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September 22, 2019

Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli.

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.

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September 22, 2019

A large, refractory nosocomial outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli demonstrates carbapenemase gene outbreaks involving sink sites require novel approaches to infection control.

Carbapenem-resistant Enterobacteriaceae (CRE) represent a health threat, but effective control interventions remain unclear. Hospital wastewater sites are increasingly being highlighted as important potential reservoirs. We investigated a large Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli outbreak and wider CRE incidence trends in the Central Manchester University Hospital NHS Foundation Trust (CMFT) (United Kingdom) over 8 years, to determine the impact of infection prevention and control measures. Bacteriology and patient administration data (2009 to 2017) were linked, and a subset of CMFT or regional hospital KPC-producing E. coli isolates (n = 268) were sequenced. Control interventions followed international guidelines and included cohorting, rectal screening (n = 184,539 screens), environmental sampling, enhanced cleaning, and ward closure and plumbing replacement. Segmented regression of time trends for CRE detections was used to evaluate the impact of interventions on CRE incidence. Genomic analysis (n = 268 isolates) identified the spread of a KPC-producing E. coli outbreak clone (strain A, sequence type 216 [ST216]; n = 125) among patients and in the environment, particularly on 2 cardiac wards (wards 3 and 4), despite control measures. ST216 strain A had caused an antecedent outbreak and shared its KPC plasmids with other E. coli lineages and Enterobacteriaceae species. CRE acquisition incidence declined after closure of wards 3 and 4 and plumbing replacement, suggesting an environmental contribution. However, ward 3/ward 4 wastewater sites were rapidly recolonized with CRE and patient CRE acquisitions recurred, albeit at lower rates. Patient relocation and plumbing replacement were associated with control of a clonal KPC-producing E. coli outbreak; however, environmental contamination with CRE and patient CRE acquisitions recurred rapidly following this intervention. The large numbers of cases and the persistence of blaKPC in E. coli, including pathogenic lineages, are of concern. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Achieving Accurate Sequence and Annotation Data for Caulobacter vibrioides CB13.

Annotated sequence data are instrumental in nearly all realms of biology. However, the advent of next-generation sequencing has rapidly facilitated an imbalance between accurate sequence data and accurate annotation data. To increase the annotation accuracy of the Caulobacter vibrioides CB13b1a (CB13) genome, we compared the PGAP and RAST annotations of the CB13 genome. A total of 64 unique genes were identified in the PGAP annotation that were either completely or partially absent in the RAST annotation, and a total of 16 genes were identified in the RAST annotation that were not included in the PGAP annotation. Moreover, PGAP identified 73 frameshifted genes and 22 genes with an internal stop. In contrast, RAST annotated the larger segment of these frameshifted genes without indicating a change in reading frame may have occurred. The RAST annotation did not include any genes with internal stop codons, since it chose start codons that were after the internal stop. To confirm the discrepancies between the two annotations and verify the accuracy of the CB13 genome sequence data, we re-sequenced and re-annotated the entire genome and obtained an identical sequence, except in a small number of homopolymer regions. A genome sequence comparison between the two versions allowed us to determine the correct number of bases in each homopolymer region, which eliminated frameshifts for 31 genes annotated as frameshifted genes and removed 24 pseudogenes from the PGAP annotation. Both annotation systems correctly identified genes that were missed by the other system. In addition, PGAP identified conserved gene fragments that represented the beginning of genes, but it employed no corrective method to adjust the reading frame of frameshifted genes or the start sites of genes harboring an internal stop codon. In doing so, the PGAP annotation identified a large number of pseudogenes, which may reflect evolutionary history but likely do not produce gene products. These results demonstrate that re-sequencing and annotation comparisons can be used to increase the accuracy of genomic data and the corresponding gene annotation.

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September 22, 2019

The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: a population-level genomic study.

Vancomycin-resistant Enterococcus faecium (VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteraemia associated with the vanA genotype, prompting investigation into the genomic epidemiology of VREfm.A population-level study of VREfm (10 November-9 December 2015) was conducted. A total of 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid.vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely related within hospitals versus between them [core SNPs 10 (IQR 1-357) versus 356 (179-416), respectively], suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796. Different vanA-harbouring plasmids were found across STs. With the exception of ST78 and ST796, Tn1546 transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission.Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

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September 22, 2019

Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation.

Acetogens synthesize acetyl-CoA via CO2 or CO fixation, producing organic compounds. Despite their ecological and industrial importance, their transcriptional and post-transcriptional regulation has not been systematically studied. With completion of the genome sequence of Acetobacterium bakii (4.28-Mb), we measured changes in the transcriptome of this psychrotolerant acetogen in response to temperature variations under autotrophic and heterotrophic growth conditions. Unexpectedly, acetogenesis genes were highly up-regulated at low temperatures under heterotrophic, as well as autotrophic, growth conditions. To mechanistically understand the transcriptional regulation of acetogenesis genes via changes in RNA secondary structures of 5′-untranslated regions (5′-UTR), the primary transcriptome was experimentally determined, and 1379 transcription start sites (TSS) and 1100 5′-UTR were found. Interestingly, acetogenesis genes contained longer 5′-UTR with lower RNA-folding free energy than other genes, revealing that the 5′-UTRs control the RNA abundance of the acetogenesis genes under low temperature conditions. Our findings suggest that post-transcriptional regulation via RNA conformational changes of 5′-UTRs is necessary for cold-adaptive acetogenesis.© 2018 Shin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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September 22, 2019

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

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September 22, 2019

Density-dependent enhanced replication of a densovirus in Wolbachia-infected Aedes cells is associated with production of piRNAs and higher virus-derived siRNAs.

The endosymbiotic bacterium Wolbachia pipientis has been shown to restrict a range of RNA viruses in Drosophila melanogaster and transinfected dengue mosquito, Aedes aegypti. Here, we show that Wolbachia infection enhances replication of Aedes albopictus densovirus (AalDNV-1), a single stranded DNA virus, in Aedes cell lines in a density-dependent manner. Analysis of previously produced small RNAs of Aag2 cells showed that Wolbachia-infected cells produced greater absolute abundance of virus-derived short interfering RNAs compared to uninfected cells. Additionally, we found production of virus-derived PIWI-like RNAs (vpiRNA) produced in response to AalDNV-1 infection. Nuclear fractions of Aag2 cells produced a primary vpiRNA signature U1 bias whereas the typical “ping-pong” signature (U1 – A10) was evident in vpiRNAs from the cytoplasmic fractions. This is the first report of the density-dependent enhancement of DNA viruses by Wolbachia. Further, we report the generation of vpiRNAs in a DNA virus-host interaction for the first time. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae with chromosomally encoded blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Discovery of the actinoplanic acid pathway in Streptomyces rapamycinicus reveals a genetically conserved synergism with rapamycin.

Actinobacteria possess a great wealth of pathways for production of bioactive compounds. Following advances in genome mining, dozens of natural product (NP) gene clusters are routinely found in each actinobacterial genome; however, the modus operandi of this large arsenal is poorly understood. During investigations of the secondary metabolome of Streptomyces rapamycinicus, the producer of rapamycin, we observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its demethyl analogue. Actinoplanic acids (APLs) are potent inhibitors of Ras farnesyltransferase and therefore represent bioactive compounds of medicinal interest. Supported with the unique structure of these polyketides and using genome mining, we identified a gene cluster responsible for their biosynthesis in S. rapamycinicus Based on experimental evidence and genetic organization of the cluster, we propose a stepwise biosynthesis of APL, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP. Although phylogenetically distant, the pathway shares some of the biosynthetic principles with the mycotoxins fumonisins. Namely, the core polyketide is acylated with the tricarballylate by an atypical nonribosomal peptide synthetase-catalyzed ester formation. Finally, motivated by the conserved colocalization of the rapamycin and APL pathway clusters in S. rapamycinicus and all other rapamycin-producing actinobacteria, we confirmed a strong synergism of these compounds in antifungal assays. Mining for such evolutionarily conserved coharboring of pathways would likely reveal further examples of NP sets, attacking multiple targets on the same foe. These could then serve as a guide for development of new combination therapies.© 2018 Mrak et al.

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September 22, 2019

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

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September 22, 2019

Paenibacillus seodonensis sp. nov., isolated from a plant of the genus Campanula.

Strain DCT-19T, representing a Gram-stain-positive, rodshaped, aerobic bacterium, was isolated from a native plant belonging to the genus Campanula on Dokdo, the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence showed that this strain was closely related to Paenibacillus amylolyticus NRRL NRS-290T (98.6%, 16S rRNA gene sequence similarity), Paenibacillus tundrae A10bT (98.1%), and Paenibacillus xylanexedens NRRL B-51090T (97.6%). DNADNA hybridization indicated that this strain had relatively low levels of DNA-DNA relatedness with P. amylolyticus NRRL NRS-290T (30.0%), P. xylanexedens NRRL B-51090T (29.0%), and P. tundrae A10bT (24.5%). Additionally, the genomic DNA G + C content of DCT-19T was 44.8%. The isolated strain grew at pH 6.0-8.0 (optimum, pH 7.0), 0-4% (w/v) NaCl (optimum, 0%), and a temperature of 15-45°C (optimum 25-30°C). The sole respiratory quinone in the strain was menaquinone-7, and the predominant fatty acids were C15:0 anteiso, C16:0 iso, and C16:0. In addition, the major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Based on its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain DCT-19T is proposed as a novel species in the genus Paenibacillus, for which the name Paenibacillus seodonensis sp. nov. is proposed (=KCTC 43009T =LMG 30888T). The type strain of Paenibacillus seodonensis is DCT-19T.

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September 22, 2019

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain’s inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

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September 22, 2019

The genome of the tegu lizard Salvator merianae: combining Illumina, PacBio, and optical mapping data to generate a highly contiguous assembly.

Reptiles are a species-rich group with great phenotypic and life history diversity but are highly underrepresented among the vertebrate species with sequenced genomes.Here, we report a high-quality genome assembly of the tegu lizard, Salvator merianae, the first lacertoid with a sequenced genome. We combined 74X Illumina short-read, 29.8X Pacific Biosciences long-read, and optical mapping data to generate a high-quality assembly with a scaffold N50 value of 55.4 Mb. The contig N50 value of this assembly is 521 Kb, making it the most contiguous reptile assembly so far. We show that the tegu assembly has the highest completeness of coding genes and conserved non-exonic elements (CNEs) compared to other reptiles. Furthermore, the tegu assembly has the highest number of evolutionarily conserved CNE pairs, corroborating a high assembly contiguity in intergenic regions. As in other reptiles, long interspersed nuclear elements comprise the most abundant transposon class. We used transcriptomic data, homology- and de novo gene predictions to annotate 22,413 coding genes, of which 16,995 (76%) likely have human orthologs as inferred by CESAR-derived gene mappings. Finally, we generated a multiple genome alignment comprising 10 squamates and 7 other amniote species and identified conserved regions that are under evolutionary constraint. CNEs cover 38 Mb (1.8%) of the tegu genome, with 3.3 Mb in these elements being squamate specific. In contrast to placental mammal-specific CNEs, very few of these squamate-specific CNEs (<20 Kb) overlap transposons, highlighting a difference in how lineage-specific CNEs originated in these two clades.The tegu lizard genome together with the multiple genome alignment and comprehensive conserved element datasets provide a valuable resource for comparative genomic studies of reptiles and other amniotes.

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September 22, 2019

Hypervirulent group A Streptococcus emergence in an acaspular background is associated with marked remodeling of the bacterial cell surface

Inactivating mutations in the control of virulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated–a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface.

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September 22, 2019

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ~2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.© 2018 Brashear et al.; Published by Cold Spring Harbor Laboratory Press.

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