Diversity Of Isolates Research Articles

Roles of fragment crystallizable-mediated effector functions in broadly neutralizing antibody activity against HIV.

'Broadly neutralizing antibodies' (bNAbs), are rare HIV-specific antibodies which exhibit the atypical ability to potently neutralize diverse viral isolates. While efforts to elicit bNAbs through vaccination have yet to succeed, recent years have seen remarkable preclinical and clinical advancements of passive immunization approaches targeting both HIV prevention and cure. We focus here on the potential to build upon this success by moving beyond neutralization to additionally harness the diverse effector functionalities available to antibodies via fragment crystallizable-effector (Fc) functions. Recent studies have leveraged the ability to engineer bNAb Fc domains to either enhance or abrogate particular effector functions to demonstrate that activities such as antibody-dependent cell-mediated cytotoxicity contribute substantially to in-vivo antiviral activity. Intriguingly, recent studies in both nonhuman primates and in humans have suggested that passive bNAb infusion can lead to durable immunity by enhancing virus-specific T-cell responses through a 'vaccinal effect'. The combination of antibody engineering strategies designed to enhance effector functions, with the broad and potent antigen recognition profile of bNAbs, has the potential to give rise to powerful new therapeutics for HIV. We aim to provide a timely review of recent advances to catalyze this development.

First Report on the Occurrence and Subtypes of Blastocystis in Pigs in Poland Using Sequence-Tagged-Site PCR and Barcode Region Sequencing.

Blastocystis is an enteric microorganism commonly found in humans and animals worldwide. Its pathogenic role in humans and transmission patterns has not been fully explained. However, nine subtypes (ST1–8, ST12) are considered as potentially zoonotic. Studies from various regions of the world show that pigs are mainly infected with ST5. Although pigs are important farmed animals in Poland, the question of Blastocystis infection in these animals has not yet been investigated. Herein, 149 pig stool samples from 10 Polish pig farms were analyzed using sequence-tagged-site PCR and barcode region sequencing. The percentage of samples in which Blastocystis was identified using each method separately was similar: 38.25% and 37.58%, respectively. However, the percentage of positive results obtained by combining both methods was 46.97%, which means that, depending on the method used, the number of undetected samples varied between 8.72% and 9.39%. This shows the methodological limitations of up-to-date molecular approaches commonly used in Blastocystis research. A moderate infection rate (44.4–50%) observed in different pig age groups with a vital predominance of ST5 (94.28%) in every age group shows that pigs are a likely natural host of ST5. A small percentage of mixed infections, namely ST5/ST1 (5.26%), ST5/ST3 (1.75%), and ST3/ST1 (1.75%), was observed only in animals of older age, suggesting that ST3 and ST1 can be acquired by pigs during contact with humans. This study provides the first data on the prevalence and Blastocystis subtypes (STs) distribution in pigs in Poland. The results also highlight the need for the development of new methods capable of detecting highly genetically diverse Blastocystis isolates and mixed infections.

Expandable and reversible copy number amplification drives rapid adaptation to antifungal drugs.

Previously, we identified long repeat sequences that are frequently associated with genome rearrangements, including copy number variation (CNV), in many diverse isolates of the human fungal pathogen Candida albicans (Todd et al., 2019). Here, we describe the rapid acquisition of novel, high copy number CNVs during adaptation to azole antifungal drugs. Single-cell karyotype analysis indicates that these CNVs appear to arise via a dicentric chromosome intermediate and breakage-fusion-bridge cycles that are repaired using multiple distinct long inverted repeat sequences. Subsequent removal of the antifungal drug can lead to a dramatic loss of the CNV and reversion to the progenitor genotype and drug susceptibility phenotype. These findings support a novel mechanism for the rapid acquisition of antifungal drug resistance and provide genomic evidence for the heterogeneity frequently observed in clinical settings.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Assessment of Antibacterial Activity of Levonadifloxacin against Contemporary Gram-positive Clinical Isolates Collected from Various Indian Hospitals using Disk-Diffusion Assay

AbstractObjectives: Levonadifloxacin is a novel benzoquinolizine subclass of quinolone with broad-spectrum activities against problematic pathogens such as methicillin-resistant Staphylococcus aureus, quinolone-resistant S. aureus, vancomycin intermediate S. aureus, and vancomycin-resistant S. aureus. Levonadifloxacin and its oral prodrug, alalevonadifloxacin, have been recently approved in India for the treatment of acute bacterial skin and skin structure infections, including concurrent bacteraemia and diabetic foot infections. The aim of the study is to assess the activity of levonadifloxacin against Gram-positive clinical isolates collected from various Indian hospitals using the disc-diffusion method. Materials and Methods: Nonduplicate isolates of S. aureus and other Gram-positive isolates collected from June 2019 to March 2020 were subjected to levonadifloxacin susceptibility testing (disk diffusion method) as per the Clinical and Laboratory Standards Institute guidelines (Year 2019). Levonadifloxacin 10 μg impregnated disks were used during the testing. Results: A total of 664 diverse Gram-positive clinical isolates collected from six different hospitals in India were analyzed. Majority (65.5%) of the isolates were S. aureus. All the S. aureus and other Gram-positive isolates were found to be susceptible to levonadifloxacin as per the prespecified interpretive criteria identified based on population pharmacokinetic model and Monte Carlo simulation enabled probability of pharmacodynamic target attainment analysis. Conclusions: The present study showed that levonadifloxacin was highly active against contemporary Gram-positive pathogens and furthermore demonstrated that levonadifloxacin susceptibilities can be reliably determined using the disc-diffusion method.

Bacterial metabolism rescues the inhibition of intestinal drug absorption by food and drug additives

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

Key Positions of HIV-1 Env and Signatures of Vaccine Efficacy Show Gradual Reduction of Population Founder Effects at the Clade and Regional Levels.

HIV-1 group M was transmitted to humans nearly one century ago. The virus has since evolved to form distinct clades, which spread to different regions of the world. The envelope glycoproteins (Envs) of HIV-1 have rapidly diversified in all infected populations. We examined whether key antigenic sites of Env and signatures of vaccine efficacy are evolving toward similar or distinct structural forms in different populations worldwide. Patterns of amino acid variants that emerged at each position of Env were compared between diverse HIV-1 clades and isolates from different geographic regions. Interestingly, at each Env position, the amino acid in the clade ancestral or regional-founder virus was replaced by a unique frequency distribution (FD) of amino acids. FDs are highly conserved in populations from different regions worldwide and in paraphyletic and monophyletic subclade groups. Remarkably, founder effects of Env mutations at the clade and regional levels have gradually decreased during the pandemic by evolution of each site toward the unique combination of variants. Therefore, HIV-1 Env is evolving at a population level toward well-defined "target" states; these states are not specific amino acids but rather specific distributions of amino acid frequencies. Our findings reveal the powerful nature of the forces that guide evolution of Env and their conservation across different populations. Such forces have caused a gradual decrease in the interpopulation diversity of Env despite an increasing intrapopulation diversity.IMPORTANCE The Env protein of HIV-1 is the primary target in AIDS vaccine design. Frequent mutations in the virus increase the number of Env forms in each population, limiting the efficacy of AIDS vaccines. Comparison of newly emerging forms in different populations showed that each position of Env is evolving toward a specific combination of amino acids. Similar changes are occurring in different HIV-1 subtypes and geographic regions toward the same position-specific combinations of amino acids, often from distinct ancestral sequences. The predictable nature of HIV-1 Env evolution, as shown here, provides a new framework for designing vaccines that are tailored to the unique combination of variants expected to emerge in each virus subtype and geographic region.

Parallel Evolution of Tobramycin Resistance across Species and Environments.

Different species exposed to a common stress may adapt by mutations in shared pathways or in unique systems, depending on how past environments have molded their genomes. Understanding how diverse bacterial pathogens evolve in response to an antimicrobial treatment is a pressing example of this problem, where discovery of molecular parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in environments containing antibiotics, combined with periodic whole-population genome sequencing, can be used to identify many contending routes to antimicrobial resistance. We separately propagated two clinically relevant Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing concentrations of tobramycin in two different environments each: planktonic and biofilm. Independently of the pathogen, the populations adapted to tobramycin selection by parallel evolution of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate phosphotransferase. As neither gene is a direct target of this aminoglycoside, mutations to either are unexpected and underreported causes of resistance. Additionally, both species acquired antibiotic resistance-associated mutations that were more prevalent in the biofilm lifestyle than in the planktonic lifestyle; these mutations were in electron transport chain components in A. baumannii and lipopolysaccharide biosynthesis enzymes in P. aeruginosa populations. Using existing databases, we discovered site-specific parallelism of fusA1 mutations that extends across bacterial phyla and clinical isolates. This study suggests that strong selective pressures, such as antibiotic treatment, may result in high levels of predictability in molecular targets of evolution, despite differences between organisms' genetic backgrounds and environments.IMPORTANCE The rise of antimicrobial resistance is a leading medical threat, motivating efforts to forecast both its evolutionary dynamics and its genetic causes. Aminoglycosides are a major class of antibiotics that disrupt translation, but resistance may occur by a number of mechanisms. Here, we show the repeated evolution of resistance to the aminoglycoside tobramycin in both P. aeruginosa and A. baumannii via mutations in fusA1, encoding elongation factor G, and ptsP, encoding the nitrogen-specific phosphotransferase system. Laboratory evolution and whole-population genome sequencing were used to identify these targets, but mutations at identical amino acid positions were also found in published genomes of diverse bacterial species and clinical isolates. We also identified other resistance mechanisms associated with growth in biofilms that likely interfere with drug binding or uptake. Characterizing the evolution of multiple species in the presence of antibiotics can identify new, repeatable causes of resistance that may be predicted and counteracted by alternative treatment.

A potent enzybiotic against methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is one of the most dreadful infectious agents, responsible for high mortality and morbidity in both humans and animals. The increased prevalence of multidrug-resistant (MDR) Staphylococcus aureus strains has limited the number of available treatment options, which calls for the development of alternative and effective modalities against MDR S. aureus. Endolysins are bacteriophage-derived antibacterials, which attack essential conserved elements of peptidoglycan that are vital for bacterial survival, making them promising alternatives or complements to existing antibiotics for tackling such infections. For developing endolysin lysin-methicillin-resistant-5 (LysMR-5) as an effective antimicrobial agent, we evaluated its physical and chemical characteristics, and its intrinsic antibacterial activity against staphylococcal strains, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we cloned, expressed, and purified LysMR-5 from S. aureus phage MR-5. In silico analysis revealed that LysMR-5 harbors two catalytic and one cell wall-binding domain. Biochemical characterization and LC-MS analysis showed that both catalytic domains were active and had no dependence on divalent ions for their action, Zn2+ exerted a negative effect. The optimal lytic activity of the endolysin was at 37°C/pH 7.0 and in the presence of ≥ 300mM concentration of NaCl. Circular dichroism (CD) demonstrated a loss in secondary structure with an increase in temperature confirming the thermosensitive nature of endolysin. Antibacterial assays revealed that LysMR-5 was active against diverse clinical isolates of staphylococci. It showed high lytic efficacy against S. aureus ATCC 43300, as an endolysin concentration as low as 15µg/ml was sufficient to achieve maximum lytic activity within 30min and it was further confirmed by scanning electron microscopy. Our results indicate that rapid and strong bactericidal activity of LysMR-5 makes it a valuable candidate for eradicating multidrug-resistant S. aureus.

Pervasive Phenotypic Impact of a Large Nonrecombining Introgressed Region in Yeast.

To explore the origin of the diversity observed in natural populations, many studies have investigated the relationship between genotype and phenotype. In yeast species, especially in Saccharomyces cerevisiae, these studies are mainly conducted using recombinant offspring derived from two genetically diverse isolates, allowing to define the phenotypic effect of genetic variants. However, large genomic variants such as interspecies introgressions are usually overlooked even if they are known to modify the genotype–phenotype relationship. To have a better insight into the overall phenotypic impact of introgressions, we took advantage of the presence of a 1-Mb introgressed region, which lacks recombination and contains the mating-type determinant in the Lachancea kluyveri budding yeast. By performing linkage mapping analyses in this species, we identified a total of 89 loci affecting growth fitness in a large number of conditions and 2,187 loci affecting gene expression mostly grouped into two major hotspots, one being the introgressed region carrying the mating-type locus. Because of the absence of recombination, our results highlight the presence of a sexual dimorphism in a budding yeast for the first time. Overall, by describing the phenotype–genotype relationship in the Lachancea kluyveri species, we expanded our knowledge on how genetic characteristics of large introgression events can affect the phenotypic landscape.

The Histological, Effectoromic, and Transcriptomic Analyses of Solanum pinnatisectum Reveal an Upregulation of Multiple NBS-LRR Genes Suppressing Phytophthora infestans Infection.

Utilization of disease resistance components from wild potatoes is a promising and sustainable approach to control Phytophthora blight. Here, we combined avirulence (Avr) genes screen with RNA-seq analysis to discover the potential mechanism of resistance in Mexican wild potato species, Solanum pinnatisectum. Histological characterization displayed that hyphal expansion was significantly restricted in epidermal cells and mesophyll cell death was predominant, indicating that a typical defense response was initiated in S. pinnatisectum. Inoculation of S. pinnatisectum with diverse Phytophthora infestans isolates showed distinct resistance patterns, suggesting that S. pinnatisectum has complex genetic resistance to most of the prevalent races of P. infestans in northwestern China. Further analysis by Avr gene screens and comparative transcriptomic profiling revealed the presence and upregulation of multiple plant NBS-LRR genes corresponding to biotic stresses. Six NBS-LRR alleles of R1, R2, R3a, R3b, R4, and Rpi-smira2 were detected, and over 60% of the 112 detected NLR proteins were significantly induced in S. pinnatisectum. On the contrary, despite the expression of the Rpi-blb1, Rpi-vnt1, and Rpi-smira1 alleles, fewer NLR proteins were expressed in susceptible Solanum cardophyllum. Thus, the enriched NLR genes in S. pinnatisectum make it an ideal genetic resource for the discovery and deployment of resistance genes for potato breeding.

Allelic polymorphism shapes community function in evolving Pseudomonas aeruginosa populations.

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of individuals with cystic fibrosis (CF) by forming antibiotic-resistant biofilms. Emergence of phenotypically diverse isolates within CF P. aeruginosa populations has previously been reported; however, the impact of heterogeneity on social behaviors and community function is poorly understood. Here we describe how this heterogeneity impacts on behavioral traits by evolving the strain PAO1 in biofilms grown in a synthetic sputum medium for 50 days. We measured social trait production and antibiotic tolerance, and used a metagenomic approach to analyze and assess genomic changes over the duration of the evolution experiment. We found that (i) evolutionary trajectories were reproducible in independently evolving populations; (ii) over 60% of genomic diversity occurred within the first 10 days of selection. We then focused on quorum sensing (QS), a well-studied P. aeruginosa trait that is commonly mutated in strains isolated from CF lungs. We found that at the population level, (i) evolution in sputum medium selected for decreased the production of QS and QS-dependent traits; (ii) there was a significant correlation between lasR mutant frequency, the loss of protease, and the 3O-C12-HSL signal, and an increase in resistance to clinically relevant β-lactam antibiotics, despite no previous antibiotic exposure. Overall, our findings provide insights into the effect of allelic polymorphism on community functions in diverse P. aeruginosa populations. Further, we demonstrate that P. aeruginosa population and evolutionary dynamics can impact on traits important for virulence and can lead to increased tolerance to β-lactam antibiotics.

Time-Course RNAseq Reveals Exserohilum turcicum Effectors and Pathogenicity Determinants.

Exserohilum turcicum (sexual stage Setosphaeria turcica) is the hemibiotrophic causal agent of northern leaf blight of maize and sorghum. This study aimed to identify the genes involved in host colonization during the biotrophic and necrotrophic phases of infection. It also aimed to identify race-specific differences in gene expression. RNAseq of maize seedlings inoculated with a race 13N or 23N E. turcicum isolate was conducted before inoculation and at 2, 5, 7, and 13 days post-inoculation (dpi). Biological replicates were pooled per time point for each race and sequenced. A bioinformatics pipeline was used to identify candidate effectors, and expression was validated for selected candidates. Fungal biomass was positively correlated with the percentages of E. turcicum reads mapped, which were low at early time points (2–7 dpi) with a significant increase at 13 dpi, indicating a lifestyle switch from biotrophy to necrotrophy between 7 and 13 dpi. AVRHt1 is the putative E. turcicum effector recognized by the maize resistance gene Ht1. Consistent with this, AVRHt1 was expressed in planta by race 23N, but transcripts were absent in race 13N. In addition, specific transposable elements were expressed in 23N only. Genes encoding the virulence-associated peptidases leupeptin-inhibiting protein 1 and fungalysin were expressed in planta. Transcriptional profiles of genes involved in secondary metabolite synthesis or cell wall degradation revealed the importance of these genes during late stages of infection (13 dpi). A total of 346 expressed candidate effectors were identified, including Ecp6 and proteins similar to the secreted in xylem (SIX) effectors common to formae speciales of Fusarium oxysporum, SIX13 and SIX5. Expression profiling of Ecp6 and SIX13-like indicated a peak in expression at 5 and 7 dpi compared to 2 and 13 dpi. Sequencing of SIX13-like from diverse isolates of E. turcicum revealed host-specific polymorphisms that were mostly non-synonymous, resulting in two groups of SIX13-like proteins that corresponded to the maize or sorghum origin of each isolate. This study suggests putative mechanisms whereby E. turcicum causes disease. Identification of the candidate effector SIX13-like is consistent with the infection mode of E. turcicum through the xylem of susceptible hosts.

Surveillance of Salmonella spp. in the environment of public hospitals in KwaZulu-Natal, South Africa

To investigate the dissemination of Salmonella spp. within four levels of government hospitals in KwaZulu-Natal, South Africa. The identification of Salmonella spp. was performed by amplification of the invA gene. Isolates were subjected to antimicrobial susceptibility testing and molecular characterization of eight resistance genes (qnrA, qnrB, qnrS, tetA, tetB, tetC, tetG, ermB) and three virulence genes (sitC, spvA, spv). Genetic relatedness between isolates was determined using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction. Ninety-four isolates were obtained. The largest source of isolates was the regional hospital. Paediatric wards had the highest prevalence of isolates. Nurses' tables contained the most isolates out of all sites sampled. Twenty-two clusters indicating diverse isolates were obtained via molecular typing. Four main ERIC types were identified, each unique to a specific hospital. A possibility of dissemination across the wards was noted as highly related isolates were present at various sites within the wards. Many of these sites were highly trafficked areas by healthcare staff. Ten multi-drug-resistant isolates were found. This study suggests that infection prevention and control strategies that involve environmental cleaning and decontamination may not be enough, or adhered to sufficiently, to prevent the dissemination of Salmonella spp.

Complete Genome Characterization and Coat Protein Genealogy of Isolates of Maize dwarf mosaic virus from Johnsongrass and Maize in Oklahoma and Missouri.

Maize dwarf mosaic virus (MDMV) significantly affects maize production worldwide, including the United States. This study describes the distribution and biological and molecular characterization of MDMV isolates from Johnsongrass and maize. A total of 262 samples (symptomatic = 214, asymptomatic = 48) were collected in Oklahoma and Missouri during 2016, 2017, and 2019 growing seasons. Based on a dot-immunobinding assay (DIBA), the average incidence of maize dwarf mosaic disease varied from ∼71% (79/111) in 2016, ∼76% (81/106) in 2017, and 62% (28/45) in 2019. Sixty-five DIBA-positive samples for MDMV were further confirmed by RT-PCR, and the complete coat protein (CP) gene was cloned and sequenced. Phylogenetic analysis of 132 isolates (This study = 65; GenBank = 67) revealed two main groups (G1 and G2) of MDMV isolates. All 65 MDMV isolates contained a 39-nucleotide insertion in the N-terminal region of CP genes and clustered in G1 which were different from the isolates in G2, without 39-nucleotide insertion. The first complete genome (9,563 nucleotides) of a MDMV isolate (Bixby1) from Johnsongrass was sequenced, which was distantly related to eight previously reported MDMV isolates from maize. The dN/dS ratio showed mostly purifying selection on each of cistrons except 6K1 being subjected to the diversifying selection. Further analyses revealed three putative recombination events between MDMV-Bixby1 and MDMV isolates from other countries. The successful mechanical and aphid transmission of MDMV-Bixby1 onto maize cultivars was achieved. Altogether, this information showed that Johnsongrass harbors genetically diverse MDMV isolates, which could pose a threat to cultivated crops such as maize and sorghum.

Contributions of Spore Secondary Metabolites to UV-C Protection and Virulence Vary in Different Aspergillus fumigatus Strains.

Fungi are versatile organisms which thrive in hostile environments, including the International Space Station (ISS). Several isolates of the human pathogen Aspergillus fumigatus have been found contaminating the ISS, an environment with increased exposure to UV radiation. Secondary metabolites (SMs) in spores, such as melanins, have been shown to protect spores from UV radiation in other fungi. To test the hypothesis that melanin and other known spore SMs provide UV protection to A. fumigatus isolates, we subjected SM spore mutants to UV-C radiation. We found that 1,8-dihydroxynaphthalene (DHN)-melanin mutants of two clinical A. fumigatus strains (Af293 and CEA17) but not an ISS-isolated strain (IF1SW-F4) were more sensitive to UV-C than their respective wild-type (WT) strains. Because DHN-melanin has been shown to shield A. fumigatus from the host immune system, we examined all DHN mutants for virulence in the zebrafish model of invasive aspergillosis. Following recent studies highlighting the pathogenic variability of different A. fumigatus isolates, we found DHN-melanin to be a virulence factor in CEA17 and IF1SW-F4 but not Af293. Three additional spore metabolites were examined in Af293, where fumiquinazoline also showed UV-C-protective properties, but two other spore metabolites, monomethylsulochrin and fumigaclavine, provided no UV-C-protective properties. Virulence tests of these three SM spore mutants indicated a slight increase in virulence of the monomethylsulochrin deletion strain. Taken together, this work suggests differential roles of specific spore metabolites across Aspergillus isolates and by types of environmental stress.IMPORTANCE Fungal spores contain secondary metabolites that can protect them from a multitude of abiotic and biotic stresses. Conidia (asexual spores) of the human pathogen Aspergillus fumigatus synthesize several metabolites, including melanin, which has been reported to be important for virulence in this species and to be protective against UV radiation in other fungi. Here, we investigate the role of melanin in diverse isolates of A. fumigatus and find variability in its ability to protect spores from UV-C radiation or impact virulence in a zebrafish model of invasive aspergillosis in two clinical strains and one ISS strain. Further, we assess the role of other spore metabolites in a clinical strain of A. fumigatus and identify fumiquinazoline as an additional UV-C-protective molecule but not a virulence determinant. The results show differential roles of secondary metabolites in spore protection dependent on the environmental stress and strain of A. fumigatus As protection from elevated levels of radiation is of paramount importance for future human outer space explorations, the discovery of small molecules with radiation-protective potential may result in developing novel safety measures for astronauts.

Genetic Diversity of Cyprinid Herpesvirus 3 from Different Geographical Locations during 1999-2019 in the United States of America.

Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is an important pathogen in common and koi carp Cyprinus carpio, varieties. Two main genotypes of KHV have been reported worldwide that are associated with Asian and European origins. In the USA, outbreaks of KHV diseases have been reported in different states since the early 1990s; however, the diversity of KHV is unknown. In the current study, 67 DNA samples that were extracted from clinical cases of koi tissues that were submitted for diagnosis during KHV outbreaks from 10 different states in the USA from 1999 to 2019 were used to investigate their genetic diversity. The thymidine kinase gene was amplified, sequenced, and used for phylogenetic analysis. Our results showed that the KHV isolates that were collected from the different states were clustered in the two known KHV genogroups, where 31 isolates belonged to the Asian genotype branch and 36 to the European genotype branch. The spatiotemporal analysis demonstrated fluctuation of KHV genotypes in the USA, as the main KHV genotype that was detected in koi in the USA from 1999 to 2013 was the European genotype, whereas the Asian KHV genotype appeared to emerge in the USA in 2008, increasing in incidence until 2019. The current study provides evidence on the genetic diversity of KHV in the USA. Future studies that evaluate the virulence of these genetically diverse isolates is warranted to obtain a better understanding of the epidemiology of this re-emerging pathogen. This may provide an improved awareness of the current status of KHV and help to control the disease in the koi population in the USA.

Genomic diversity affects the accuracy of bacterial single-nucleotide polymorphism-calling pipelines.

BackgroundAccurately identifying single-nucleotide polymorphisms (SNPs) from bacterial sequencing data is an essential requirement for using genomics to track transmission and predict important phenotypes such as antimicrobial resistance. However, most previous performance evaluations of SNP calling have been restricted to eukaryotic (human) data. Additionally, bacterial SNP calling requires choosing an appropriate reference genome to align reads to, which, together with the bioinformatic pipeline, affects the accuracy and completeness of a set of SNP calls obtained. This study evaluates the performance of 209 SNP-calling pipelines using a combination of simulated data from 254 strains of 10 clinically common bacteria and real data from environmentally sourced and genomically diverse isolates within the genera Citrobacter, Enterobacter, Escherichia, and Klebsiella.ResultsWe evaluated the performance of 209 SNP-calling pipelines, aligning reads to genomes of the same or a divergent strain. Irrespective of pipeline, a principal determinant of reliable SNP calling was reference genome selection. Across multiple taxa, there was a strong inverse relationship between pipeline sensitivity and precision, and the Mash distance (a proxy for average nucleotide divergence) between reads and reference genome. The effect was especially pronounced for diverse, recombinogenic bacteria such as Escherichia coli but less dominant for clonal species such as Mycobacterium tuberculosis.ConclusionsThe accuracy of SNP calling for a given species is compromised by increasing intra-species diversity. When reads were aligned to the same genome from which they were sequenced, among the highest-performing pipelines was Novoalign/GATK. By contrast, when reads were aligned to particularly divergent genomes, the highest-performing pipelines often used the aligners NextGenMap or SMALT, and/or the variant callers LoFreq, mpileup, or Strelka.

Insight into the resilience and susceptibility of marine bacteria to T6SS attack by Vibrio cholerae and Vibrio coralliilyticus.

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.

Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61–98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.