Oxford Nanopore MinION Research Articles

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

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. 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. 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. 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. 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.

Emergence of B.1.1.318 SARS-CoV-2 viral lineage and high incidence of alpha B.1.1.7 variant of concern in the Republic of Gabon

ObjectiveVariants of concern (VOCs) associated with relatively high transmissibility appear to be rapidly spreading in Gabon. Therefore, it is imperative to understand the distribution of several VOCs in the population, which could have implications for transmissibility and vaccine efficacy. MethodsBetween February and May 2021, SARS-CoV-2 genomes were sequenced using the Oxford nanopore MinION method and the respective genome diversity was elucidated. Phylogenetic analysis was performed and genomes were classified using pangolin lineages. ResultsThe results highlighted an increase (46%) in the alpha VOC (B.1.1.7) in the Gabonese population over the study period. In addition, an increase (31%) in the B.1.1.318 lineage, which is associated with high transmission and impaired vaccine efficacy (D614G+E484K+Y144del), was detected. ConclusionWith the second wave ongoing, these findings highlight the need for surveillance of the SARS-CoV-2 genome in the Republic of Gabon and should provide useful guidance to policymakers in selecting an appropriate vaccine for this population.

Sequencing of SARS-CoV-2 in local transmission cases through Oxford Nanopore MinION platform from Karachi Pakistan.

The first case of severe acute respiratory syndrome 2 (SARS-CoV-2) was imported to Pakistan in February 2020, since then 8,260 deaths have been witnessed. The virus has been constantly mutating and local transmission cases from different countries vary due to host dependent viral adaptation. Many distinct clusters of variant SARS-CoV-2 have been defined globally. In this study, the epidemiology of SARS-CoV-2 was studied and locally transmitted SARS-CoV-2 isolates from Karachi were sequenced to compared and identify any possible variants. The real time PCR was performed on nasopharyngeal specimen to confirm SARS-CoV-2 with Orf 1ab and E gene as targets. The virus isolates were sequenced through oxford nanopore technology MinION platform. Isolates from the first and second wave of COVID-19 outbreak in Karachi were compared. The overall positivity rate for PCR was 26.24% with the highest number of positive cases in June. Approximately, 37.45% PCR positive subjects aged between 19-40 years. All the isolates belonged to GH clade and shared missense mutation D614G in spike protein linked to increased transmission rate worldwide. Another spike protein mutation A222V coexisted with D614G in the virus from the second wave of COVID-19. Based on the present findings it is suggested that the locally transmitted virus from Karachi varies from those reported from other parts of Pakistan. Slight variability was also observed between viruses from the first and second wave. Variability in any potential vaccine target may result in failed trials, therefore information on any local viral variants is always useful for effective vaccine design and/or selection.

Investigating changes in β-adrenergic gene expression (ADRB1 and ADRB2) in Takotsubo (stress) cardiomyopathy syndrome; a pilot study.

BackgroundTakotsubo Cardiomyopathy (TC) is a rare disorder that is mostly caused by stress and is often misdiagnosed. We aimed to analyze Takotsubo Syndrome at the molecular level by using the Oxford Nanopore Minion Device and its protocol.Methods and resultsTen patients who were previously diagnosed with Takotsubo Syndrome (increased after decrease in ejection fraction and without critical stenosis in coronary arteries) and 10 healthy individuals in the control group were included in our project. The mean age was 53 ± 12.2 for the patient group and 52.4 ± 9.9 for the control group, and the left ventricular ejection fraction was 50.3 ± 11.5 for the patient group and 64.2 ± 2.8 for the control group (p < 0.05). Peripheral blood of patients and healthy individuals was taken and their DNA was obtained. By making long reads throughout the genome, the most studied regions responsible for β-adrenergic signaling pathways; The gene expression level of cardiac β-1 ADRB1 (rs1801253-ENST00000369295.4), G > C, (Gly389Arg) and cardiac β-2 ADRB2 (rs1800888-ENSG00000169252), C > T, (Thr165Ile) adrenoceptors was investigated. As a result; no structural variation was detected leading to Takotsubo Cardiomyopathy. The results obtained from the bioinformatics analysis were also checked from the VarSome Tools and similar results were found.ConclusionsMany publications in TC susceptibility have that may lead to adrenergic pathway dysregulation, most studied adrenergic receptor genes in the similar literatures too. We searched for genetic variants in b1AR and b2AR genes in our study and however we could not find any variants in this study, we think larger numbers of cohort studies are needed.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-021-06816-w.

Genome Sequences of SARS-CoV-2 Sublineage B.1.617.2 Strains from 12 Children in Chattogram, Bangladesh

ABSTRACTWe announce the complete genome sequences of 12 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sublineage B.1.617.2 strains (Delta variant) obtained from nasopharyngeal and oropharyngeal swab samples from 12 pediatric patients in Chittagong, Bangladesh, displaying COVID-19 symptoms. Oxford Nanopore MinION sequencing technology was used to generate the genomic sequences.

Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses.

Simple SummaryPeste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR), in sheep and goats, which is targeted for global control and eradication. However, in many developing countries, access to expensive sequencing technologies is limited and is compounded by difficulties in transporting clinical samples across international borders. Oxford nanopore MinION is a relatively cheap sequencing technology using portable devices that require minimal supporting laboratory infrastructure or technical expertise for sample preparation and rapid sequencing. In this study, Oxford nanopore MinION sequencing was carried out to generate complete genomes of PPRV from archived PPRV-positive samples collected from PPR outbreaks in goats in Ngorongoro and Momba districts in Tanzania during 2016 and 2018, respectively. Complete genomes of PPRV of 15,948 nucleotides long were generated within four hours of sequencing. The phylogenetic analysis of the complete genomes revealed a high nucleotide identity (96.19–99.24%) with lineage III PPR viruses currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in developing countries in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed between the matrix (M) and fusion (F) genes. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.

Complete Circular Genome Sequences of Three Bacillus cereus Group Strains Isolated from Positive Blood Cultures from Preterm and Immunocompromised Infants Hospitalized in France.

ABSTRACTWe report here the complete genome sequences of three Bacillus cereus group strains isolated from blood cultures from premature and immunocompromised infants hospitalized in intensive care units in three French hospitals. These complete genome sequences were obtained from a combination of Illumina HiSeq X Ten short reads and Oxford Nanopore MinION long reads.

Genomic and phylogenetic analysis of a multidrug-resistant mcr-1-carrying Klebsiella pneumoniae recovered from a urinary tract infection in China

The emergence and dissemination of colistin-resistant Enterobacterales has become a major global public-health threat. Here we investigated the genomic and phylogenetic characteristics of a multidrug-resistant Klebsiella pneumoniae strain (KP4823) carrying the mcr-1 gene recovered from a urinary tract infection in China. Antimicrobial susceptibility of K. pneumoniae KP4823 was determined by broth microdilution. Whole genomic DNA was extracted and sequenced using Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. Hybrid assembly with long and short reads was performed using Unicycler, and the genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The sequence type (ST), capsular type, and antimicrobial resistance and virulence genes were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis was performed by BacWGSTdb 2.0 server. Klebsiella pneumoniae KP4823 was resistant to colistin, ceftazidime, cefepime, cefotaxime, fosfomycin and aztreonam. The complete genome sequence of KP4823 consists of five contigs comprising 5 445 519 bp, including one chromosome and four plasmids. The isolate was assigned to ST101 with capsular serotype KL106. Several antimicrobial resistance genes were identified, including the colistin resistance gene mcr-1, which was located on a 34 685-bp IncX4 plasmid. The closest relative of K. pneumoniae KP4823 was another ST101 isolate (08EU827) recovered from Sweden in 2008, which differed by 191 cgMLST loci. Our study reports the genome sequence of a multidrug-resistant mcr-1-carrying K. pneumoniae in China. These data may help to understand the antimicrobial resistance mechanisms, genomic features and transmission dynamics of colistin resistance in clinical settings.

Direct Nanopore Sequencing of Individual Full Length tRNA Strands.

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

Complete Plastome of Three Korean Asarum (Aristolochiaceae): Confirmation Tripartite Structure within Korean Asarum and Comparative Analyses.

The genus Asarum (Aristolochiaceae) is a well-known resource of medicinal and ornamental plants. However, the taxonomy of Korean Asarum is ambiguous due to their considerable morphological variations. Previously, a unique plastome structure has been reported from this genus. Therefore, we investigated the structural change in the plastomes within three Korean Asarum species and inferred their phylogenetic relationships. The plastome sizes of Asarum species assembled here range from 190,168 to 193,356 bp, which are longer than a typical plastome size (160 kb). This is due to the incorporation and duplication of the small single copy into the inverted repeat, which resulted in a unique tripartite structure. We first verified this unique structure using the Illumina Miseq and Oxford Nanopore MinION platforms. We also investigated the phylogeny of 26 Aristolochiaceae species based on 79 plastid protein-coding genes, which supports the monophyly of Korean Asarum species. Although the 79 plastid protein-coding gene data set showed some limitations in supporting the previous classification, it exhibits its effectiveness in delineating some sections and species. Thus, it can serve as an effective tool for resolving species-level phylogeny in Aristolochiaceae. Last, we evaluated variable sites and simple sequence repeats in the plastome as potential molecular markers for species delimitation.

A draft genome assembly for the eastern fox squirrel, Sciurus niger.

The eastern fox squirrel, Sciurus niger, exhibits marked geographic variation in size and coat color, is a model organism for studies of behavior and ecology, and a potential model for investigating physiological solutions to human porphyrias. We assembled a genome using Illumina HiSeq, PacBio SMRT, and Oxford Nanopore MinION sequencing platforms. Together, the sequencing data resulted in a draft genome of 2.99 Gb, containing 32,830 scaffolds with an average size of 90.9 Kb and N50 of 183.8 Kb. Genome completeness was estimated to be 93.78%. A total of 24,443 protein-encoding genes were predicted from the assembly and 23,079 (94.42%) were annotated. Repeat elements comprised an estimated 38.49% of the genome, with the majority being LINEs (13.92%), SINEs (6.04%), and LTR elements. The topology of the species tree reconstructed using maximum-likelihood phylogenetic analysis was congruent with those of previous studies. This genome assembly can prove useful for comparative studies of genome structure and function in this rapidly diversifying lineage of mammals, for studies of population genomics and adaptation, and for biomedical research. Predicted amino acid sequence alignments for genes affecting heme biosynthesis, color vision, and hibernation showed point mutations and indels that may affect protein function and ecological adaptation.

Nanopore-Based Surveillance of Zoonotic Bacterial Pathogens in Farm-Dwelling Peridomestic Rodents.

The effective control of rodent populations on farms is crucial for food safety, as rodents are reservoirs and vectors for several zoonotic pathogens. Clear links have been identified between rodents and farm-level outbreaks of pathogens throughout Europe and Asia; however, comparatively little research has been devoted to studying the rodent–agricultural interface in the USA. Here, we address this knowledge gap by metabarcoding bacterial communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of putative zoonotic foodborne pathogens, among others. Rodents were live trapped (n = 90) from three dairy and mixed animal farms. DNA extraction was performed on 63 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed. Our farm-level rodent-metabarcoding data indicate the presence of multiple foodborne pathogens, including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many mastitis pathogens circulating within five rodent species (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and a shrew (Blarina brevicauda). Interestingly, we observed a higher abundance of enteric pathogens (e.g., Salmonella) in shrew feces compared to the rodents analyzed in our study. Knowledge gained from our research efforts will directly inform and improve farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units.

Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets

BackgroundThe direct use of medical zinc oxide in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders.ResultsThis study investigated the effect of using rapeseed-seaweed blend (rapeseed meal added two brown macroalgae species Ascophylum nodosum and Saccharina latissima) fermented by lactobacilli (FRS) as feed ingredients in piglet weaning. From d 28 of life to d 85, the piglets were fed one of three different feeding regimens (n = 230 each) with inclusion of 0%, 2.5% and 5% FRS. In this period, no significant difference of piglet performance was found among the three groups. From a subset of piglets (n = 10 from each treatment), blood samples for hematology, biochemistry and immunoglobulin analysis, colon digesta for microbiome analysis, and jejunum and colon tissues for histopathological analyses were collected. The piglets fed with 2.5% FRS manifested alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier relative to the 0% FRS group. The colon microbiota composition was determined using V3 and V1-V8 region 16S rRNA gene amplicon sequencing by Illumina NextSeq and Oxford Nanopore MinION, respectively. The two amplicon sequencing strategies showed high consistency between the detected bacteria. Both sequencing strategies indicated that inclusion of FRS reshaped the colon microbiome of weaned piglets with increased Shannon diversity. Prevotella stercorea was verified by both methods to be more abundant in the piglets supplied with FRS feed, and its abundance was positively correlated with colonic mucosa thickness but negatively correlated with blood concentrations of leucocytes and IgG.ConclusionsFRS supplementation relieved the gut lymphocyte infiltration of the weaned piglets, improved the colon mucosa barrier with altered microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not lead to further improvements.

High-molecular weight DNA extraction, clean-up and size selection for long-read sequencing.

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30-50 kb, with reads exceeding 200 kb and outputs ranging from 15-30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients

BackgroundThird-generation cephalosporin-resistant Gram-negatives (3GCR-GN) and vancomycin-resistant enterococci (VRE) are common causes of multi-drug resistant healthcare-associated infections, for which gut colonisation is considered a prerequisite. However, there remains a key knowledge gap about colonisation and infection dynamics in high-risk settings such as the intensive care unit (ICU), thus hampering infection prevention efforts.MethodsWe performed a three-month prospective genomic survey of infecting and gut-colonising 3GCR-GN and VRE among patients admitted to an Australian ICU. Bacteria were isolated from rectal swabs (n = 287 and n = 103 patients ≤2 and > 2 days from admission, respectively) and diagnostic clinical specimens between Dec 2013 and March 2014. Isolates were subjected to Illumina whole-genome sequencing (n = 127 3GCR-GN, n = 41 VRE). Multi-locus sequence types (STs) and antimicrobial resistance determinants were identified from de novo assemblies. Twenty-three isolates were selected for sequencing on the Oxford Nanopore MinION device to generate completed reference genomes (one for each ST isolated from ≥2 patients). Single nucleotide variants (SNVs) were identified by read mapping and variant calling against these references.ResultsAmong 287 patients screened on admission, 17.4 and 8.4% were colonised by 3GCR-GN and VRE, respectively. Escherichia coli was the most common species (n = 36 episodes, 58.1%) and the most common cause of 3GCR-GN infection. Only two VRE infections were identified. The rate of infection among patients colonised with E. coli was low, but higher than those who were not colonised on admission (n = 2/33, 6% vs n = 4/254, 2%, respectively, p = 0.3). While few patients were colonised with 3GCR- Klebsiella pneumoniae or Pseudomonas aeruginosa on admission (n = 4), all such patients developed infections with the colonising strain. Genomic analyses revealed 10 putative nosocomial transmission clusters (≤20 SNVs for 3GCR-GN, ≤3 SNVs for VRE): four VRE, six 3GCR-GN, with epidemiologically linked clusters accounting for 21 and 6% of episodes, respectively (OR 4.3, p = 0.02).Conclusions3GCR-E. coli and VRE were the most common gut colonisers. E. coli was the most common cause of 3GCR-GN infection, but other 3GCR-GN species showed greater risk for infection in colonised patients. Larger studies are warranted to elucidate the relative risks of different colonisers and guide the use of screening in ICU infection control.

Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Draft Genome Sequences of Multiple Streptomyces Isolates from Arizona.

ABSTRACTStreptomyces strains are bacteria that are well known for their distinctive physiology, behaviors, and ecology, as well as for being prodigious producers of diverse antibiotics. Here, we report draft genome sequences for eight Streptomyces strains that were isolated from multiple sky islands in Arizona and sequenced using an Oxford Nanopore Technologies Flongle adapter and MinION system.

Variation in type two taste receptor genes is associated with bitter tasting phenylthiocarbamide consumption in mature Targhee and Rambouillet rams.

Bitter taste perception in sheep can lead to avoidance of specific types of forage, such as sagebrush, which is present on many rangeland grazing systems in the Intermountain West. In humans, bitter taste perception is influenced by variation in several TAS2R genes, including more extensively studied TAS2R38 and TAS2R16. We hypothesize that variation in taste receptor genes in sheep is associated with bitter taste. Therefore, the objective of this study was to examine variation in TAS2R genes in relation to consumption of a bitter tasting compound phenylthiocarbamide (PTC) which determines bitter “taster” and “non-taster” status in humans. Rambouillet and Targhee rams (n = 26) were offered various concentrations of PTC solution (0.2–12.29 mM) and water in a side-by-side presentation during two experiments. Blood was collected for DNA isolation and sequencing. Nineteen TAS2R genes were amplified and sequenced with long read Oxford Nanopore MinION technology. A total of 1,049 single nucleotide polymorphisms (SNPs) and 26 haplotypes were identified in these genes. Of these, 24 SNPs and 11 haplotypes were significantly (P < 0.05) associated with PTC consumption in TAS2R3, TAS2R5, TAS2R8, TAS2R9, TAS2R16, TAS2R31-like, TAS2R38, TAS2R39, and TAS2R42-like. Over 50% of the SNPs resulted in a change in amino acid sequence and several resided in potential regulatory regions, which could have downstream functional consequences and influence bitter taste perception in sheep. Further research is needed to validate these associations and elucidate the mechanisms that link variation in TAS2R genes to bitter taste perception in sheep. This may enable producers to select sheep more likely to consume bitter forage such as sagebrush as a flock and rangeland management strategy.