Scalable and versatile container-based pipelines for de novo genome assembly and bacterial annotation. [version 1; peer review: 2 approved, 1 approved with reservations]

Background: Advancements in DNA sequencing technology have transformed the field of bacterial genomics, allowing for faster and more cost effective chromosome level assemblies compared to a decade ago. However, transforming raw reads into a complete genome model is a significant computational challenge due to the varying quality and quantity of data obtained from different sequencing instruments, as well as intrinsic characteristics of the genome and desired analyses. To address this issue, we have developed a set of container-based pipelines using Nextflow, offering both common workflows for inexperienced users and high levels of customization for experienced ones. Their processing strategies are adaptable based on the sequencing data type, and their modularity enables the incorporation of new components to address the community’s evolving needs. Methods: These pipelines consist of three parts: quality control, de novo genome assembly, and bacterial genome annotation. In particular, the genome annotation pipeline provides a comprehensive overview of the genome, including standard gene prediction and functional inference, as well as predictions relevant to clinical applications such as virulence and resistance gene annotation, secondary metabolite detection, prophage and plasmid prediction, and more. Results: The annotation results are presented in reports, genome browsers, and a web-based application that enables users to explore and interact with the genome annotation results. Conclusions: Overall, our user-friendly pipelines offer a seamless integration of computational tools to facilitate routine bacterial genomics research. The effectiveness of these is illustrated by examining the sequencing data of a clinical sample of Klebsiella pneumoniae

Ocorrência de seqüências relacionadas com a virulência e análise filogenética de estirpes comensais e patogênicas de Escherichia coli aviário (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.533540Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A New Complex of Palladium(II) With 2-Furoic Hydrazide: Synthesis, Characterization, Theoretical Calculations and Biological Studies

A new complex of palladium was isolated with 2-furoic hydrazide (FH) and characterized by spectroscopic methods. The results show that the ligand is coordinated to palladium by the basic nitrogen of NH2 group and has a general structure of type cis-[Pd(FH)2Cl2]. The structure of palladium(II) complex was optimized and theoretical data show good agreement with the experimental results. The cytotoxic activity was evaluated in a chronic myelogenous leukemia cell line, which revealed that the compound is less active than cisplatin or carboplatin. At 300 g mL−1, the complex presented antimicrobial activity more efficient than ampicillin, chloranfenicol and kanamicyn. (doi: 10.5562/cca2151

A Fatal Bacteremia Caused by Hypermucousviscous KPC-2 Producing Extensively Drug-Resistant K64-ST11 Klebsiella pneumoniae in Brazil

We report a fatal bacteremia caused by Klebsiella pneumoniae in a 60–70-year-old patient from Brazil. The genomic analysis of three isolates (from blood culture, nasal and anal swabs) showed that the bacteremia was caused by a KPC-2 producing extensively drug-resistant K64-ST11 hypermucousviscous K. pneumoniae (hmKP) harboring several virulence and antimicrobial resistance genes. Although the isolates did not present virulence markers associated with hypervirulent K. pneumoniae (hvKP), they showed invasion and toxicity to epithelial Hep-2 cells; resistance to cell microbicidal mechanisms; and blood and human serum survival, evidencing their pathogenic potential. This study highlights the risk of infection caused by hmKp strains not characterized as hvKP as well as the clinical implications and difficulty of treatment, especially in elderly or immunocompromised patients

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD50 of 4.0 × 105 CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD50 of 1.2 × 1012 CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes

Clonal and biological characterization of avian Escherichia coli

Orientador: Wanderley Dias da SilveiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Quarenta e nove linhagens de Escherichia coli isoladas de aves isoladas de aves doentes (24 de aves com septicemia - S, 14 de aves com síndrome da cabeça inchada SHS e 11 de aves com onfalite - O) e 30 linhagens isoladas da região cloacal de aves saudáveis (C) foram analisadas em relação à presença de genes de virulência, grupos filogenéticos de E. coli, similaridade do gene mc. Estes dados foram comparados a um estudo populacional previamente obtido a partir de ERIC PCR (Silveira et al.,2002b), para a caracterização de possíveis dones patogênicos presentes nesta população. Os resultados demonstraram que os genes de virulência analisados foram mais freqüentes entre as linhagens isoladas de aves doentes. Linhagens isoladas de aves saudáveis, de aves com onfalite e de aves com septicemia derivam-se de grupos clonais não patogênicos de E. coli (A, e 81). Por 'outro lado, linhagens SHS e com septicemia derivam-se de grupos clonais patogênicos (82 e D). A comparação destes dados com os dados de ERIC-PCR demonstrou que estas linhagens constituem de três grupos principais: (i) 1 não patogênico, formado por linhagens isoladas de aves com onfalite e de aves saudáveis ambas derivadas principalmente de grupos clonais não patogênicos de E. coli (A); (ii) outro composto por linhagens SHS, derivadas principalmente do grupo clonal patogênico de E. coli (grupo D); e um terceiro grupo (iii) formado por linhagens isoladas por aves com septicemia onde 46% derivaram-se do grupo não patogênico A e outros 46% do grupo patogênico D. Estes dados sugerem que linhagens SHS provavelmente constituem um grupo clonal patogênico dentro da população analisada. Por outro lado, linhagens S possivelmente apresentam uma origem polifilética de dois ancestrais diferentes (um patogênico e outro não patogênico) onde a aquisição de genes de virulência através de transmissão horizontal possivelmente seja responsável por eventos que conduziram a um processo de evolução convergente. O agrupamento das linhagens isoladas de aves saudáveis e de linhagens O, sugere que a onfalite seja causada por linhagens não patogênicas. Os dados de similaridade do gene fIíC demonstram que houve a formação de dois grupos: um formado por linhagens isoladas de aves doentes e outro formada por linhagens isoladas de aves saudáveis. Estes dados sugerem que o gene fliC pode estar associado à diferenciação de possíveis dones patogênicos e não patogênico em linhagens de E. coli de origem aviária, assim como o observado em E. calí patogênicas para humanosAbstract: Forty and nine Escherichía coli strains isolated frem sick chickens (24 frem septicaemia - S, 14 frem swollen head syndrem - SHS, and 11 frem omphalitis - O), and 30 strains isolated frem the cloacae region (C) of healthy chickens were analyzed in relation of the presence of virulence-related genes, E. colí phylogenetical groups, fliC gene similarity. These data were compared to a ERIC-PCR study realized of the same strains (Silveira et a/.,2002b) to characterize possible pathogenic clones present in this population. The results demonstrated that the virulence genes were more frequent among E. colí strains isolated from sick chicken. Strains isolated frem healthy chicken, frem omphalitis and from septicemic chickens were derived frem E. colí non pathogenic greups (A and B 1). SHS and septicemic strains belonged to E. coli pathogenic greups (B2 and D). The comparison of these data with the ERIC-PCR study demonstrated that these strains compounded three main clusters: (i) one non pathogenic, comp<?unded by E. colí strains isolated frem healthy chickens and frem omphalitis; (ii) one compounded by SHS strains belonged to D phylogenetical group; (iii) one compounded by septicemic strains where 46% belonged to A E. colí greup and 46% belonged to D E. calí greup. These data suggests the SHS strains prebably form a pathogenic clonal group inside the population analyzed. By contrast, septicemic strains may have a poliphyletical origin frem two ancestors (one pathogenic and another non pathogenic) where the horizontal acquisition of virulence genes may be respansible for events that conduced to a convergent evolution. The clustering of omphalitis and strains isolated frem healthy chickens suggests that omphalitis is caused by non pathogenic E. colí strains. The mc similarity data demonstrated that were two clusters one compounded by strains isolated frem healthy chickens and other compounded by strains isolated frem sick chickens. These data suggest that f/iC gene can be associated to pathogenic and non pathogenic clones differentiation like described among E. colí strains pathogenic for humanDoutoradoMicrobiologiaDoutor em Genetica e Biologia Molecula

Estrutura clonal e fatores de colonização de linhagens de Escherichia coli de origem aviaria

Orientador : Wanderley Dias da SilveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaMestrad

Virulence factors of avian pathogenic Escherichia coli (APEC) Fatores de virulência de Escherichia coli aviária patogênica (APEC)

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.<br>Linhagens de Escherichia coli patogênicas para aves (APEC) causam uma grande diversidade de doenças em aves e são responsáveis por grandes prejuízos na indústria aviária. Nos últimos anos, vários estudos foram realizados para melhor entender a patogênese de linhagens APEC e para desenvolver ferramentas que podem prevenir as perdas econômicas causadas por estas linhagens. Esta revisão discute os fatores de virulência descritos nestas linhagens e os avanços realizados para entender e identificar os determinantes de virulência presentes em APEC

Caracterização biológica e molecular de linhagens de Streptococcus crista isoladas do biofilme dental de seres humanos através de iniciadores arbitrários - PCR (AP-PCR)

Streptococcus ssp are important components of the dental biofilm and Streptococcus crista is considered to be an interesting model of bacterial interactions taking place in this biofilm. In the present work, S. crista strains were isolated from the dental biofilm of Brazilian individuals and studied with respect to their biological characteristics and their molecular profile by means of AP-PCR techniques, using the RR2, 434, OPR2, OPR8, and OPR13 primers. Results allowed us to build a similarity dendrogram. Analysis of the similarity dendrogram allowed the separation of the studied strains into similarity groups. All isolates presented fibril tufts by Transmission Electron Microscopy (TEM). These isolates were able to bind to salivary amylase and to adhere to mouth epithelial cells. Some strains displaying fibril tufts and positive adherence were not able to co-aggregate with Fusobacterium nucleatum, suggesting that different adhesin groups are present in these strains.Streptococcus spp são importantes componentes do biofilme dental sendo Streptococus crista considerado um interessante modelo de interações bacterianas que nele ocorrem. No presente trabalho linhagens de S. crista, foram isoladas do biofilme dental de indivíduos brasileiros, e estudadas em relação a suas características biológicas e ao seu perfil molecular através da técnica do AP-PCR, usando-se os iniciadores RR2, 434, OPR2, OPR8 e OPR13. Os resultados nos permitiram construir um dendrograma de similaridade. A análise do dendrograma de similaridade permitiu a separação das linhagens estudadas em grupos de similaridade. Todos os isolados apresentaram tufo de fibrilas, quando estudados por Microscopia Eletrônica de Transmissão (MET). Estes isolados foram capazes de se ligar à amilase salivar e de se aderir a células epiteliais bucais. Algumas linhagens, que apresentam tufo de fibrilas e aderência positiva, não foram capazes de coagregar com a Fusobacterium nucleatum, sugerindo que diferentes grupos de adesinas estão presentes nestas amostras.119126Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq