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Resistance to insecticides inAnophelesmosquitoes threatens the effectiveness of the most widespread tools currently used to control malaria. The genetic underpinnings of resistance are still only partially understood, with much of the variance in resistance phenotype left unexplained. We performed a multi-country large scale genome-wide association study of resistance to two insecticides widely used in malaria control: deltamethrin and pirimiphos-methyl. Using a bioassay methodology designed to maximise the phenotypic difference between resistant and susceptible samples, we sequenced 969 phenotyped femaleAn. gambiaeandAn. coluzziifrom ten locations across four countries in West Africa (Benin, Côte d’Ivoire, Ghana and Togo), identifying single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) segregating in the populations. The patterns of resistance association were highly multiallelic and variable between populations, with different genomic regions contributing to resistance, as well as different mutations within a given region. While the strongest and most consistent association with deltamethrin resistance came from the region aroundCyp6aa1, this resistance was based on a combination of several independent CNVs inAn. coluzzii, and on a non-CNV bearing haplotype inAn. gambiae. Further signals involved a range of cytochrome P450, mitochondrial, and immunity genes. Similarly, for pirimiphos-methyl, while the strongest signal came from the region ofAce1, more widespread signals included cytochrome P450s, glutathione S-transferases, and a subunit of thenAChRtarget site of neonicotinoid insecticides. The regions aroundCyp9k1and theTepfamily of immune genes were associated with resistance to both insecticide classes, suggesting possible cross-resistance mechanisms. These locally-varying, multigenic and multiallelic patterns highlight the challenges involved in genomic monitoring and surveillance of resistance, and form the basis for improvement of methods used to detect and predict resistance. Based on simulations of resistance variants, we recommend that yet larger scale studies, exceeding 500 phenotyped samples per population, are required to better identify associated genomic regions.

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis, divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles (A.) gambiae and A. funestus complexes appear at low density based solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within A. demeilloni and A. moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here we utilise genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size. We further confirm the presence of cytoplasmic incompatibility factor genes, despite noticeably few prophage regions. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, and unlikely to be by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes that enable us to study the relationship between natural strains Plasmodium malaria parasites and their Anopheline hosts.Impact statementWolbachia naturally infects a wide range of arthropod species, including insect vectors of human pathogens, where they may play a role in inhibiting their replication. These bacteria have been commonly found within Aedes (Ae.) albopictus and Culex pipiens mosquitoes but have been noticeably absent in the Anopheles mosquito genera, which includes all species responsible for malaria transmission. Recent PCR-based methods have suggested the potential for natural Wolbachia strains within the A. gambiae species complex, which includes major malaria vector species including A. gambiae s.s., A. coluzzii and A. arabiensis. We recently reported the presence of stable Wolbachia strains naturally occurring within two different Anopheles species (A. demeilloni and A. moucheti). In this study, we perform comparative genomic analysis of these two Wolbachia genomes against each other and published Wolbachia strains. The current assemblies are some of the smallest sequenced Wolbachia strains of insects, although their metabolic pathway repertoire is comparable to other strains. Interestingly, prophage fragments were identified within only one of the two strains. The findings of this study will be of significant interest to researchers investigating Wolbachia as a potential malaria biocontrol strategy, giving greater insight into the evolution and diversity of this obligate intracellular endosymbiont.Data summarySequence data generated and used for this analysis are available in the National Centre for Biotechnology Information Sequence Read Archive (NCBI SRA bioproject number PRJNA642000). The two assembled Wolbachia genomes are available with genome accession numbers GCA_018491735.2 and GCA_018491625.2. Additional Wolbachia genomes used for comparative analysis are described in the supplementary material.The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Additional supplementary data files used to generate several figures can be found at: https://figshare.com/projects/Wolbachia_endosymbionts_in_two_Anopheles_species_indicates_independent_acquisitions_and_lack_of_prophage_elements/126533

K. pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes.

IncX-type plasmids have achieved clinical significance for their contribution in the dissemination of genes confering resistance to carbapenems (most bla

Vívian D’Afonseca1,*, Siomar C Soares1,*, Amjad Ali1, Anderson R Santos1, Anne C Pinto1, Aryane AC Magalhães1, Cássio de Jesus Faria1, Eudes Barbosa1, Luis C Guimarães1, Marcus Eslabão2, Sintia S Almeida1, Vinicius AC Abreu1, Adhemar Zerlotini3,4, Adriana R Carneiro5, Louise T Cerdeira5, Rommel TJ Ramos5, Raphael Hirata Jr6, Ana L Mattos-Guaraldi6, Eva Trost7, Andreas Tauch7, Artur Silva5, Maria P Schneider5, Anderson Miyoshi1, Vasco Azevedo11Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; 2Federal University of Pelotas, Pelotas, Rio Grande do Sul, Brazil; 3FIOCRUZ - CEBIO, Belo Horizonte, Minas Gerais, Brazil; 4EMBRAPA - CNPTIA, Campinas, São Paulo, Brazil; 5Federal University of Pará, Belém, Pará, Brazil; 6Rio de Janeiro State University, Rio de Janeiro, Brazil; 7Center for Biotechnology, Bielefeld University, Bielefeld, Germany *These authors contributed equally to this workBackground: The reannotation of genomes already on file is a new approach to discovering new genetic elements and to make the genomes more descriptive and current with relevant features regarding the organism’s lifestyle. Within this approach, the present study aimed to reannotate the genome of the Gram-positive human pathogen Corynebacterium diphtheriae, which causes diphtheria. The deposit of massive amounts of information linked to other species of the genus Corynebacterium has facilitated the updating of the genomic interpretation of this microorganism. Additionally, the emergence of invasive disease by nontoxigenic strains of C. diphtheriae and the reemergence of diphtheria in partially immunized populations have given impetus to new studies in relation to its structural and functional genome.Results: In relation to structural genomics, 23 coding regions (coding sequences) were deleted and 71 new genes were added to the genome annotation. Nevertheless, all the pseudogenes were validated and ten new pseudogenes were created. In relation to functional genomics, about 57% of the genome annotation was updated and became functionally more informative. The product descriptions of 41% (973 proteins) were updated. Among them, 370 that were previously annotated as “hypothetical proteins,” now have more informative descriptions. With the new annotation, the plasticity of the genome became evident, which shows improvements in the annotation of 13 pathogenicity islands already described in the literature. In addition, the large number of transposases and the presence of structural genes of bacteriophages make their genomic versatility evident. Contrasting with this reality, it also allowed the clarification of some aspects concerned with mechanisms used by C. diphtheriae to stop the invasion of the genome by bacteriophages, mediated by the clustered regularly interspaced short palindromic repeats region.Conclusion: The reannotation of the C. diphtheriae genome provided an improvement in annotation of the C. diphtheriae genome in several aspects, such as virulence characteristics and plasticity events. Moreover, the protocol used here can be extended to various other pathogens in order to improve the genomic information already on file in public databases and to minimize propagating errors. The reannotated archive and updated archive are available at: http://lgcm.icb.ufmg.br/pub/C_diphtheriae_reannotation.embl.Keywords: Corynebacterium diphtheriae, diphtheria, reannotation, CRISPR, pathogenicity islands, genome

Made available in DSpace on 2013-09-27T14:53:33Z (GMT). No. of bitstreams: 1 WOS000289620100009.pdf: 394055 bytes, checksum: 9a23eb5b1d44e130bf137337fc894796 (MD5) Previous issue date: 2011-04-18 Made available in DSpace on 2013-09-30T18:01:03Z (GMT). No. of bitstreams: 1 WOS000289620100009.pdf: 394055 bytes, checksum: 9a23eb5b1d44e130bf137337fc894796 (MD5) Previous issue date: 2011-04-18 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:17:54Z No. of bitstreams: 1 WOS000289620100009.pdf: 394055 bytes, checksum: 9a23eb5b1d44e130bf137337fc894796 (MD5) Made available in DSpace on 2014-05-20T13:17:54Z (GMT). No. of bitstreams: 1 WOS000289620100009.pdf: 394055 bytes, checksum: 9a23eb5b1d44e130bf137337fc894796 (MD5) Previous issue date: 2011-04-18 Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) NIH (National Institutes of Health) - Fogarty Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829. Fundação Oswaldo Cruz, Res Ctr Rene Rachou, Ctr Excellence Bioinformat, Natl Inst Sci & Technol, Belo Horizonte, MG, Brazil Universidade Federal de Minas Gerais (UFMG), Dept Gen Biol, Belo Horizonte, MG, Brazil Fed Univ Para, Dept Genet, BR-66059 Belem, Para, Brazil Univ Fed Sao Joao del Rei, Hlth Sci Ctr, Divinopilis, MG, Brazil Univ Estadual Ceara, Dept Vet Med, Fortaleza, Ceara, Brazil Univ Bielefeld, Dept Genet, CeBiTech, Bielefeld, Nordrhein Westf, Germany Max Planck Inst Informat, Dept Comp Sci, Saarbrucken, Saarlan, Germany Univ Fed Ouro Preto, Dept Pharmaceut Sci, Ouro Preto, MG, Brazil Univ Fed Ouro Preto, Dept Phis, Ouro Preto, MG, Brazil Univ Fed Triangulo Mineiro, Dept Biol Sci, Uberaba, MG, Brazil Universidade Federal de Uberlândia (UFU), Dept Biochem & Genet, BR-38400 Uberlandia, MG, Brazil Brazilian Agr Res Corp Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Sete Lagoas, MG, Brazil Universidade Federal de Minas Gerais (UFMG), Dept Biochem & Immunol, Belo Horizonte, MG, Brazil Universidade Federal de Minas Gerais (UFMG), Dept Parasitol, Belo Horizonte, MG, Brazil Univ Fed Ouro Preto, Dept Pharm, Ouro Preto, MG, Brazil State Univ São Paulo, Dept Technol, Jaboticabal, SP, Brazil Universidade Federal de Lavras (UFLA), Dept Chem, Lavras, MG, Brazil Brazilian Agr Res Corp Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Campinas, SP, Brazil Universidade Federal de Viçosa (UFV), Dept Plant Pathol, Vicosa, MG, Brazil Universidade Federal da Bahia (UFBA), Dept Biointeract Sci, Salvador, BA, Brazil CSIRO Livestock Ind, Clayton, Vic, Australia State Univ São Paulo, Dept Technol, Jaboticabal, SP, Brazil FAPEMIG: CBB-1181/0 FAPEMIG: REDE-186/08 NIH - Fogarty: TW007012