Annotated Genome Sequence of Aspergillus tanneri NIH1004.

The annotated genome of Aspergillus tanneri, a recently discovered drug-resistant pathogen, was determined by employing the Oxford Nanopore MinION platform and the Funannotate pipeline. The genome size and the number of protein-coding genes are notably larger than those of the most common etiological agent of aspergillosis, Aspergillus fumigatus.

rmtA-Dependent Transcriptome and Its Role in Secondary Metabolism, Environmental Stress, and Virulence in Aspergillus flavus.

Aspergillus flavus colonizes numerous oil seed crops such as maize, peanuts, treenuts and cottonseed worldwide, contaminating them with aflatoxins and other harmful toxins. Previously our lab characterized the gene rmtA, which encodes an arginine methyltransferase in A. flavus, and demonstrated its role governing the expression of regulators in the aflatoxin gene cluster and subsequent synthesis of toxin. Furthermore, our studies revealed that rmtA also controls conidial and sclerotial development implicating it as an epigenetic regulator in A. flavus To confirm this, we performed a RNA sequencing analysis to ascertain the extent of rmtA's influence on the transcriptome of A. flavus In this analysis we identified over 2000 genes that were rmtA-dependent, including over 200 transcription factor genes, as well as an uncharacterized secondary metabolite gene cluster possibly responsible for the synthesis of an epidithiodiketopiperazine-like compound. Our results also revealed rmtA-dependent genes involved in multiple types of abiotic stress response in A. flavus Importantly, hundreds of genes active during maize infection were also regulated by rmtA In addition, in the animal infection model, rmtA was dispensable for virulence, however forced overexpression of rmtA increased mortality with respect to the wild type.

A MarR family transcriptional regulator and subinhibitory antibiotics regulate type VI secretion gene clusters in Burkholderia pseudomallei.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2-lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6-lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2-lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.

Whole genome comparison of Aspergillus flavus L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70.

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 µm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 µm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.

Strain Level Streptococcus Colonization Patterns during the First Year of Life.

Pneumococcal pneumonia has decreased significantly since the implementation of the pneumococcal conjugate vaccine (PCV), nevertheless, in many developing countries pneumonia mortality in infants remains high. We have undertaken a study of the nasopharyngeal (NP) microbiome during the first year of life in infants from The Philippines and South Africa. The study entailed the determination of the Streptococcus sp. carriage using a lytA qPCR assay, whole metagenomic sequencing, and in silico serotyping of Streptococcus pneumoniae, as well as 16S rRNA amplicon based community profiling. The lytA carriage in both populations increased with infant age and lytA+ samples ranged from 24 to 85% of the samples at each sampling time point. We next developed informatic tools for determining Streptococcus community composition and pneumococcal serotype from metagenomic sequences derived from a subset of longitudinal lytA-positive Streptococcus enrichment cultures from The Philippines (n = 26 infants, 50% vaccinated) and South African (n = 7 infants, 100% vaccinated). NP samples from infants were passaged in enrichment media, and metagenomic DNA was purified and sequenced. In silico capsular serotyping of these 51 metagenomic assemblies assigned known serotypes in 28 samples, and the co-occurrence of serotypes in 5 samples. Eighteen samples were not typeable using known serotypes but did encode for capsule biosynthetic cluster genes similar to non-encapsulated reference sequences. In addition, we performed metagenomic assembly and 16S rRNA amplicon profiling to understand co-colonization dynamics of Streptococcus sp. and other NP genera, revealing the presence of multiple Streptococcus species as well as potential respiratory pathogens in healthy infants. A range of virulence and drug resistant elements were identified as circulating in the NP microbiomes of these infants. This study revealed the frequent co-occurrence of multiple S. pneumoniae strains along with Streptococcus sp. and other potential pathogens such as S. aureus in the NP microbiome of these infants. In addition, the in silico serotype analysis proved powerful in determining the serotypes in S. pneumoniae carriage, and may lead to developing better targeted vaccines to prevent invasive pneumococcal disease (IPD) in these countries. These findings suggest that NP colonization by S. pneumoniae during the first years of life is a dynamic process involving multiple serotypes and species.

Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

Acquisition and loss of virulence-associated factors during genome evolution and speciation in three clades of Bordetella species.

BACKGROUND: The genus Bordetella consists of nine species that include important respiratory pathogens such as the 'classical' species B. bronchiseptica, B. pertussis and B. parapertussis and six more distantly related and less extensively studied species. Here we analyze sequence diversity and gene content of 128 genome sequences from all nine species with focus on the evolution of virulence-associated factors. RESULTS: Both genome-wide sequence-based and gene content-based phylogenetic trees divide the genus into three species clades. The phylogenies are congruent between species suggesting genus-wide co-evolution of sequence diversity and gene content, but less correlated within species, mainly because of strain-specific presence of many different prophages. We compared the genomes with focus on virulence-associated genes and identified multiple clade-specific, species-specific and strain-specific events of gene acquisition and gene loss, including genes encoding O-antigens, protein secretion systems and bacterial toxins. Gene loss was more frequent than gene gain throughout the evolution, and loss of hundreds of genes was associated with the origin of several species, including the recently evolved human-restricted B. pertussis and B. holmesii, B. parapertussis and the avian pathogen B. avium. CONCLUSIONS: Acquisition and loss of multiple genes drive the evolution and speciation in the genus Bordetella, including large scale gene loss associated with the origin of several species. Recent loss and functional inactivation of genes, including those encoding pertussis vaccine components and bacterial toxins, in individual strains emphasize ongoing evolution.

Candida albicans Transcriptional Profiling Within Biliary Fluid From a Patient With Cholangitis, Before and After Antifungal Treatment and Surgical Drainage.

We used ribonucleic acid sequencing to profile Candida albicans transcription within biliary fluid from a patient with cholangitis; samples were collected before and after treatment with fluconazole and drainage. Candida albicans transcriptomes at the infection site distinguished treated from untreated cholangitis. After treatment, 1131 C. albicans genes were differentially expressed in biliary fluid. Up-regulated genes were enriched in hyphal growth, cell wall organization, adhesion, oxidation reduction, biofilm, and fatty acid and ergosterol biosynthesis. This is the first study to define Candida global gene expression during deep-seated human infection. Successful treatment of cholangitis induced C. albicans genes involved in fluconazole responses and pathogenesis.

Expert Panel Recommendations on Lower Urinary Tract Health of Women Across Their Life Span.

Urologic and kidney problems are common in women across their life span and affect their daily life, including physical activity, sexual relations, social life, and future health. Urological health in women is still understudied and the underlying mechanisms of female urological dysfunctions are not fully understood. The Society for Women's Health Research (SWHR®) recognized the need to have a roundtable discussion where researchers and clinicians would define the current state of knowledge, gaps, and recommendations for future research directions to transform women's urological health. This report summarizes the discussions, which focused on epidemiology, clinical presentation, basic science, prevention strategies, and efficacy of current therapies. Experts around the table agreed on a set of research, education, and policy recommendations that have the potential to dramatically increase awareness and improve women's urological health at all stages of life.

Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids.

The genomes of a diverse set of Escherichia coli, including many Shiga toxin-producing strains of various serotypes were determined. A total of 39 plasmids were identified among these strains, and many carried virulence or putative virulence genes of Shiga toxin-producing E. coli strains, virulence genes for other pathogenic E. coli groups, and some had combinations of these genes. Among the novel plasmids identified were eight that carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines and resistance to heavy metals. Two of the plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. The results of this study showed that plasmids carrying diverse resistance and virulence genes of various pathogenic E. coli groups can be found in E. coli strains and serotypes regardless of the isolate's source and therefore, is consistent with the premise that these mobile elements carrying these traits may be broadly disseminated among E. coli.

The In vitro Antibiotic Tolerant Persister Population in Burkholderia pseudomallei is Altered by Environmental Factors.

Bacterial persistence due to antibiotic tolerance is a critical aspect of antibiotic treatment failure, disease latency, and chronic or reemergent infections. The levels of persisters is especially notable for the opportunistic Gram-negative pathogens from the Burkholderia and Pseudomonas genera. We examined the rate of drug tolerant persisters in Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia cepacia complex organisms, and Pseudomonas aeruginosa at mid-log growth in LB broth culture. We found that a fraction of the antibiotic-sensitive cells from every species were tolerant to a 24 h high-dose antibiotic challenge. All tested Burkholderia strains demonstrated a drug tolerant persister population at a rate that was at least 100-500 times higher than P. aeruginosa. When challenged with at least a 10X minimum inhibitory concentration (MIC) 24 h exposure to three different antibiotics with different modes of action we found that in B. pseudomallei Bp82 each of the tree antibiotics revealed different persister fractions at each of two different growth states. This observation suggests that our assay is detecting heterogeneous persister subpopulations. Persistence in B. pseudomallei Bp82 was highly dependent on growth stage, with a surprisingly high persister fraction of >64% of the late stationary phase cells being antibiotic tolerant to 100XMIC cefotaxime. Adaptation of B. pseudomallei to distilled water storage resulted in a population of drug tolerant cells up to 100% of the non-drug-challenged viable cell count in the same cefotaxime assay. Cultivation of B. pseudomallei with a sub-inhibitory concentration of several antibiotics resulted in altered persister fractions within the population relative to cultures lacking the antibiotic. Our study provides insight into the sensitivity of the persister fraction within the population of B. pseudomallei due to environmental variables and suggests diversity within the persister population revealed by different challenge antibiotics.

Genome Sequence of Aspergillus flavus NRRL 3357, a Strain That Causes Aflatoxin Contamination of Food and Feed.

Aflatoxin contamination of food and livestock feed results in significant annual crop losses internationally. Aspergillus flavus is the major fungus responsible for this loss. Additionally, A. flavus is the second leading cause of aspergillosis in immunocompromised human patients. Here, we report the genome sequence of strain NRRL 3357.

Draft Genome Sequences of 53 Genetically Distinct Isolates of Bordetella bronchiseptica Representing 11 Terrestrial and Aquatic Hosts.

Bordetella bronchiseptica infects a variety of mammalian and avian hosts. Here, we report the genome sequences of 53 genetically distinct isolates acquired from a broad range of terrestrial and aquatic animals. These data will greatly facilitate ongoing efforts to better understand the evolution, host adaptation, and virulence mechanisms of B. bronchiseptica.

Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Draft Genome Sequences of Six Bordetella hinzii Isolates Acquired from Avian and Mammalian Hosts.

Bordetella hinzii is a Gram-negative bacterium known to infect poultry, humans, rabbits, and rodents. It is an opportunistic pathogen in immunocompromised humans, and some strains cause mild to moderate respiratory disease in turkeys. Little is known as to the degree of genetic diversity within the species or the genetic basis for virulence. Here, we report the genome sequences of six isolates of B. hinzii acquired from humans, rabbits, or turkeys. These data provide a framework for refining the population structure of the genus, establishing relationships among genetically distinct isolates, and developing an understanding of the possible virulence mechanisms of the bacterium.

Using Bayesian modelling to investigate factors governing antibiotic-induced Candida albicans colonization of the GI tract.

Receipt of broad-spectrum antibiotics enhances Candida albicans colonization of the GI tract, a risk factor for haematogenously-disseminated candidiasis. To understand how antibiotics influence C. albicans colonization, we treated mice orally with vancomycin or a combination of penicillin, streptomycin, and gentamicin (PSG) and then inoculated them with C. albicans by gavage. Only PSG treatment resulted in sustained, high-level GI colonization with C. albicans. Furthermore, PSG reduced bacterial diversity in the colon much more than vancomycin. Both antibiotic regimens significantly reduced IL-17A, IL-21, IL-22 and IFN-γ mRNA levels in the terminal ileum but had limited effect on the GI fungal microbiome. Through a series of models that employed Bayesian model averaging, we investigated the associations between antibiotic treatment, GI microbiota, and host immune response and their collective impact on C. albicans colonization. Our analysis revealed that bacterial genera were typically associated with either C. albicans colonization or altered cytokine expression but not with both. The only exception was Veillonella, which was associated with both increased C. albicans colonization and reduced IL-21 expression. Overall, antibiotic-induced changes in the bacterial microbiome were much more consistent determinants of C. albicans colonization than either the GI fungal microbiota or the GI immune response.

A systematic evaluation of high-dimensional, ensemble-based regression for exploring large model spaces in microbiome analyses.

BACKGROUND: Microbiome studies incorporate next-generation sequencing to obtain profiles of microbial communities. Data generated from these experiments are high-dimensional with a rich correlation structure but modest sample sizes. A statistical model that utilizes these microbiome profiles to explain a clinical or biological endpoint needs to tackle high-dimensionality resulting from the very large space of variable configurations. Ensemble models are a class of approaches that can address high-dimensionality by aggregating information across large model spaces. Although such models are popular in fields as diverse as economics and genetics, their performance on microbiome data has been largely unexplored. RESULTS: We developed a simulation framework that accurately captures the constraints of experimental microbiome data. Using this setup, we systematically evaluated a selection of both frequentist and Bayesian regression modeling ensembles. These are represented by variants of stability selection in conjunction with elastic net and spike-and-slab Bayesian model averaging (BMA), respectively. BMA ensembles that explore a larger space of models relative to stability selection variants performed better and had lower variability across simulations. However, stability selection ensembles were able to match the performance of BMA in scenarios of low sparsity where several variables had large regression coefficients. CONCLUSIONS: Given a microbiome dataset of interest, we present a methodology to generate simulated data that closely mimics its characteristics in a manner that enables meaningful evaluation of analytical strategies. Our evaluation demonstrates that the largest ensembles yield the strongest performance on microbiome data with modest sample sizes and high-dimensional measurements. We also demonstrate the ability of these ensembles to identify microbiome signatures that are associated with opportunistic Candida albicans colonization during antibiotic exposure. As the focus of microbiome research evolves from pilot to translational studies, we anticipate that our strategy will aid investigators in making evaluation-based decisions for selecting appropriate analytical methods.

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping.

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources.

Draft Genome Sequence of the Plant-Pathogenic Soil Fungus Rhizoctonia solani Anastomosis Group 3 Strain Rhs1AP.

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus.