Levels Of Virulence Research Articles

Host range and molecular characterization of the four races of Fusarium oxysporum f. sp. lactucae.

Vascular wilt of lettuce is caused by strains of the Fusarium oxysporum species complex. According to their biological activity, these strains are members of the forma specialis lactucae (FOL). The aim of this study was to characterize strains of Fusarium oxysporum isolated from lettuce in Italy and other countries, belonging to the four currently known races. Strains were analyzed based on pathogenicity on different hosts and a combined phylogenetic analysis of rpb2, tef-1α, cmdA and tub2 genes. For uncharacterized strains, the race was determined through pathogenicity test with a set of differential lettuce cultivars for races 1 and 4. To assess the host range, pathogenicity tests were performed on five species: lettuce (Lactuca sativa), lamb's lettuce (Valerianella locusta), spinach (Spinacia oleracea), wild rocket (Diplotaxis tenuifolia) and cultivated rocket (Eruca sativa). Most FOL strains were pathogenic on lettuce with different levels of disease severity; in particular, FOL races 3 and 4 showed the highest level of virulence on lettuce. FOL races 3 and 4 were pathogenic both on lettuce and lamb's lettuce, whereas races 1 and 2 only on lettuce. Furthermore, this study identified the first occurrence of FOL race 4 in Italy to 2016, as the strain FOL 1/16 was assigned to race 4. Results of multigene phylogenetic analysis showed that FOL races 1 and 4 cluster with the species F. curvatum, strains of race 2 with F. curvatum and F. odoratissimum and strains of race 3 with F. cugenangense. Phylogenetically, race 4 is very close to race 1, but it showed more similarities to race 3 for host range and virulence. This study shows for the first time a broader host range for the races 3 and 4 of FOL. The results demonstrate that phylogenetic analysis permits the separation of some races of FOL, but phylogenetic classification alone is insufficient for conclusive separation of races, therefore it should be accompanied by biological assays based on pathogenicity.

Single-cell transcriptomics of bronchoalveolar lavage during PRRSV infection with different virulence

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the global swine industry due to its high genetic diversity and different virulence levels, which complicate disease management and vaccine development. This study evaluated longitudinal changes in the immune cell composition of bronchoalveolar lavage fluid and the clinical outcomes across PRRSV strains with varying virulence, using techniques including single-cell transcriptomics. In highly virulent infection, faster viral replication results in an earlier peak lung-damage time point, marked by significant interstitial pneumonia, a significant decrease in macrophages, and an influx of lymphocytes. Viral tracking reveals less than 5% of macrophages are directly infected, and further analysis indicates bystander cell death, likely regulated by exosomal microRNAs as a significant factor. In contrast, the peak intermediate infection shows a delayed lung-damage time point with fewer cell population modifications. Furthermore, anti-inflammatory M2-like macrophages (SPP1-CXCL14high) are identified and their counts increase during the peak lung-damage time point, likely contributing to local defense and lung recovery, which is not observed in high virulent infection. These findings provide a comprehensive description of the immune cellular landscape and differential PRRSV virulence mechanisms, which will help build new hypotheses to understand PRRSV pathogenesis and other respiratory infections.

Characterization of Listeria monocytogenes strains isolated from soil under organic carrot farming.

Listeria monocytogenes are Gram-positive, non-spore-producing rods that are the etiological agent of listeriosis. L. monocytogenes is isolated from soil, water, sewage, rotting vegetation, and the main source of these rods for humans is food (fish, unpasteurized dairy products, or raw fruits and vegetables). In recent years, there has been an increase in consumer interest in healthy food, especially organically grown. The use of natural fertilizers during organic farming can be a source of pathogens, including L. monocytogenes in the soil and finally in vegetables and fruits. The aim of this study was to assess the prevalence of L. monocytogenes in soil samples from organic carrot crops (Poland) and to characterize the tested strains. Microbial contamination of the soil has a direct impact on the safety of the crops grown on it. This is an important aspect in organic farming, where products are chosen as a healthier option and consumed by children and the elderly. The isolates were subjected to genetic similarity assessment (PFGE method), and the tested strains were evaluated for antibiotic susceptibility (disc-diffusion method), invasiveness (HT-29 line human colon cancer cell line), coaggregation with Salmonella Enteritidis, biofilm-forming ability and the effect of disinfectants on the biofilm. Twenty-seven isolates of L. monocytogenes were isolated from 250 soil samples, 10 of which were genetically different. 80% of the tested strains were sensitive to the tested antibiotics. Antibiotic resistance was demonstrated in two strains (strain 11 - resistant to penicillin and cotrimoxazole, strain 22 - resistant to penicillin). The highest invasiveness against HT-29 cells at 23.2% was shown for strain 11. However, invasiveness of less than 1% was demonstrated for three strains, and strain 13 showed no ability to invade HT-29 human colon cancer cells. The level of coaggregation between the tested strains and S. Enteritidis ranged from 22.2 to 39.1%. The number of biofilm-isolated rods from the stainless steel surface was 6.37 to 7.10 log colony-forming unit (CFU)/cm2, while on polypropylene it was from 6.75 to 8.06 log CFU/cm2. The effectiveness of the disinfectants used depended on the duration of action and the concentration of the disinfectant. Chlorosol was shown to be the disinfectant causing stronger biofilm eradication on each of the tested surfaces. It has been shown that soils and thus food from organic farming can be a source of L. monocytogenes. These rods can vary in phenotypic characteristics and virulence levels. The research conducted allows to draw attention to the occurrence of pathogens, including L. monocytogenes in crops from organic farming. In addition, the results presented can help to introduce standards regulating the safety of organic farming, taking into account the occurrence of antibiotic-resistant or highly invasive strains, thus maintaining food safety.

Comparative Analysis of In Vivo and In Vitro Virulence Among Foodborne and Clinical Listeria monocytogenes Strains.

Listeria monocytogenes is one of the most important foodborne pathogens that can cause invasive listeriosis. In this study, the virulence levels of 26 strains of L. monocytogenes isolated from food and clinical samples in Shanghai, China, between 2020 and 2022 were analyzed. There were significant differences among isolates in terms of their mortality rate in Galleria mellonella, cytotoxicity to JEG-3 cells, hemolytic activity, and expression of important virulence genes. Compared with other STs, both the ST121 (food source) and ST1930 (clinic source) strains exhibited higher G. mellonella mortality. The 48 h mortality in G. mellonella of lineage II strains was significantly higher than that in lineage I. Compared with other STs, ST1930, ST3, ST5, and ST1032 exhibited higher cytotoxicity to JEG-3 cells. Based on the classification of sources (food and clinical strains) and serogroups (II a, II b, and II c), there were no significant differences observed in terms of G. mellonella mortality, cytotoxicity, and hemolytic activity. In addition, ST121 exhibited significantly higher hly, inlA, inlB, prfA, plcA, and plcB gene expression compared with other STs. A gray relation analysis showed a high correlation between the toxicity of G. mellonella and the expression of the hly and inlB genes; in addition, L. monocytogenes may have a consistent virulence mechanism involving hemolysis activity and cytotoxicity. Through the integration of in vivo and in vitro infection models with information on the expression of virulence factor genes, the differences in virulence between strains or subtypes can be better understood.

New Strains of the Entomopathogenic Nematodes Steinernema scarabaei, S. glaseri, and S. cubanum for White Grub Management.

White grubs possess natural defense mechanisms against entomopathogenic nematodes (EPNs). Hence, EPN isolates that naturally infect white grubs tend to be among the most effective biological control agents of white grubs. We tested the virulence of four EPN isolates recently isolated from infected white grubs in turfgrass areas in central New Jersey, USA against third-instar larvae of Anomala orientalis, Popillia japonica, and Cyclocephala borealis, which are pests of turfgrass and ornamental plants in the northeastern USA. Against A. orientalis and P. japonica larvae, the Steinernema scarabaei Ad and S. scarabaei SL isolates were highly virulent in laboratory tests, whereas S. cubanum HF was less virulent and S. glaseri SH the least virulent. None of the isolates caused high mortality of C. borealis larvae. EPN efficacy in greenhouse tests against A. orientalis followed the same pattern. The original isolate of S. scarabaei, the AMK001 strain, that had been maintained in the laboratory on A. orientalis and P. japonica larvae for 19 years showed the same virulence level against A. orientalis larvae as it did soon after its first isolation and was also as virulent as the fresh S. scarabaei Ad isolate; both isolates were more virulent than S. scarabaei SL. Future tests should determine the ability of these white grub-adapted isolates to provide long-term suppression of white grub populations.

Variability analysis of Phytophthora meadii - A major causal agent of capsule rot (Azhukal) disease in small cardamom

Capsule rot disease, caused by Phytophthora spp., pose a significant challenge to the cultivation of cardamom and other spices and plantation crops, leading to considerable yield losses. In the present study, the impact of P. meadii on small cardamom was assessed in the primary cardamom-growing regions of Tamil Nadu and Kerala through a roving survey conducted during 2023–2024. The survey revealed varying levels of disease severity, ranging from 8.23% to 52.80%. The highest incidence was recorded in the Udumbanchola region (52.80%), while the lowest was observed in Thandikudi (8.32%). A total of eight isolates of Phytophthora (designated as PHY-1 to PHY-8) were collected from diseased samples and purified using the single hyphal tip method. Pathogenicity studies were conducted to evaluate the virulence of these isolates through two different methods: a detached capsule assay and an in-planta assay. Among the 8 pathogenic isolates, PHY-4 exhibited the highest level of virulence (75%) and presented typical symptoms of capsule rot disease. The isolates were analyzed for cultural and morphological variability. All eight isolates displayed distinct variations in growth patterns and sporangial morphology. Optimal growth and development of P. meadii were observed at temperatures ranging from 25–30ºC. Scanning electron microscopy (SEM) analyses revealed specific morphological characteristics of the isolates, including hyaline, coenocytic mycelium and distinct sporangial structures.

Molecular dissection of laboratory contamination between two schistosome populations

BackgroundGenomic analysis has revealed extensive contamination among laboratory-maintained microbes including malaria parasites, Mycobacterium tuberculosis, and Salmonella spp. Here, we provide direct evidence for recent contamination of a laboratory schistosome parasite population, and we investigate its genomic consequences. The Brazilian Schistosoma mansoni population SmBRE has several distinctive phenotypes, showing poor infectivity, reduced sporocyst number, low levels of cercarial shedding and low virulence in the intermediate snail host, and low worm burden and low fecundity in the vertebrate rodent host. In 2021 we observed a rapid change in SmBRE parasite phenotypes, with a 10-fold increase in cercarial production and fourfold increase in worm burden.MethodsTo determine the underlying genomic cause of these changes, we sequenced pools of SmBRE adults collected during parasite maintenance between 2015 and 2023. We also sequenced another parasite population (SmLE) maintained alongside SmBRE without phenotypic changes.ResultsWhile SmLE allele frequencies remained stable over the 8-year period, we observed sudden changes in allele frequency across the genome in SmBRE between July 2021 and February 2023, consistent with expectations of laboratory contamination. (i) SmLE-specific alleles increased in the SmBRE population from 0 to 41–46% across the genome between September and October 2021, reflecting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s ≅0.23) drove the replacement of low-fitness SmBRE with high-fitness SmLE alleles. (iii) Allele frequency changed rapidly across the whole genome, except for a region on chromosome 4, where SmBRE alleles remained at high frequency.ConclusionsWe were able to detect contamination in this case because SmBRE shows distinctive phenotypes. However, this would likely have been missed with phenotypically similar parasites. These results provide a cautionary tale about the importance of tracking the identity of parasite populations, but also showcase a simple approach to monitor changes within populations using molecular profiling of pooled population samples to characterize single-nucleotide polymorphisms. We also show that genetic drift results in continuous change even in the absence of contamination, causing parasites maintained in different labs (or sampled from the same lab at different times) to diverge.Graphical

Functional Characterization of FgAsp, a Gene Coding an Aspartic Acid Protease in Fusarium graminearum.

Aspartic proteases (APs), hydrolases with aspartic acid residues as catalytic active sites, are closely associated with processes such as plant growth and development and fungal and bacterial pathogenesis. F. graminearum is the dominant pathogenic fungus that causes Fusarium head blight (FHB) in wheat. However, the relationship of APs to the growth, development, and pathogenesis of F. graminearum is not clear. Therefore, we selected the FGSG_09558 gene, whose function annotation is aspartate protease, for further study. In this study, FGSG_09558 was found to contain a conserved structural domain and signal peptide sequence of aspartic acid protease and was therefore named FgAsp. The function of FgAsp in F. graminearum was investigated by constructing the knockout and complementation mutants of this gene. The results showed that with respect to the wild type (PH-1), the knockout mutant showed a significant reduction in mycelial growth, asexual spore production, and sexual spore formation, highlighting the key role of FgAsp in the growth and development of F. graminearum. In addition, the mutants showed a significant reduction in the virulence and accumulation level of deoxynivalenol (DON) content on maize whiskers, wheat germ sheaths, and wheat ears. DON, as a key factor of virulence, plays an important role in the F. graminearum infection of wheat ears, suggesting that FgAsp is involved in the regulation of F. graminearum pathogenicity by affecting the accumulation of the DON toxin. FgAsp had a significant effect on the ability of F. graminearum to utilize various sugars, especially arabinose. In response to the stress, hydrogen peroxide inhibited the growth of the mutant most significantly, indicating the important function of FgAsp in the strain's response to environmental stress. Finally, FgAsp plays a key role in the regulation of F. graminearum growth and development, pathogenicity, and environmental stress response.

Transcriptomic profiling of "brain-eating amoeba" Naegleria fowleri infection in mice: the host and the protozoa perspectives.

The free-living amoeba Naegleria fowleri (NF) causes a rare but lethal parasitic meningoencephalitis (PAM) in humans. Currently, this disease lacks effective treatments and the specific molecular mechanisms that govern NF pathogenesis and host brain response remain unknown. To address some of these issues, we sought to explore naturally existing virulence diversity within environmental NF isolates. Herein, we purified two new NF environmental isolates (NF45 and NF1) and tested their in vivo virulence using experimental infection in mice. We found that NF45 was highly virulent (NF45_HV) compared with NF1 (low virulence, NF1_LV), based on in vivo amoeba growth kinetics and mouse survival. To identify underlying differences, we conducted RNA-seq and bioinformatics analyses from the infected mouse brains. Our results showed that NF1_LV and NF45_HV modulated the expression of their genes during mouse brain infection. Differentially expressed genes (DEGs) in NF1_LV were mostly involved in Translational protein, Protein-binding activity modulator, Protein modifying enzyme, while DEGs in NF45_HV were related to DNA metabolism, Cytoskeletal protein, Protein-binding activity modulator. Proteases (namely the virulence factor Cathepsin B) were upregulated in NF1_LV, while downregulated in NF45_HV. When analyzing the host response against infection by these two NF strains, enrichment analyses uncovered genes and mechanisms related to the host immune responses and nervous systems. We detected more DEGs in NF1_LV infected mice compared to NF45_HV, related to blood brain barrier leakage, immune cell recruitment, cytokine production (including IL-6, IFN-Ɣ and TNFα), inflammation of astrocytes and microglia, and oligodendrocyte and neurons degeneration. Increased expression of neuromotor-related genes such as Adam22, Cacnb4 and Zic1 (activated by NF1_LV infection) and ChAt (activated by NF45_LV infection) could explain PAM symptoms such as muscle weakness and seizures. Globally, our results showed that NF isolated from the environment can have different levels of virulence and differentially modulate their gene expression during brain infection. We also provided, for the first time, a comprehensive information for the molecular mechanisms of neuro-immune and host-pathogen interactions during PAM disease. As the host and the protozoa are strongly implicated in PAM lethality, new therapies targeting both the parasite, and the host should be considered to treat PAM infection.

Intra- and Interpopulation Diversity of the Phytopathogenic Fungi of the Microdochium nivale Species.

Our study investigates the intra- and interpopulation diversity of phytopathogenic fungi, using Microdochium nivale as a model organism. A total of 136 strains, representing two populations, were collected from different winter cereal crops (rye, wheat, and triticale) across two agrocenoses. In these strains, we analyzed and compared genetic and phenotypic traits, exploring potential relationships between them. Significant genetic and phenotypic diversity was revealed among the assayed strains, with most exhibiting distinctive genetic features compared to previously characterized strains from other countries. Both populations included highly virulent and lowly virulent strains, with the majority displaying similar virulence levels across different cereal crops. Some strains demonstrated differential virulence toward different crops, and some even exhibited a plant-beneficial effect on rye while being virulent to wheat and triticale. The percentages of strains with specific genetic traits were different in the two analyzed populations. Our study lays the groundwork for identifying genetic markers associated with phenotypic traits in the studied strains, which will aid in developing reliable diagnostics for predicting crop loss risk and improving disease management. Furthermore, it will assist in identifying reliable sources of resistance to M. nivale-induced diseases within the winter cereal gene pool.

Guardians of resistance and virulence: detection of mec, femA, Van, pvl, hlg and spa genes in methicillin and vancomycin-resistant Staphylococcus aureus from clinical and food samples in Southwestern Nigeria.

Staphylococcus aureus strains are highly virulent and associated with an eclectic range of severe nosocomial and community-acquired infections. This study assessed methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA/VRSA) from clinical and ready-to-eat (RTE) food sources, screened for antibiotic resistance; and molecular determinants of antibiotic and virulence genes. Altogether, 465 clinical and RTE food samples were analyzed via conventional microbiological techniques and S. aureus identification was confirmed by nuc gene detection. Phenotypic screening for methicillin and vancomycin-resistance was by agar-screen cum micro-broth dilution respectively, while antibiotic susceptibility testing was done by the disc-diffusion technique. VanA/vanB/VanC1, femA, mecA/mecC; pvl/hlg and spa gene detection was via Polymerase chain reaction. Phenotypically, 211 Staphylococcal isolates were recovered, 138 (65.4%) of them carrying the nuc gene - all 138 (100.0%) were VRSA, while 59/138 (42.8%) were MRSA phenotypically. Overall, 114/138 (82.6%), 7/138 (5.1%), and 6/138 (4.3%) of isolates had the femA, mecA, and mecC genes, while van genes were detected in only 3 (2.2%) isolates, with virulence determinants pvl, hlg, and spa gene carriage in 8 (5.8%), 10 (7.2%), and 77 (55.8%) isolates respectively. In all, 11.6% carried resistance-associated genes, 55.8% carried virulence genes, and co-detection of resistance and virulence genes was observed in 12.3%. Overall, 96/138 (69.6%) were multidrug-resistant (MDR), while one strain was extremely drug-resistant (XDR). MAR Indices ≥ 0.2 was observed in 83.3% of isolates. This study highlights virulence levels of MRSA and VRSA circulating strains in Osogbo, contributing to their sustained surveillance, and improving available data for successive epidemiology investigations. This study also reports the occurrence of the mecC gene in S. aureus isolates from RTE foods and human samples in Southwestern Nigeria.

Emergence of Nigrospora Leaf Blight as Major Biotic Stress in South Eastern Coastal Region of India

Background: Nigrospora is one of the diversified pathogens with wide host range. Endophytic nature of Nigrospora oryzae has been reported previously. Daily variations in the climate led to the emergence of virulent infections from previously expressed weak ones. Nigrospora oryzae infestation comes as a major biotic stress under this present investigation. Methods: A study was conducted to examine the variability of Nigrospora oryzae by collecting isolates from various locations in the south coastal region of Odisha. The level of virulence was assessed and analyzed between 2021 and 2023 to anticipate the emergence and progression of disease. The pathogen was identified using molecular techniques through ITS-r DNA sequencing method. Result: Disease incidence was observed as a critical blast-like appearance, but greater in size, distinguishing it from blast. In the experiment, incomplete filling of grains was highest in MTU-7029 (39.73 plant-1) followed by Niranjana and Gobindobhog; disease incidence was recorded to be highest in Niranjana (60.03%) followed by MTU-7029 and Barsha; and grain yield plant-1 was observed to be highly affected in Gobindobhog (14.58 g plant-1 followed by MTU-7029 and Niranjana. Molecular confirmation was established by ITS-rDNA sequencing, resulted sequence NO 9 (528 bp)- OP592217 and NO 15 (519 bp)- OP592218, exactly matches gene bank accession numbers- MH748173 (India) and ON514037 (India) of Nigrospora database, with 99% accuracy.

To be a good killer: Evaluation of morphometry and nematodes-bacteria complex effect on entomopathogenic nematodes virulence against wireworms

Entomopathogenic nematodes (EPNs) have emerged as a promising tool for controlling soil-dwelling crop pests. However, their efficacy varies according to EPN populations and targeted hosts. Wireworms are polyphagous insects causing significant crop losses, especially since the ban on pesticides previously used for their control. They are highly resistant to EPN populations and require high number of infective juveniles (IJs) to achieve optimal mortality rates. In this research, we collected and compared the virulence of 16 EPN populations, of foreign origin, purchased or collected from wireworms infested fields. Then, we have tested two hypotheses: (i) smaller nematodes induce heightened mortality rates against wireworms; (ii) virulence levels can be linked to nematodes-bacteria complex. Mortality rates scaled from three to 43 % after 56 days of continuous exposure across the 16 tested EPN populations (Heterorhabditis spp. and Steinernema spp.). Morphometric analysis of IJs revealed both intra- and interspecific variations in length and diameter among populations. Interestingly, while EPN length influence mortality at three days post-inoculation. We found leaner IJs (< 25 µm) to induce higher mortality rates at 56 days post-inoculation. To better determine the structure and dimensions of the primary entry routes utilized by EPNs, we provide optical microscope micrographs of wireworm Agriotes spp. spiracle, anal sclerotized coating anus and anal muscles. Symbiotic bacteria of each EPN population were identified, and a biochemical characterization was performed using Analytical Profile Index tests. The symbiotic bacteria belong to the species Photorhabdus antumapuensis, P. laumondii subsp. laumondii, P. thracensis, Xenorhabdus bovienii and X. nematophila. Bacteria biochemical profiles did not reflect the differences in virulence of nematodes-bacteria complex against wireworms. These findings highlight the importance of considering EPN morphometry and intraspecific variability in designing applications to control wireworms.

Colletotrichum species associated with anthracnose disease on mango (Mangifera indica L.) cv. Azúcar in Colombia

The role of some Colletotrichum species is uncertain in crops of economic importance for Colombia such as mango (Mangifera indica L.) cv. Azúcar. Eight Colletotrichum isolates from asymptomatic plant tissues were characterized by their pathogenicity on fruits and identified by phylogenetic analysis of the sequences of the ITS, GADPH, Tub2, CHs-1 and Act genomic regions. Colletotrichum asianum, C. gloeosporioides and C. tropicale of the C. gloeosporioides species complex and C. karstii of the C. boninense species complex were identified. The highest virulence was observed for isolates of C. asianum, that was the most prevalent species, and for C. tropicale, that showed high and intermediate virulence levels, indicating diversity in pathogenicity within the same species. The isolate of C. karstii, which displayed intermediate virulence, is the first report on mango in Colombia. Colletotrichum gloeosporioides presented intermediate virulence and was isolated at a low frequency in the present research. The mycelial growth rate did not show differences between species and ranged between 6.1 and 8.3 mm per day. Colletotrichum asianum recorded higher values of conidia germination and appressoria formation than other species, at 77% and 79%, respectively (p < 0.001). The shape and size of conidia did not vary between species. Appressoria exhibited different shapes between isolates and the largest average diameter was for C. karstii with 7.6 µm (p < 0.001). There was a diversity of Colletotrichum species associated with mango cv. Azúcar in Colombia, with differential virulence among isolates of the same species.

Rapid phenotypic and genotypic change in a laboratory schistosome population.

Genomic analysis has revealed extensive contamination among laboratory-maintained microbes including malaria parasites, Mycobacterium tuberculosis and Salmonella spp. Here, we provide direct evidence for recent contamination of a laboratory schistosome parasite population, and we investigate its genomic consequences. The Brazilian Schistosoma mansoni population SmBRE has several distinctive phenotypes, showing poor infectivity, reduced sporocysts number, low levels of cercarial shedding and low virulence in the intermediate snail host, and low worm burden and low fecundity in the vertebrate rodent host. In 2021 we observed a rapid change in SmBRE parasite phenotypes, with a~10x increase in cercarial production and ~ 4x increase in worm burden. To determine the underlying genomic cause of these changes, we sequenced pools of SmBRE adults collected during parasite maintenance between 2015 and 2023. We also sequenced another parasite population (SmLE) maintained alongside SmBRE without phenotypic changes. While SmLE allele frequencies remained stable over the eight-year period, we observed sudden changes in allele frequency across the genome in SmBRE between July 2021 and February 2023, consistent with expectations of laboratory contamination. (i) SmLE-specific alleles rose in the SmBRE population from 0 to 41-46% across the genome between September and October 2021, documenting the timing and magnitude of the contamination event. (ii) After contamination, strong selection (s=~ 0.23) drove replacement of low fitness SmBRE with high fitness SmLE alleles. (iii) Allele frequency changed rapidly across the whole genome, except for a region on chromosome 4 where SmBRE alleles remained at high frequency. We were able to detect contamination in this case because SmBRE shows distinctive phenotypes. However, this would likely have been missed with phenotypically similar parasites. These results provide a cautionary tale about the importance of tracking the identity of parasite populations, but also showcase a simple approach to monitor changes within populations using molecular profiling of pooled population samples to characterize fixed single nucleotide polymorphisms. We also show that genetic drift results in continuous change even in the absence of contamination, causing parasites maintained in different labs (or sampled from the same lab at different times) to diverge.

Adaptations to cationic biocide exposure differentially influence virulence factors and pathogenicity in Pseudomonas aeruginosa

ABSTRACT Cationic biocides (CBs), which include quaternary ammonium compounds (QACs), are employed to mitigate the spread of infectious bacteria, but resistance to such surface disinfectants is rising. CB exposure can have profound phenotypic implications that extend beyond allowing microorganisms to persist on surfaces. Pseudomonas aeruginosa is a deadly bacterial pathogen that is intrinsically tolerant to a wide variety of antimicrobials and is commonly spread in healthcare settings. In this study, we pursued resistance selection assays to the QAC benzalkonium chloride and quaternary phosphonium compound P6P–10,10 to assess the phenotypic effects of CB exposure in P. aeruginosa PAO1 and four genetically diverse, drug-resistant clinical isolates. In particular, we sought to examine how CB exposure affects defensive strategies and the virulence-associated “offensive” strategies in P. aeruginosa. We demonstrated that development of resistance to BAC is associated with increased production of virulence-associated pigments and alginate as well as pellicle formation. In an in vivo infection model, CB-resistant PAO1 exhibited a decreased level of virulence compared to wild type, potentially due to an observed fitness cost in these strains. Taken together, these results illustrate the significant consequence CB resistance exerts on the virulence-associated phenotypes of P. aeruginosa.

Comparative Genomic Analyses of Colletotrichum lindemuthianum Pathotypes with Different Virulence Levels and Lifestyles.

Colletotrichum lindemuthianum is the most frequent pathogenic fungus of the common bean Phaseolus vulgaris. This filamentous fungus employs a hemibiotrophic nutrition/infection strategy, which is characteristic of many Colletotrichum species. Due to host-pathogen coevolution, C. lindemuthianum includes pathotypes with a diversity of virulence against differential common bean varieties. In this study, we performed comparative genomic analyses on three pathotypes with different virulence levels and a non-pathogenic pathotype, isolated from different geographical areas in Mexico. Our results revealed large genomes with high transposable element contents that have undergone expansions, generating intraspecific diversity. All the pathotypes exhibited a similar number of clusters of orthologous genes (COGs) and Gene Ontology (GO) terms. TFomes contain families that are typical in fungal genomes; however, they show different contents between pathotypes, mainly in transcription factors with the fungal-specific TF and Zn2Cys6 domains. Peptidase families mainly contain abundant serine peptidases, metallopeptidases, and cysteine peptidases. In the secretomes, the number of genes differed between the pathotypes, with a high percentage of candidate effectors. Both the virulence gene and CAZyme gene content for each pathotype was abundant and diverse, and the latter was enriched in hemicellulolytic enzymes. We provide new insights into the nature of intraspecific diversity among C. lindemuthianum pathotypes and the origin of their ability to rapidly adapt to genetic changes in its host and environmental conditions.

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.

Transcriptomic Analyses to Unravel Cronobacter sakazakii Resistance Pathways.

The proliferation of antibiotic usage has precipitated the emergence of drug-resistant variants of bacteria, thereby augmenting their capacity to withstand pharmaceutical interventions. Among these variants, Cronobacter sakazakii (C. sakazakii), prevalent in powdered infant formula (PIF), poses a grave threat to the well-being of infants. Presently, global contamination by C. sakazakii is being observed. Consequently, research endeavors have been initiated to explore the strain's drug resistance capabilities, alterations in virulence levels, and resistance mechanisms. The primary objective of this study is to investigate the resistance mechanisms and virulence levels of C. sakazakii induced by five distinct antibiotics, while concurrently conducting transcriptomic analyses. Compared to the susceptible strains prior to induction, the drug-resistant strains exhibited differential gene expression, resulting in modifications in the activity of relevant enzymes and biofilm secretion. Transcriptomic studies have shown that the expression of glutathione S-transferase and other genes were significantly upregulated after induction, leading to a notable enhancement in biofilm formation ability, alongside the existence of antibiotic resistance mechanisms associated with efflux pumps, cationic antimicrobial peptides, and biofilm formation pathways. These alterations significantly influence the strain's resistance profile.

Replication Kinetics and Infectivity of African Swine Fever Virus (ASFV) Variants with Different Genotypes or Levels of Virulence in Cell Culture Models of Primary Porcine Macrophages

African Swine Fever (ASF) is a devastating viral hemorrhagic disease that causes high morbidity and mortality in domestic pigs and wild boars, severely impacting the swine industry. The etiologic agent, African Swine Fever virus (ASFV), mainly infects myeloid cells of the swine mononuclear phagocytic system (MPS). For other porcine viruses, in vitro culture models with primary cells are widely used as they mimic the in vivo viral replication behavior better compared to continuous cell lines. Our study validates this possible correlation for ASFV using cell culture models established for three different porcine macrophages, isolated from the lungs (porcine alveolar macrophages), blood (monocyte-derived macrophages) and spleen (spleen macrophages). The cells were infected with two genotype I and two genotype II strains with different pathogenic potential in vivo. The highly virulent strains replicated better in general than the low-virulent strains. This was most pronounced in monocyte-derived macrophages, although only statistically significant 18 h post-infection (hpi) in the intracellular genomic ASFV copies between E70 and the low-virulent strains. For this reason, we conclude that the different replication characteristics between the strains with different virulence do not proportionally represent the differences in pathology seen between the strains in vivo. Additionally, ASFV-positive cells were observed earlier in monocyte-derived macrophages (MDMs) compared to the alveolar and spleen macrophages, subsequently leading to an earlier rise in extracellular virus, and, ultimately, more MDMs were infected at the end of sampling. For these reasons, we propose MDMs as the best-suited cell type to study ASFV.