Microbiota profiling with long amplicons using Nanopore sequencing : full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice

Novel canine high-quality metagenome-assembled genomes, prophages and host-associated plasmids provided by long-read metagenomics together with Hi-C proximity ligation

The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues - short-read MAGs from public datasets - for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog's gastrointestinal environment. Most of them belonged to , followed by and . We also assembled one and one MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition

Diverse Populations of Staphylococcus pseudintermedius Colonize the Skin of Healthy Dogs

Staphylococcus pseudintermedius is a commensal bacterium of the canine skin but is also a key opportunistic pathogen that is responsible for most cases of pyoderma in dogs. The current paradigm indicates that infection arises when predisposing factors alter the healthy skin barrier. Despite their importance, the characteristics of the S. pseudintermedius populations colonizing the skin of healthy dogs are yet largely unknown. Here, we retrieved 67 complete circular genomes and 19 associated plasmids from S. pseudintermedius isolated from the skin of 9 healthy dogs via long-reads Nanopore sequencing. Within the S. pseudintermedius populations isolated from healthy skin, multilocus sequence typing (MLST) detected 10 different STs, distributed mainly by the host. 39% of the 18 representative genomes isolated herein were methicillin-resistant S. pseudintermedius (MRSP), and they showed, on average, a higher number of antibiotic resistance genes and prophages than did the methicillin-sensitive (MSSP). In summary, our results revealed that the S. pseudintermedius populations inhabiting the skin of healthy dogs are relatively diverse and heterogeneous in terms of MLST and methicillin resistance. In this study, all of the 67 commensal S. pseudintermedius populations that were isolated from healthy dogs contained antibiotic resistance genes, indicating the extent and severity of the problem of antimicrobial resistance in staphylococci with zoonotic potential.info:eu-repo/semantics/publishedVersio

Concordance between Antimicrobial Resistance Phenotype and Genotype of Staphylococcus pseudintermedius from Healthy Dogs

Staphylococcus pseudintermedius, a common commensal canine bacterium, is the main cause of skin infections in dogs and is a potential zoonotic pathogen. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has compromised the treatment of infections caused by these bacteria. In this study, we compared the phenotypic results obtained by minimum inhibitory concentration (MICs) for 67 S. pseudintermedius isolates from the skin of nine healthy dogs versus the genotypic data obtained with Nanopore sequencing. A total of 17 antibiotic resistance genes (ARGs) were detected among the isolates. A good correlation between phenotype and genotype was observed for some antimicrobial classes, such as ciprofloxacin (fluoroquinolone), macrolides, or tetracycline. However, for oxacillin (beta-lactam) or aminoglycosides the correlation was low. Two antibiotic resistance genes were located on plasmids integrated in the chromosome, and a third one was in a circular plasmid. To our knowledge, this is the first study assessing the correlation between phenotype and genotype regarding antimicrobial resistance of S. pseudintermedius from healthy dogs using Nanopore sequencing technology.info:eu-repo/semantics/publishedVersio

Concordance between Antimicrobial Resistance Phenotype and Genotype of Staphylococcus pseudintermedius from Healthy Dogs

Staphylococcus pseudintermedius, a common commensal canine bacterium, is the main cause of skin infections in dogs and is a potential zoonotic pathogen. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has compromised the treatment of infections caused by these bacteria. In this study, we compared the phenotypic results obtained by minimum inhibitory concentration (MICs) for 67 S. pseudintermedius isolates from the skin of nine healthy dogs versus the genotypic data obtained with Nanopore sequencing. A total of 17 antibiotic resistance genes (ARGs) were detected among the isolates. A good correlation between phenotype and genotype was observed for some antimicrobial classes, such as ciprofloxacin (fluoroquinolone), macrolides, or tetracycline. However, for oxacillin (beta-lactam) or aminoglycosides the correlation was low. Two antibiotic resistance genes were located on plasmids integrated in the chromosome, and a third one was in a circular plasmid. To our knowledge, this is the first study assessing the correlation between phenotype and genotype regarding antimicrobial resistance of S. pseudintermedius from healthy dogs using Nanopore sequencing technology

Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1-mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential.info:eu-repo/semantics/publishedVersio

Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1-mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential

Whole genome sequencing and de novo assembly of Staphylococcus pseudintermedius: a pangenome approach to unravelling pathogenesis of canine pyoderma

Background Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms. Hypothesis/Objectives Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas. Methods and materials Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi’o 6.1. Results Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages. Conclusions and clinical importance This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.info:eu-repo/semantics/publishedVersio

Whole genome sequencing and de novo assembly of Staphylococcus pseudintermedius: a pangenome approach to unravelling pathogenesis of canine pyoderma

Background: Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms. Hypothesis/Objectives: Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas. Methods and materials: Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi’o 6.1. Results: Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages. Conclusions and clinical importance: This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.Fil: Ferrer, Lluís. Universitat Autònoma de Barcelona; EspañaFil: García Fonticoba, Rocío. Universitat Autònoma de Barcelona; EspañaFil: Pérez, Daniel. Universitat Autònoma de Barcelona; EspañaFil: Viñes, Joaquim. Universitat Autònoma de Barcelona; EspañaFil: Fàbregas, Norma. Universitat Autònoma de Barcelona; EspañaFil: Madroñero, Sergi. Universitat Autònoma de Barcelona; EspañaFil: Meroni, Gabriele. No especifíca;Fil: Martino, Piera A.. No especifíca;Fil: Martínez, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Maté, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Sanchez Bruni, Sergio Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Cuscó, Anna. Universitat Autònoma de Barcelona; EspañaFil: Migura García, Lourdes. Universitat Autònoma de Barcelona; EspañaFil: Francino, Olga. Universitat Autònoma de Barcelona; Españ

From the clinic to the farm: Applying sequencing and de novo assembly to characterize One-Health bacterial pathogens

Les infeccions causades per bacteris resistents a múltiples antibiòtics son una amenaça tant per a la salut animal com la humana degut a la limitació en el tractament, la qual pot portar a complicacions clíniques severes, estades hospitalàries més llargues o, fins i tot, a la mort. Els bacteris resistents a múltiples antibiòtics poden ser transmesos al medi ambient, a altres animals o a humans a través de diferents vies, tals com la contaminació fecal o per la cadena alimentària. En aquest escenari, l’aproximació One-Health considera la salut com una entitat global, incloent la humana, l’animal i la mediambiental. L’objectiu d’aquesta tesis és optimitzar l’ús de reads llargs en la seqüenciació de genoma complet (whole-genome sequencing) per a la caracterització del cromosoma i plasmidis bacterians, incloent la presència de gens de resistència a antibiòtics, elements genètics mòbils, i factors de virulència. Mitjançant l’ús de seqüenciació de genoma complet amb reads llargs hem muntat de novo cromosomes i plasmidis en contigs únics per a Staphylococcus pseudintermedius aïllats en gossos, Escherichia coli d’animals de producció i el granger, i Klebsiella pneumoniae d’humans. Aquesta aproximació ha permès localitzar els gens de resistència a antibiòtics en plasmidis o cromosoma, i ha permès seqüenciar elements genètics mòbils sencers, superant així els desavantatges associats al reads curts. A més, hem pogut descriure la transmissió de plasmidis similars que duien gens de resistència a antibiòtics per a Escherichia coli resistent a colistina en una granja mixta, i per a Klebisella pneumoniae resistent a carbapenems en un brot interhospitalari. Els perfils de resistència a antibiòtics i factors de virulència d’Staphylococcus pseudintermedius aïllats de gossos i Escherichia coli d’animals de producció ressalten el rol dels animals domèstics com a un reservori de patògens amb alt potencial zoonòtic.Las infecciones causadas por bacterias resistentes a múltiples antibióticos son una amenaza tanto para la salud animal como la humana debido a la limitación en el tratamiento, la cual puede conllevar complicaciones clínicas severas, estancias hospitalarias más largar o incluso la muerte. Las bacterias resistentes a múltiples antibióticos pueden ser transmitidos al medio ambiente, otros animales o humanos mediante diferentes vías, tales como la contaminación fecal o a través de la cadena alimentaria. En este escenario, la aproximación One-Health considera la salud como una entidad global, incluyendo la humana, la animal y la medioambiental. El objetivo de esta tesis es optimizar el uso de reads largos en la secuenciación de genoma completo (whole-genome sequencing) para la caracterización del cromosoma y plásmidos bacterianos, incluyendo la presencia de genes de resistencia a antibióticos, elementos genéticos móviles, y factores de virulencia. Mediante el uso de secuenciación de genoma completo con reads largos hemos montado de novo cromosomas y plásmidos en contigs únicos para Staphylococcus pseudintermedius aislados de perros, Escherichia coli de animales de producción y el granjero, i Klebsiella pneumoniae de humanos. Esta aproximación ha permitido localizar los genes de resistencia a antibióticos en plásmidos o el cromosoma, y ha permitido secuenciar elementos genéticos móviles completos, superando así las desventajas asociadas a reads cortos. Además, hemos podido describir la transmisión de plásmidos similares que llevaban genes de resistencia a antibióticos para Escherichia coli resistente a colistina en una granja mixta, y para Klebsiella pneumoniae resistente a carbapenems en un brote interhospitalario. Los perfiles de resistencia a antibióticos y factores de virulencia de Staphylococcus pseudintermedius aislados de perros y Escherichia coli de animales de producción resaltan el rol de los animales domésticos como reservorio de patógenos con alto potencial zoonótico.Multi-drug resistant bacteria infections are a threat to animal and human health due to the limitation in treatment, which can lead to severe clinical complications, longer hospital stays, or even death. Multi-drug resistant bacteria can be transmitted to the environment, other animals, or humans via different ways, such as fecal contamination or food-chain. In this scenario, One-Health is an approach that considers health as a global entity, including human, animal, and the environment. The aim of this thesis is to optimize the use of long reads in whole-genome sequencing approaches for characterizing the bacterial genome and plasmids, including the presence of antibiotic resistance genes, mobile genetic elements, and virulence factors. By using long-read whole-genome sequencing, we de novo assembled complete chromosomes and plasmids as single contigs for Staphylococcus pseudintermedius from dogs, Escherichia coli from livestock and the farmer, and Klebsiella pneumoniae from humans. This approach located the antibiotic resistance genes in plasmids or chromosomes and spanned mobile genetic elements, thus overcoming the pitfalls associated with short reads. Moreover, we unraveled the transmission of similar plasmids harboring antibiotic resistance genes for colistin-resistant Escherichia coli in a mixed farm and for carbapenem-resistant Klebsiella pneumoniae in an inter-hospital outbreak. The antibiotic resistance and virulence factor genes profiles of Staphylococcus pseudintermedius from dogs and Escherichia coli from livestock highlight the role of domestic animals as a reservoir of pathogens with highly zoonotic potential.Universitat Autònoma de Barcelona. Programa de Doctorat en Producció Anima