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September 22, 2019

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.

Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan.KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features.A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain.We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.

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September 22, 2019

Transcriptome analysis of Neisseria gonorrhoeae during natural infection reveals differential expression of antibiotic resistance determinants between men and women.

Neisseria gonorrhoeae is a bacterial pathogen responsible for the sexually transmitted infection gonorrhea. Emergence of antimicrobial resistance (AMR) of N. gonorrhoeae worldwide has resulted in limited therapeutic choices for this infection. Men who seek treatment often have symptomatic urethritis; in contrast, gonococcal cervicitis in women is usually minimally symptomatic, but may progress to pelvic inflammatory disease. Previously, we reported the first analysis of gonococcal transcriptome expression determined in secretions from women with cervical infection. Here, we defined gonococcal global transcriptional responses in urethral specimens from men with symptomatic urethritis and compared these with transcriptional responses in specimens obtained from women with cervical infections and in vitro-grown N. gonorrhoeae isolates. This is the first comprehensive comparison of gonococcal gene expression in infected men and women. RNA sequencing analysis revealed that 9.4% of gonococcal genes showed increased expression exclusively in men and included genes involved in host immune cell interactions, while 4.3% showed increased expression exclusively in women and included phage-associated genes. Infected men and women displayed comparable antibiotic-resistant genotypes and in vitro phenotypes, but a 4-fold higher expression of the Mtr efflux pump-related genes was observed in men. These results suggest that expression of AMR genes is programed genotypically and also driven by sex-specific environments. Collectively, our results indicate that distinct N. gonorrhoeae gene expression signatures are detected during genital infection in men and women. We propose that therapeutic strategies could target sex-specific differences in expression of antibiotic resistance genes.IMPORTANCE Recent emergence of antimicrobial resistance of Neisseria gonorrhoeae worldwide has resulted in limited therapeutic choices for treatment of infections caused by this organism. We performed global transcriptomic analysis of N. gonorrhoeae in subjects with gonorrhea who attended a Nanjing, China, sexually transmitted infection (STI) clinic, where antimicrobial resistance of N. gonorrhoeae is high and increasing. We found that N. gonorrhoeae transcriptional responses to infection differed in genital specimens taken from men and women, particularly antibiotic resistance gene expression, which was increased in men. These sex-specific findings may provide a new approach to guide therapeutic interventions and preventive measures that are also sex specific while providing additional insight to address antimicrobial resistance of N. gonorrhoeae. Copyright © 2018 Nudel et al.

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September 22, 2019

Genomic analysis of the insect-killing fungus Beauveria bassiana JEF-007 as a biopesticide.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.

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September 22, 2019

Orphan legumes growing in dry environments: Marama bean as a case study.

Plants have developed morphological, physiological, biochemical, cellular, and molecular mechanisms to survive in drought-stricken environments with little or no water caused by below-average precipitation. In this mini-review, we highlight the characteristics that allows marama bean [Tylosema esculentum (Burchell) Schreiber], an example of an orphan legume native to arid regions of southwestern Southern Africa, to flourish under an inhospitable climate and dry soil conditions where no other agricultural crop competes in this agro-ecological zone. Orphan legumes are often better suited to withstand such harsh growth environments due to development of survival strategies using a combination of different traits and responses. Recent findings on questions on marama bean speciation, hybridization, population dynamics, and the evolutionary history of the bean and mechanisms by which the bean is able to extract and conserve water and nutrients from its environment as well as aspects of morphological and physiological adaptation will be reviewed. The importance of the soil microbiome and the genetic diversity in this species, and their interplay, as a reservoir for improvement will also be considered. In particular, the application of the newly established marama bean genome sequence will facilitate both the identification of important genes involved in the interaction with the soil microbiome and the identification of the diversity within the wild germplasm for genes involved drought tolerance. Since predicted future changes in climatic conditions, with less water availability for plant growth, will severely affect agricultural productivity, an understanding of the mechanisms of unique adaptations in marama bean to such conditions may also provide insights as to how to improve the performance of the major crops.

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September 22, 2019

Opposite polarity monospore genome de novo sequencing and comparative analysis reveal the possible heterothallic life cycle of Morchella importuna.

Morchella is a popular edible fungus worldwide due to its rich nutrition and unique flavor. Many research efforts were made on the domestication and cultivation of Morchella all over the world. In recent years, the cultivation of Morchella was successfully commercialized in China. However, the biology is not well understood, which restricts the further development of the morel fungus cultivation industry. In this paper, we performed de novo sequencing and assembly of the genomes of two monospores with a different mating type (M04M24 and M04M26) isolated from the commercially cultivated strain M04. Gene annotation and comparative genome analysis were performed to study differences in CAZyme (Carbohydrate-active enzyme) enzyme content, transcription factors, duplicated sequences, structure of mating type sites, and differences at the gene and functional levels between the two monospore strains of M. importuna. Results showed that the de novo assembled haploid M04M24 and M04M26 genomes were 48.98 and 51.07 Mb, respectively. A complete fine physical map of M. importuna was obtained from genome coverage and gene completeness evaluation. A total of 10,852 and 10,902 common genes and 667 and 868 endemic genes were identified from the two monospore strains, respectively. The Gene Ontology (GO) and KAAS (KEGG Automatic Annotation Serve) enrichment analyses showed that the endemic genes performed different functions. The two monospore strains had 99.22% collinearity with each other, accompanied with certain position and rearrangement events. Analysis of complete mating-type loci revealed that the two monospore M. importuna strains contained an independent mating-type structure and remained conserved in sequence and location. The phylogenetic and divergence time of M. importuna was analyzed at the whole-genome level for the first time. The bifurcation time of morel and tuber was estimated to be 201.14 million years ago (Mya); the two monospore strains with a different mating type represented the evolution of different nuclei, and the single copy homologous genes between them were also different due to a genetic differentiation distance about 0.65 Mya. Compared with truffles, M. importuna had an extension of 28 clusters of orthologous genes (COGs) and a contraction of two COGs. The two different polar nuclei with different degrees of contraction and expansion suggested that they might have undergone different evolutionary processes. The different mating-type structures, together with the functional clustering and enrichment analysis results of the endemic genes of the two different polar nuclei, imply that M. importuna might be a heterothallic fungus and the interaction between the endemic genes may be necessary for its complete life history. Studies on the genome of M. importuna facilitate a better understanding of morel biology and evolution.

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September 22, 2019

Distinct genomic features characterize two clades of Corynebacterium diphtheriae: Proposal of Corynebacterium diphtheriae subsp. diphtheriae subsp. nov. and Corynebacterium diphtheriae subsp. lausannense subsp. nov.

Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

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September 22, 2019

Functional and genome sequence-driven characterization of tal effector gene repertoires reveals novel variants with altered specificities in closely related Malian Xanthomonas oryzae pv. oryzae strains.

Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TalF (formerly Tal5) or TalC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that talF and talC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either talC- or talF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TalF and TalC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related tal effector genes. Notably, a new TalF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TalB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TalB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in tal repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

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September 22, 2019

Natural selection in bats with historical exposure to white-nose syndrome

Hibernation allows animals to survive periods of resource scarcity by reducing their energy expenditure through decreased metabolism. However, hibernators become susceptible to psychrophilic pathogens if they cannot mount an efficient immune response to infection. While Nearctic bats infected with white-nose syndrome (WNS) suffer high mortality, related Palearctic taxa are better able to survive the disease than their Nearctic counterparts. We hypothesised that WNS exerted historical selective pressure in Palearctic bats, resulting in genomic changes that promote infection tolerance.

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September 22, 2019

Long-term colonization dynamics of Enterococcus faecalis in implanted devices in research macaques.

Enterococcus faecalis is a common opportunistic pathogen that colonizes cephalic recording chambers (CRCs) of macaques used in cognitive neuroscience research. We previously characterized 15 E. faecalis strains isolated from macaques at the Massachusetts Institute of Technology (MIT) in 2011. The goal of this study was to examine how a 2014 protocol change prohibiting the use of antimicrobials within CRCs affected colonizing E. faecalis strains. We collected 20 E. faecalis isolates from 10 macaques between 2013 and 2017 for comparison to 4 isolates previously characterized in 2011 with respect to the sequence type (ST) distribution, antimicrobial resistance, biofilm formation, and changes in genes that might confer a survival advantage. ST4 and ST55 were predominant among the isolates characterized in 2011, whereas the less antimicrobial-resistant lineage ST48 emerged to dominance after 2013. Two macaques remained colonized by ST4 and ST55 strains for 5 and 4 years, respectively. While the antimicrobial resistance and virulence factors identified in these ST4 and ST55 strains remained relatively stable, we detected an increase in biofilm formation ability over time in both isolates. We also found that ST48 strains were typically robust biofilm formers, which could explain why this ST increased in prevalence. Finally, we identified mutations in the DNA mismatch repair genes mutS and mutL in separate ST55 and ST4 strains and confirmed that strains bearing these mutations displayed a hypermutator phenotype. The presence of a hypermutator phenotype may complicate future antimicrobial treatment for clinically relevant E. faecalis infections in macaques.IMPORTANCEEnterococcus faecalis is a common cause of health care-associated infections in humans, largely due to its ability to persist in the hospital environment, colonize patients, acquire antimicrobial resistance, and form biofilms. Understanding how enterococci evolve in health care settings provides insight into factors affecting enterococcal survival and persistence. Macaques used in neuroscience research have long-term cranial implants that, despite best practices, often become colonized by E. faecalis This provides a unique opportunity to noninvasively examine the evolution of enterococci on a long-term indwelling device. We collected E. faecalis strains from cephalic implants over a 7-year period and characterized the sequence type, antimicrobial resistance, virulence factors, biofilm production, and hypermutator phenotypes. Improved antimicrobial stewardship allowed a less-antimicrobial-resistant E. faecalis strain to predominate at the implant interface, potentially improving antimicrobial treatment outcomes if future clinical infections occur. Biofilm formation appears to play an important role in the persistence of the E. faecalis strains associated with these implants. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d’Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Exploring benzimidazole resistance in Haemonchus contortus by next generation sequencing and droplet digital PCR.

Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect resistance-predicting alleles. Here we propose a novel droplet digital PCR (ddPCR) assay for rapid and precise identification of H. contortus strains as being resistant or susceptible to benzimidazole drugs based on the presence or absence of the most common resistance-conferring mutation F200Y (TAC) in the ß tubulin isotype 1 gene. The newly developed ddPCR assay was first optimized and validated utilizing DNA templates from single-worm samples, which were previously sequenced using the next generation PacBio RSII Sequencing (NGS) platform. Subsequent NGS results for faecal larval cultures were then used as a reference to compare the obtained values for fractional abundances of the resistance-determining mutant allele between ddPCR and NGS techniques in each sample. Both methods managed to produce highly similar results and ddPCR proved to be a reliable tool which, when utilized at full capacity, can be used to create a powerful mutation detection and quantification assay. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

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September 22, 2019

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen.We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence.Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

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September 22, 2019

Draft genome sequence of wild Prunus yedoensis reveals massive inter-specific hybridization between sympatric flowering cherries.

Hybridization is an important evolutionary process that results in increased plant diversity. Flowering Prunus includes popular cherry species that are appreciated worldwide for their flowers. The ornamental characteristics were acquired both naturally and through artificially hybridizing species with heterozygous genomes. Therefore, the genome of hybrid flowering Prunus presents important challenges both in plant genomics and evolutionary biology.We use long reads to sequence and analyze the highly heterozygous genome of wild Prunus yedoensis. The genome assembly covers >?93% of the gene space; annotation identified 41,294 protein-coding genes. Comparative analysis of the genome with 16 accessions of six related taxa shows that 41% of the genes were assigned into the maternal or paternal state. This indicates that wild P. yedoensis is an F1 hybrid originating from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura, and it can be clearly distinguished from its confusing taxon, Yoshino cherry. A focused analysis of the S-locus haplotypes of closely related taxa distributed in a sympatric natural habitat suggests that reduced restriction of inter-specific hybridization due to strong gametophytic self-incompatibility is likely to promote complex hybridization of wild Prunus species and the development of a hybrid swarm.We report the draft genome assembly of a natural hybrid Prunus species using long-read sequencing and sequence phasing. Based on a comprehensive comparative genome analysis with related taxa, it appears that cross-species hybridization in sympatric habitats is an ongoing process that facilitates the diversification of flowering Prunus.

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September 22, 2019

Genomic approaches for studying crop evolution.

Understanding how crop plants evolved from their wild relatives and spread around the world can inform about the origins of agriculture. Here, we review how the rapid development of genomic resources and tools has made it possible to conduct genetic mapping and population genetic studies to unravel the molecular underpinnings of domestication and crop evolution in diverse crop species. We propose three future avenues for the study of crop evolution: establishment of high-quality reference genomes for crops and their wild relatives; genomic characterization of germplasm collections; and the adoption of novel methodologies such as archaeogenetics, epigenomics, and genome editing.

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September 22, 2019

Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix.

The date palm tree is a commercially important member of the genus Phoenix whose 14 species are dioecious with separate male and female individuals. To identify sex determining genes we sequenced the genomes of 15 female and 13 male Phoenix trees representing all 14 species. We identified male-specific sequences and extended them using phased single-molecule sequencing or BAC clones. We observed that only four genes contained sequences conserved in all analyzed Phoenix males. Most of these sequences showed similarity to a single genomic locus in the closely related monoecious oil palm. CYP703 and GPAT3, two single copy genes present in males and critical for male flower development in other monocots, were absent in females. A LOG-like gene appears translocated into the Y-linked region and is suggested to play a role in suppressing female flowers. Our data are consistent with a two-mutation model for the evolution of dioecy in Phoenix.

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