 were untypeable, and the 41 strains with 28 different sequence types were clustered into three clonal complexes (BG1, BG2, and BG3).In conclusion, the CRPA isolates from our hospital exhibit high genetic diversity, with the deletion of the OprD2 gene possibly being the primary determinant of carbapenem resistance in Pseudomonas aeruginosa.Moreover, Las and RhI systems play a key role in quorum sensing signal system. Further research and development of drugs targeting quorum sensing signaling system may provide valuable guidance for the treatment of CRPA.

Virulence variation of Israeli populations of Puccinia graminis f. sp. tritici during the period 2009 – 2019

This paper is dedicated to the memory of the APS Fellow Prof. Yehoshua Anikster (1934 -2023). A total of 336 urediniospore isolates of Puccinia graminis f. sp. tritici (Pgt) were derived from samples collected in Israel from 2009 to 2019 and analyzed for virulence with the standard set of 20 differentials. Seventy-four virulence phenotypes were identified during the survey. Two Pgt phenotypes (TKTTF, TTTTF) were found in nine annual populations while 57 appeared in only one year, in most of the cases (51) only once. The yearly pathogen collections of 2009 – 2014 differed from the collections of 2015-2018, and the 2019 collection diverged from all others. No virulence to Sr24 and Sr31 indicators of UG99 was detected. When comparing the 2009 – 2014 and 2015 – 2018 periods, virulence frequencies declined for Sr17, 30, and 38 genes from 0.85—0.98 to 0.31 – 0.59, while the frequency for Sr36 rose (0.42 vs. 0.87). The average relative virulence complexity of Pgt phenotypes decreased from 0.83 (2009—2014) and 0.79 (2015 – 2018) to 0.67 in 2019. Variability within the annual populations gradually increased over time. The Pgt collections of isolates in 2009 – 2014 and 2015 – 2018 were significantly different (p = 0.01). The effective number of different annual populations in 2009 – 2018 was 2.04 (β-variation = 0.12). Since Pgt does not over-summer in Israel, the northern source of inoculum from Turkey and Russia seems the most probable.

A food color-based colorimetric assay for Cryptococcus neoformans laccase activity.

Cryptococcus neoformans is a fungal pathogen that causes cryptococcosis primarily in immunocompromised patients, such as those with HIV/AIDS. One survival mechanism of C. neoformans during infection is melanin production, which catalyzed by laccase and protects fungal cells against immune attack. Hence, the comparative assessment of laccase activity is useful for characterizing cryptococcal strains. We serendipitously observed that culturing C. neoformans with food coloring resulted in degradation of some dyes with phenolic structures. Consequently, we investigated the color changes for the food dyes metabolized by C. neoformans laccase and by using this effect explored the development of a colorimetric assay to measure laccase activity. We developed several versions of a food dye-based colorimetric laccase assay that can be used to compare the relative laccase activities between different C. neoformans strains. We found that phenolic color degradation was glucose-dependent, which may reflect changes in the reduction properties of the media. Our food color-based colorimetric assay has several advantages, including lower cost, irreversibility, and not requiring constant monitoring , over the commonly used 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay for determining laccase activity. This method has potential applications to bioremediation of water pollutants in addition to its use in determining laccase virulence factor expression.IMPORTANCECryptococcus neoformans is present in the environment, and while infection is common, disease occurs mostly in immunocompromised individuals. C. neoformans infection in the lungs results in symptoms like pneumonia, and consequently, cryptococcal meningitis occurs if the fungal infection spreads to the brain. The laccase enzyme catalyzes the melanization reaction that serves as a virulence factor for C. neoformans. Developing a simple and less costly assay to determine the laccase activity in C. neoformans strains can be useful for a variety of procedures ranging from studying the relative virulence of cryptococci to environmental pollution studies.

Pathological and molecular characteristics of a new infectious Streptococcus agalactiae from farmed Schizopygopsis pylzovi in southern China

During the months of November and December 2022, a severe disease outbreak led to substantial mortality among Schizopygopsis pylzovi (S. pylzovi) in the Manwan reservoir of Lincang, China. The prominent clinical symptoms observed were exophthalmos with cataract, iris congestion, splenomegaly, and a pale liver. To elucidate the etiology, bacterial isolation was performed, followed by identification using Diff-Quik staining, 16s rDNA sequencing, and specific PCR. The results revealed that all three isolates were a non-hemolytic streptococcus with 93% identity to the 16S rDNA sequence of Streptococcus agalactiae (S. agalactiae), and further specific PCR demonstrated that it belonged to S. agalactiae. In addition, to further elucidate the molecular characteristics of the pathogen, a comprehensive analysis was conducted, encompassing pathological diagnosis, serotyping, multilocus sequence typing (MLST), physiological and biochemical identification, as well as the detection of related virulence genes. The findings showed that the isolates were S. agalactiae serotype Ib ST-891, exhibiting significant damage to the eye, spleen, and brain. Notably, its virulence gene profile and physiological and biochemical characteristics exhibited a significant degree of similarity to those from S. agalactiae serotype Ib ST261. Finally, experimental infection revealed that mortality rates were 100% and 75%, respectively, after exposure to 1 × 108 CFU/mL and 1 × 107 CFU/mL of this isolate suspension. It is the first report of natural infection with S. agalactiae serotype Ib ST-891 in fish, thus providing evidence for the molecular genetic diversity of fish-derived S. agalactiae.

Safety assessment of a novel marine multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 according to phenotype and whole genome-sequencing analysis

The application of microorganisms as probiotics is limited due to lack of safety evaluation. Here, a novel multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified from marine mangrove microorganisms. Its safety and probiotic properties were assessed in accordance with phenotype and whole-genome sequencing analysis. Results showed that the genes and phenotypic expression of related virulence, antibiotic resistance and retroelement were rarely found. Hyphal morphogenesis genes (SIT4, HOG1, SPA2, ERK1, ICL1, CST20, HSP104, TPS1, and RHO1) and phospholipase secretion gene (VPS4) were annotated. True hyphae and phospholipase were absent. Only one retroelement (Tad1-65_BG) was found. Major biogenic amines (BAs) encoding genes were absent, except for spermidine synthase (JA9_002594), spermine synthase (JA9_004690), and tyrosine decarboxylase (inx). The production of single BAs and total BAs was far below the food-defined thresholds. GXDK6 had no resistance to common antifungal drugs. Virulence enzymes, such as gelatinase, DNase, hemolytic, lecithinase, and thrombin were absent. Acute toxicity test with mice demonstrated that GXDK6 is safe. GXDK6 has a good reproduction ability in the simulation gastrointestinal tract. GXDK6 also has a strong antioxidant ability, β-glucosidase, and inulinase activity. To sum up, GXDK6 is considered as a safe probiotic for human consumption and food fermentation.

Distinct Pathological Changes in Preweaning Mice Infected with Live-Attenuated Rift Valley Fever Virus Strains.

Rift Valley fever (RVF) is a mosquito-borne zoonotic viral disease endemic to Africa and the Middle East. Live-attenuated RVF vaccines have been studied for both veterinary and human use due to their strong immunogenicity and cost-effective manufacturing. The live-attenuated MP-12 vaccine has been conditionally approved for veterinary use in the U.S.A., and next-generation live-attenuated RVF vaccine candidates are being actively researched. Assessing the virulence phenotype of vaccine seeds or lots is crucial for managing vaccine safety. Previously, preweaning 19-day-old outbred CD1 mice have been used to evaluate the MP-12 strain. This study aimed to characterize the relative virulence of three live-attenuated RVF vaccine strains in 19-day-old inbred C57BL/6 mice: the recombinant MP-12 (rMP-12), the RVax-1, and the ∆NSs-∆NSm-rZH501 strains. Although this mouse model did not show dose-dependent pathogenesis, mice that succumbed to the infection exhibited distinct brain pathology. Mice infected with ∆NSs-∆NSm-rZH501 showed an infiltration of inflammatory cells associated with infected neurons, and focal lesions formed around virus-infected cells. In contrast, mice infected with rMP-12 or RVax-1 showed a minimal association of inflammatory cells in the brain, yet the virus spread diffusely. The preweaning model is likely useful for evaluating host responses to attenuated RVFV strains, although further refinement may be necessary to quantitate the virulence among different RVFV strains or vaccine lots.

Host resources and parasite traits interact to determine the optimal combination of host parasite-mitigation strategies.

Organisms have evolved diverse strategies to manage parasite infections. Broadly, hosts may avoid infection by altering behaviour, resist infection by targeting parasites or tolerate infection by repairing associated damage. The effectiveness of a strategy depends on interactions between, for example,resource availability, parasite traits (virulence, life-history) and the host itself (nutritional status, immunopathology). To understand how these factors shape host parasite-mitigation strategies, we developed a mathematical model of within-host, parasite-immune dynamics in the context of helminth infections. The model incorporated host nutrition and resource allocation to different mechanisms of immune response: larval parasite prevention; adult parasite clearance; damage repair (tolerance). We also considered a non-immune strategy: avoidance via anorexia, reducing intake of infective stages. Resources not allocated to immune processes promoted host condition, whereas harm due to parasites and immunopathology diminished it. Maximising condition (a proxy for fitness), we determined optimal host investment for each parasite-mitigation strategy, singly and combined, across different environmental resource levels and parasite trait values. Which strategy was optimal varied with scenario. Tolerance generally performed well, especially with high resources. Success of the different resistance strategies (larval prevention or adult clearance) tracked relative virulence of larval and adult parasites: slowly maturing, highly damaging larvae favoured prevention; rapidly maturing, less harmful larvae favoured clearance. Anorexia was viable only in the short term, due to reduced host nutrition. Combined strategies always outperformed any lone strategy: these were dominated by tolerance, with some investment in resistance. Choice of parasite mitigation strategy has profound consequences for hosts, impacting their condition, survival and reproductive success. We show that the efficacy of different strategies is highly dependent on timescale, parasite traits and resource availability. Models that integrate such factors can inform the collection and interpretation of empirical data, to understand how those drivers interact to shape host immune responses in natural systems.

Characterization of Amino Acid Mutations of Newcastle Disease Virus (NDV) In Swan Geese (Anser cygnoides) In East Java, Indonesia

Background: Newcastle disease (ND) is a highly contagious viral disease affecting the poultry industry. The NDV is classified into three strains based on their relative virulence, namely velogenic or highly virulent, mesogenic or moderately virulent, and lentogenic or lowly virulent. The clinical manifestations of the disease vary depending on many factors, such as host susceptibility and the virulence of the NDV strain. Objective: This study aims to analyze the amino acid mutations of the NDV in unvaccinated swan goose (Anser cygnoides) from various locations in Java. Methods: Samples were collected through cloacal swabs and isolated by inoculation in Specific Pathogen-Free (SPF) embryonated eggs that were nine days old. Hemagglutination and hemagglutination inhibition tests were conducted to confirm that the isolated virus was NDV. The isolated virus was processed using reverse transcription-polymerase chain reaction (RT-PCR) with primers that amplified partial sequences of the fusion (F) gene, which was analyzed to determine the pathotype. Results: The results indicated the presence of mutations in several regions. The amino acid changes occurred in 17 variable sites (7.2%) between RefSeq/JF950510 and ND/SW1/2018, 12 variable sites (5.1%) between RefSeq/JF950510 and ND/SW2/2018, 13 variable sites (5.5%) between RefSeq/JF950510 and ND/SW3/2018, and 19 variable sites (8.1%) between RefSeq/JF950510 and ND/SW4/2018. The amino acid sequences of the cleavage site of the fusion (F) protein revealed that all isolates had low virulence. Conclusion: The results indicated that mutations in the region outside the cleavage site not were incapable of altering the virulence of the virus.

Beyond the β-amino alcohols framework: identification of novel β-hydroxy pyridinium salt-decorated pterostilbene derivatives as bacterial virulence factor inhibitors.

Bacterial virulence factors are involved in various biological processes and mediate persistent bacterial infections. Focusing on virulence factors of phytopathogenic bacteria is an attractive strategy and crucial direction in pesticide discovery to prevent invasive and persistent bacterial infection. Hence, discovery and development of novel agrochemicals with high activity, low-risk, and potent anti-virulence is urgently needed to control plant bacterial diseases. A series of novel β-hydroxy pyridinium cation decorated pterostilbene derivatives were prepared and their antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo) were systematacially assessed. Among these pterostilbene derivatives, compound 4S exhibited the best antibacterial activity against Xoo in vitro, with an half maximal effective concentration (EC50) value of 0.28 μg mL-1. A series of biochemical assays including scanning electron microscopy, crystal violet staining, and analysis of biofilm formation, swimming motility, and related virulence factor gene expression levels demonstrated that compound 4S could function as a new anti-virulence factor inhibitor by interfering with the bacterial infection process. Furthermore, the pot experiments provided convinced evidence that compound 4S had the high control efficacy (curative activity: 71.4%, protective activity: 72.6%), and could be used to effectively manage rice bacterial leaf blight in vivo. Compounds 4S is an attractive virulence factor inhibitor with potential for application in treating plant bacterial diseases by suppressing production of several virulence factors. © 2024 Society of Chemical Industry.

Approbation of the Technology for Constructing Means of Express Indication of New Especially Dangerous

Catastrophic pandemic of the particularly dangerous coronavirus SARS-CoV-2 in 2020–2022 and the unexpected spread of the monkeypox pathogen from Africa in 2022, demonstrate the need for an adequate response to biological threats that have exotic infections as their source, overcome the interspecies barrier between animals and humans and have high rates of virulence and contagiousness in relation to the latter. The purpose of the article is to create a technology for constructing means for the express indication of new especially dangerous and exotic infections, which makes it possible to quickly develop a gene diagnostic tool, evaluate its characteristics and launch large-scale production. Materials and methods. The authors used technologies for constructing means for express indication of new especially dangerous and exotic infections based on real-time polymerase chain reaction (RT-PCR-RT-Flu/ Coronavirus) methods, suitable for multiplex identification of coronavirus RNA. The discussion of the results. The developed technology for constructing means for express indication of new especially dangerous and exotic infections was successfully tested at the laboratory base of the FSBЕ «48 Central Research Institute» of the Ministry of Defense of the Russian Federation using the example of designing a «Set of reagents for detecting the RNA of coronaviruses SARS-CoV, MERS-CoV, SARS-CoV-2 and virus influenza A by real-time polymerase chain reaction (RT-PCR-RT-Flu/Coronavirus)», suitable for multiplex identification of coronavirus RNA. Conclusion. As a result of the research carried out to evaluate the equipment available at the laboratory base of the FSBЕ «48 Central Research Institute» of the Ministry of Defense of the Russian Federation, the adaptation and implementation of key production processes, the development and production of express-indication reagents, as well as testing the technology for constructing express-indication means for new especially dangerous and exotic infections, using the example of designing a set of RT-PCR-RV-Flu/Coronavirus reagents, a gene diagnostic platform was created for the development of reagents for the express indication of new especially dangerous and exotic infections.

Arbutin interacts with Vibrio harveyi hemolysin to alleviate damage from associated infection

Vibrio harveyi, an opportunistic pathogen, is a major causative agent of vibriosis in aquatic organisms. Many research conducted both domestically and internationally has revealed the significant multidrug resistance exhibited by V. harveyi. Among its important virulence factors, V. harveyi hemolysin (VHH) plays a crucial role in the infection process of this bacterium within the host. Due to the limitations of traditional antimicrobial drugs and the need to prevent bacterial resistance, alternative approaches are being explored worldwide. In this study, VHH was targeted, and through drug screening, it was discovered that arbutin, a plant glycoside, effectively inhibits the hemolytic activity of the VHH. The findings indicate that arbutin does not impede the growth of V. harveyi in vitro, nor does it affect the transcription of related virulence factors such as vhh and luxR. Instead, arbutin counteracts the hemolytic activity of VHH by directly binding to key amino acid residues, including Tyr227, Asn159, and Trp200, thereby influencing the function of the cytotoxic protein. In the context of V. harveyi infection, the presence of arbutin enhances cell survival in vitro and mitigates pathological damage to the gills and kidneys of experimental animals. The results show that arbutin, has the potential to be a promising anti-virulence therapy.

Unraveling the transcriptional features and gene expression networks of pathogenic and saprotrophic Ophiostoma species during the infection of Ulmus americana.

American elm (Ulmus americana), highly prized for its ornamental value, has suffered two successive outbreaks of Dutch elm disease (DED) caused by ascomycete fungi belonging to the genus Ophiostoma. To identify the genes linked to the pathogenicity of different species and lineages of Ophiostoma, we inoculated 2-year-old U. americana saplings with six strains representing three species of DED fungi, and one strain of the saprotroph Ophiostoma quercus. Differential expression analyses were performed following RNA sequencing of fungal transcripts recovered at 3- and 10-days post-infection. Based on a total of 8,640 Ophiostoma genes, we observed a difference in fungal gene expression depending on the strain inoculated and the time of incubation in host tissue. Some genes overexpressed in the more virulent strains of Ophiostoma encode hydrolases that possibly act synergistically. A mutant of Ophiostoma novo-ulmi in which the gene encoding the ogf1 transcription factor had been deleted did not produce transcripts for the gene encoding the hydrophobin cerato-ulmin and was less virulent. Weighted gene correlation network analyses identified several candidate pathogenicity genes distributed among 13 modules of interconnected genes.IMPORTANCEOphiostoma is a genus of cosmopolitan fungi that belongs to the family Ophiostomataceae and includes the pathogens responsible for two devastating pandemics of Dutch elm disease (DED). As the mechanisms of action of DED agents remain unclear, we carried out the first comparative transcriptomic study including representative strains of the three Ophiostoma species causing DED, along with the phylogenetically close saprotrophic species Ophiostoma quercus. Statistical analyses of the fungal transcriptomes recovered at 3 and 10 days following infection of Ulmus americana saplings highlighted several candidate genes associated with virulence and host-pathogen interactions wherein each strain showed a distinct transcriptome. The results of this research underscore the importance of investigating the transcriptional behavior of different fungal taxa to understand their pathogenicity and virulence in relation to the timeline of infection.

Methyl gallate from Camellia nitidissima Chi flowers reduces quorum sensing related virulence and biofilm formation against Aeromonas hydrophila

Aeromonas hydrophila, a Gram-negative zoonotic bacterium, causes high mortality in fish farming and immunocompromised patients. This study aimed to extract methyl gallate (MG) from the flowers of Camellia nitidissima Chi and evaluate its potential as a quorum sensing inhibitor (QSI) against Aeromonas hydrophila SHAe 115. MG reduced QS-associated virulence factors, including hemolysis, protease, and lipase, while impairing swimming motility and biofilm formation. Additionally, MG down-regulated positive regulatory genes (ahyR, fleQ) and up-regulated negative regulators (litR, fleN). This highlights MG's promise as a potent QSI for A. hydrophila SHAe 115, advancing strategies against infections in aquaculture and human health.