Dogs present almost all their skin sites covered by hair, but canine skin disorders are more common in certain skin sites and breeds. The goal of our study is to characterize the composition and ...variability of the skin microbiota in healthy dogs and to evaluate the effect of the breed, the skin site, and the individual. We have analyzed eight skin sites of nine healthy dogs from three different breeds by massive sequencing of 16S rRNA gene V1-V2 hypervariable regions. The main phyla inhabiting the skin microbiota in healthy dogs are Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, and Bacteroidetes. Our results suggest that skin microbiota composition pattern is individual specific, with some dogs presenting an even representation of the main phyla and other dogs with only a major phylum. The individual is the main force driving skin microbiota composition and diversity rather than the skin site or the breed. The individual is explaining 45% of the distances among samples, whereas skin site explains 19% and breed 9%. Moreover, analysis of similarities suggests a strong dissimilarity among individuals (
= 0.79,
= 0.001) that is mainly explained by low-abundant species in each dog. Skin site also plays a role: inner pinna presents the highest diversity value, whereas perianal region presents the lowest one and the most differentiated microbiota composition.
Abstract
Objectives
To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain.
...Methods
Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids.
Results
Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring blaOXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. blaNDM-1 was carried in two related IncR plasmid populations and blaNDM-7 in a conjugative 50 kb IncX3 plasmid.
Conclusions
We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring blaNDM genes.
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.
Résumé
Contexte
Staphylococcus pseudintermedius est le principal agent étiologique de la pyodermite canine. Le séquençage du génome entier est le moyen le plus complet d'obtenir des informations génomiques pertinentes sur les micro‐organismes.
Hypothèse/Objectifs
La technologie Oxford Nanopore permet un séquençage de qualité et un assemblage de novo de l'ensemble du génome de S. pseudintermedius. L'analyse du génome entier de S. pseudintermedius peut aider à mieux comprendre la pathogénie des pyodermites canines.
Méthodes et materiel
Vingt‐deux souches de S. pseudintermedius isolées de la peau de cinq chiens sains et 33 souches isolées de la peau de 33 chiens atteints de pyodermite ont été analysées. L'ADN a été extrait et séquencé à l'aide d'Oxford Nanopore MinION, une nouvelle technologie qui offre des lectures plus longues dans un appareil portatif. Le pangénome a été analysé et visualisé avec ANVI'O 6.1.
Résultats
La technologie Nanopore a permis le séquençage et l'assemblage de novo des génomes de 55 souches de S. pseudintermedius isolées de chiens sains et de chiens atteints de pyodermite. La taille moyenne du génome de S. pseudintermedius était de 2,62 Mpb, dont 48 % étaient le génome central. Les isolats de pyodermite contenaient un nombre plus élevé de gènes de résistance aux antimicrobiens, mais le nombre total de gènes de facteurs de virulence n'a pas changé entre les isolats de chiens sains et de chiens atteints de pyodermite. Les génomes des souches de S. pseudintermedius résistantes à la méticilline (MRSP) étaient plus grands que ceux des souches sensibles à la méticilline (MSSP) (2,80 Mbp contre 2,59 Mbp), en raison d'une plus grande présence de gènes de résistance aux antimicrobiens, de phages et de prophages.
Conclusions et importance Clinique
Cette technique permet une caractérisation beaucoup plus précise et plus facile des populations canines de S. pseudintermedius et peut conduire à une meilleure compréhension de la pathogénie des pyodermites canines.
Resumen
Introducción
Staphylococcus pseudintermedius es el principal agente etiológico de la pioderma canina. La secuenciación del genoma completo es la forma más completa de obtener información genómica relevante sobre microorganismos.
Hipótesis/Objetivos
la tecnología Oxford Nanopore permite la secuenciación de calidad y el ensamblaje de novo de todo el genoma de S. pseudintermedius. El análisis del genoma completo de S. pseudintermedius puede ayudar a comprender mejor la patogenia de las piodermas caninas.
Métodos y materiales
Se analizaron veintidós cepas de S. pseudintermedius aisladas de la piel de cinco perros sanos y 33 cepas aisladas de la piel de 33 perros con pioderma. El DNA se extrajo y secuenció utilizando Oxford Nanopore MinION, una nueva tecnología que ofrece lecturas más largas en un dispositivo de mano. El pangenoma se analizó y visualizó con ANVI’O 6.1.
Resultados
la tecnología Nanopore permitió la secuenciación y el ensamblaje de novo de los genomas de 55 cepas de S. pseudintermedius aisladas de perros sanos y de perros con pioderma. El tamaño medio del genoma de S. pseudintermedius fue de 2,62 Mbp, con un 48% de genoma central. Los aislados de pioderma contenían un mayor número de genes de resistencia a los antimicrobianos, sin embargo, el número total de genes de factores de virulencia no cambió entre los aislados de perros sanos y los de perros con pioderma. Los genomas de las cepas de S. pseudintermedius resistentes a la meticilina (MRSP) fueron más grandes que los de las cepas sensibles a la meticilina (MSSP) (2,80 Mbp versus 2,59 Mbp), como consecuencia de una mayor presencia de genes de resistencia a los antimicrobianos, fagos y profagos.
Conclusiones e importancia clínica
esta técnica permite una caracterización mucho más precisa y sencilla de las poblaciones caninas de S. pseudintermedius y puede conducir a una mejor comprensión de la patogenia de las piodermas caninas.
Zusammenfassung
Hintergrund
Staphylococcus pseudintermedius stellt die hauptsächliche ätiologische Ursache für die canine Pyodermie dar. Eine Gesamtgenomsequenzierung ist der umfassendste Weg, um relevante Genominformation über Mikroorganismen zu erhalten.
Hypothese/Ziele
Die Oxford Nanopore Technologie ermöglicht eine qualitative Sequenzierung und eine de novo Anordnung des Gesamtgenoms von S. pseudintermedius. Die Gesamtgenomsequenzierung von S. pseudintermedius könnte dabei helfen, die Pathogenese der caninen Pyodermie besser zu verstehen.
Methoden und Materialien
Zweiundzwanzig Stämme von S. pseudintermedius, die von der Haut von fünf gesunden Hunden und 33 Stämme, die von 33 Hunden mit Pyodermie isoliert worden waren, wurden analysiert. DNA wurde extrahiert und mittels Oxford Nanopore MinION, einer neuen Technologie, die längere Sequenzen in einem Handgerät liefert, sequenziert. Das Pangenom wurde analysiert und mittels ANVI´O 6.1. analysiert.
Ergebnisse
Die Nanopore Technologie ermöglichte eine Sequenzierung und de novo Anordnung der Genome von 55 S. pseudintermedius Stämmen, die von gesunden Hunden und von Hunden mit Pyodermie isoliert worden waren. Die durchschnittliche Genom Länge von S. pseudintermedius betrug 2,62 Mbp, wobei 48% davon das Hauptgenom ausmachte. Pyodermie Isolate beinhalteten eine größere Anzahl an antimikrobiellen Resistenzgenen; die Gesamtzahl der Virulenzfaktorgene unterschied sich allerdings nicht zwischen den Isolaten von gesunden Hunden und von Hunden mit Pyodermie. Genome von Methicillin‐resistenten S. pseudintermedius (MRSP) Stämmen waren größer als jene der Methicillin‐sensiblen (MSSP) Stämme (2,8 Mbp versus 2,59 Mbp), was eine Folge der vermehrten Präsenz von antimikrobiellen Resistenzgenen, Phagen und Prophagen war.
Schlussfolgerungen und klinische Bedeutung
Diese Technik erlaubt eine wesentlich präzisere und einfachere Charakterisierung der caninen S. pseudintermedius Populationen und könnte zu einem besseren Verstehen der Pathogenese der caninen Pyodermien führen.
要約
背景
Staphylococcus pseudintermediusは、犬の膿皮症の主な病因です。全ゲノムシーケンスは、微生物に関する関連するゲノム情報を取得するための最も包括的な方法です。
仮説/目的
Oxford Nanoporeテクノロジーにより、S。pseudintermediusの全ゲノムの高品質なシーケンスとdenovoアセンブリが可能になります。 S. pseudintermediusの全ゲノム解析は、犬の膿皮症の病因をよりよく理解するのに役立つ可能性があります。
方法と材料
5匹の健康な犬の皮膚から分離されたS.pseudintermediusの22株と、膿皮症の33匹の犬の皮膚から分離された33株が分析されました。 DNAは、ハンドヘルドデバイスでより長い読み取りを実現する新しいテクノロジーであるOxford Nanopore MinIONを使用して抽出およびシーケンスされました。パンゲノムはANVI’O6.1で分析および視覚化されました。
結果
Nanoporeテクノロジーにより、健康な犬と膿皮症の犬から分離された55のS.pseudintermedius株のゲノムの配列決定とdenovoアセンブリが可能になりました。 S.pseudintermediusの平均ゲノムサイズは2.62Mbpで、48％がコアゲノムでした。膿皮症分離株は、より多くの抗菌薬耐性遺伝子を含んでいましたが、病原性因子遺伝子の総数は、健康な犬と膿皮症の犬からの分離株間で変化しませんでした。メチシリン耐性S.pseudintermedius（MRSP）株のゲノムは、抗菌剤耐性遺伝子、ファージ、およびプロファージの存在が大きい結果として、メチシリン感受性（MSSP）株のゲノムよりも大きかった（2.80Mbp対2.59Mbp）。
結論と臨床的重要性
この手法により、犬のS. pseudintermedius集団のより正確で簡単な特性評価が可能になり、犬の膿皮症の病因のより良い理解につながる可能性があります。
摘要
背景
假中间型葡萄球菌是犬脓皮病的主要病原。全基因组测序是获得微生物相关基因组信息最全面的方式。
假设/目的
纳米孔技术能够对假中间型葡萄球菌的全基因组进行质量测序和从头组装。对假中间型葡萄球菌进行全基因组分析可能有助于更好地了解犬脓皮病的发病机制。
方法和材料
从5只健康犬皮肤中分离的22株，以及从33只脓皮病犬皮肤中分离的33株假中间型葡萄球菌，对其进行分析。使用纳米孔 MinION提取DNA并测序，这是一种手持设备，这种新技术可传递更长的测序片段。用ANVI'O 6.1分析全基因组并将其可视化。
结果
从健康犬和脓皮病犬中分离的55株假中间型葡萄球菌，用纳米孔技术对其基因组进行测序和从头组装。假中间型葡萄球菌的平均基因组大小为2.62 Mbp，其中48%为核心基因组。脓皮病分离株含有较多数量的抗菌药物耐药基因，然而毒力因子基因的总数在健康犬和脓皮病犬的分离株之间没有变化。耐甲氧西林假中间型葡萄球菌(MRSP)菌株的基因组比甲氧西林敏感(MSSP)菌株的基因组大（2.80 Mbp与2.59 Mbp），这是由于存在更多抗菌药物耐药基因、噬菌体和前噬菌体。
结论和临床重要性
该技术可更精确和更容易地表征犬假中间型葡萄球菌种群，并可能有助于更好地了解犬脓皮病的发病机制。
Resumo
Contexto
Staphylococcus pseudintermedius é o principal agente etiológico da piodermite canina. O sequenciamento completo do genoma é a forma mais aprofundada de se obter informações genômicas relevantes sobre microrganismos.
Hipótese/Objetivos
A tecnologia de Oxford Nanopore permite o sequenciamento de boa qualidade e montagem “de novo” do genoma completo de S. pseudintermedius. A análise do genoma completo de S. pseudintermedius pode auxiliar na melhor compreensão da patogênese da piodermite canina.
Métodos e materiais
Vinte e duas cepas de S. pseudintermedius isoladas da pele de cinco cães saudáveis e 33 cepas isoladas da pele de 33 cães com piodermite foram analisadas. O DNA foi extraído e sequenciado utilizando Oxford Nanopore MinION, uma tecnologia nova que fornece leituras mais longas em um aparelho
We have
assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We ...released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing.
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.
Objectives:
The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in ...Catalonia, Spain.
Methods:
Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of
bla
genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids.
Results:
From July 2018 to February 2019, 125
Klebsiella pneumoniae
carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of
Klebsiella pneumoniae
belonging to sequence type ST15 and additional isolates of
K. pneumoniae
,
Escherichia coli
and
Enterobacter
sp. from different STs. All isolates but one carried the
bla
KPC–2
allelic variant. The
bla
KPC–2
gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn
4401
element designated NTE
KPC
-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTE
KPC
-pMC-2-1 module was highly conserved.
Conclusion:
We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15
K. pneumoniae
but also the intra- and inter-species transmission of the
bla
KPC–2
gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones.
Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) considered to be the primary sensors of pathogens in innate immunity. Genetic variants could be associated to differences in breed ...innate immune response to pathogens and thus to susceptibility to infections or autoimmune diseases. There is therefore great interest in the characterization of canine TLRs.
Polymorphisms in canine TLRs have been characterized by massive sequencing after enrichment of their exonic regions. DNAs from 335 dogs (seven different breeds) and 100 wolves (two different populations) were used in pools. The ratio of SNP discovery was 76.5% (in relation to CanFam 3.1); 155 out of 204 variants identified were new. Functional annotation identified 64 non-synonymous variants (43 new), 73 synonymous variants (56 new) and 67 modifier variants (57 new). 12 out of 64 non-synonymous variants are breed or wolf specific. TLR5 has been found to be the most polymorphic among canine TLRs. Finally, a TaqMan OpenArray® plate containing 64 SNPs with a possible functional effect in the protein (4 frameshifts and 60 non-synonymous codons) has been designed and validated.
Non-synonymous genetic variation has been characterized in exonic regions of canine Toll-like Receptors. The TaqMan OpenArray® plate developed to capture the individual variability that affects protein function will allow high-throughput genotyping either to study association to infection susceptibility or even TLR evolution in the canine genome.
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