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Asset Tag: Bacteria

July 7, 2019

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark.

From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals.VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid.Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n?=?44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon.This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Methods for genome-wide methylome profiling of Campylobacter jejuni.

Methylation has a profound role in the regulation of numerous biological processes in bacteria including virulence. The study of methylation in bacteria has greatly advanced thanks to next-generation sequencing technologies. These technologies have expedited the process of uncovering unique features of many bacterial methylomes such as characterizing previously uncharacterized methyltransferases, cataloging genome-wide DNA methylations in bacteria, identifying the frequency of methylation at particular genomic loci, and revealing regulatory roles of methylation in the biology of various bacterial species. For instance, methylation has been cited as a potential source for the pathogenicity differences observed in C. jejuni strains with syntenic genomes as seen in recent publications. Here, we describe the methodology for the use of Pacific Biosciences’ single molecule real-time (SMRT) sequencing for detecting methylation patterns in C. jejuni and bioinformatics tools to profile its methylome.

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July 7, 2019

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction.© 2016 Blackwell Verlag GmbH.

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July 7, 2019

Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

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July 7, 2019

Complete genome sequence of Lutibacter profundi LP1T isolated from an Arctic deep-sea hydrothermal vent system

Lutibacter profundi LP1T within the family Flavobacteriaceae was isolated from a biofilm growing on the surface of a black smoker chimney at the Loki’s Castle vent field, located on the Arctic Mid-Ocean Ridge. The complete genome of L. profundi LP1T is the first genome to be published within the genus Lutibacter. L. profundi LP1T consists of a single 2,966,978 bp circular chromosome with a GC content of 29.8%. The genome comprises 2,537 protein-coding genes, 40 tRNA species and 2 rRNA operons. The microaerophilic, organotrophic isolate contains genes for all central carbohydrate metabolic pathways. However, genes for the oxidative branch of the pentose-phosphate-pathway, the glyoxylate shunt of the tricarboxylic acid cycle and the ATP citrate lyase for reverse TCA are not present. L. profundi LP1T utilizes starch, sucrose and diverse proteinous carbon sources. In accordance, the genome harbours 130 proteases and 104 carbohydrate-active enzymes, indicating a specialization in degrading organic matter. Among a small arsenal of 24 glycosyl hydrolases, which offer the possibility to hydrolyse diverse poly- and oligosaccharides, a starch utilization cluster was identified. Furthermore, a variety of enzymes may be secreted via T9SS and contribute to the hydrolytic variety of the microorganism. Genes for gliding motility are present, which may enable the bacteria to move within the biofilm. A substantial number of genes encoding for extracellular polysaccharide synthesis pathways, curli fibres and attachment to surfaces could mediate adhesion in the biofilm and may contribute to the biofilm formation. In addition to aerobic respiration, the complete denitrification pathway and genes for sulphide oxidation e.g. sulphide:quinone reductase are present in the genome. sulphide:quinone reductase and denitrification may serve as detoxification systems allowing L. profundi LP1T to thrive in a sulphide and nitrate enriched environment. The information gained from the genome gives a greater insight in the functional role of L. profundi LP1T in the biofilm and its adaption strategy in an extreme environment.

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July 7, 2019

The complete genome sequence of Cronobacter sakazakii ATCC 29544(T), a food-borne pathogen, isolated from a child’s throat.

Cronobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of severe diseases: meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. However, the pathogenesis mechanism of this pathogen remains largely unknown. To determine its pathogenesis at the genomic level, the genome of C. sakazakii ATCC 29544(T) was completely sequenced and analyzed.The genomic DNA, containing a circular chromosome and three plasmids, is composed of 4,511,265 bp with a GC content of 56.71%, containing 4380 predicted open reading frames (ORFs), 22 rRNA genes, and 83 tRNA genes. The plasmids, designated pCSK29544_p1, pCSK29544_p2, and pCSK29544_p3, were 93,905-bp, 4938-bp, and 53,457-bp with GC contents of 57.02, 54.88, and 50.07%, respectively. They were also predicted to have 72, 6, and 57 ORFs without RNA genes.The strain ATCC 29544(T) genome has ompA and ibeB-homologous cusC genes, probably associated with the invasion of human brain microvascular endothelial cells (BMECs). In addition, gene clusters for siderophore production (iucABCD/iutA) and the related transport system (eitCBAD) were detected in pCSK29544_p1 plasmid, indicating better iron uptake ability for survival. Furthermore, to survive under extremely dry condition like milk powder, this genome has gene clusters for biosynthesis of capsular proteins (CSK29544_00281-00284) and cellulose (CSK29544_01124-01127) for biofilm formation and a gene cluster for utilization of sialic acid in the milk (nanKTAR). The genome information of C. sakazakii ATCC 29544(T) would provide further understanding of its pathogenesis at the molecular level for the regulation of pathogenicity and the development of a rapid detection method using biomarkers.

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July 7, 2019

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.

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July 7, 2019

The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n?=?35), imipenem (n?=?1) or ciprofloxacin (n?=?1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5?µg/ml, 4-fold below the susceptibility breakpoint (S?=?2?µg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials.

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July 7, 2019

Genomic data mining of the marine actinobacteria Streptomyces sp. H-KF8 unveils insights into multi-stress related genes and metabolic pathways involved in antimicrobial synthesis.

Streptomyces sp. H-KF8 is an actinobacterial strain isolated from marine sediments of a Chilean Patagonian fjord. Morphological characterization together with antibacterial activity was assessed in various culture media, revealing a carbon-source dependent activity mainly against Gram-positive bacteria (S. aureus and L. monocytogenes). Genome mining of this antibacterial-producing bacterium revealed the presence of 26 biosynthetic gene clusters (BGCs) for secondary metabolites, where among them, 81% have low similarities with known BGCs. In addition, a genomic search in Streptomyces sp. H-KF8 unveiled the presence of a wide variety of genetic determinants related to heavy metal resistance (49 genes), oxidative stress (69 genes) and antibiotic resistance (97 genes). This study revealed that the marine-derived Streptomyces sp. H-KF8 bacterium has the capability to tolerate a diverse set of heavy metals such as copper, cobalt, mercury, chromate and nickel; as well as the highly toxic tellurite, a feature first time described for Streptomyces. In addition, Streptomyces sp. H-KF8 possesses a major resistance towards oxidative stress, in comparison to the soil reference strain Streptomyces violaceoruber A3(2). Moreover, Streptomyces sp. H-KF8 showed resistance to 88% of the antibiotics tested, indicating overall, a strong response to several abiotic stressors. The combination of these biological traits confirms the metabolic versatility of Streptomyces sp. H-KF8, a genetically well-prepared microorganism with the ability to confront the dynamics of the fjord-unique marine environment.

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July 7, 2019

Resequencing and annotation of the Nostoc punctiforme ATTC 29133 genome: facilitating biofuel and high-value chemical production.

Cyanobacteria have the potential to produce bulk and fine chemicals and members belonging to Nostoc sp. have received particular attention due to their relatively fast growth rate and the relative ease with which they can be harvested. Nostoc punctiforme is an aerobic, motile, Gram-negative, filamentous cyanobacterium that has been studied intensively to enhance our understanding of microbial carbon and nitrogen fixation. The genome of the type strain N. punctiforme ATCC 29133 was sequenced in 2001 and the scientific community has used these genome data extensively since then. Advances in bioinformatics tools for sequence annotation and the importance of this organism prompted us to resequence and reanalyze its genome and to make both, the initial and improved annotation, available to the scientific community. The new draft genome has a total size of 9.1 Mbp and consists of 65 contiguous pieces of DNA with a GC content of 41.38% and 7664 protein-coding genes. Furthermore, the resequenced genome is slightly (5152 bp) larger and contains 987 more genes with functional prediction when compared to the previously published version. We deposited the annotation of both genomes in the Department of Energy’s IMG database to facilitate easy genome exploration by the scientific community without the need of in-depth bioinformatics skills. We expect that an facilitated access and ability to search the N. punctiforme ATCC 29133 for genes of interest will significantly facilitate metabolic engineering and genome prospecting efforts and ultimately the synthesis of biofuels and natural products from this keystone organism and closely related cyanobacteria.

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July 7, 2019

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated “Smooth” and “Rough”, under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants’ genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

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July 7, 2019

High-quality genome sequence of human pathogen Enterobacter asburiae type strain 1497-78(T).

Enterobacter asburiae belongs to the Enterobacter cloacae complex (Ecc), which comprises six heterogenic species. These bacteria can cause nosocomial infections. Moreover, they are well known for antibiotic resistance features based on overproduction of AmpC ß-lactamases. Although Ecc have clinical importance, little is known about their virulence-associated properties, and very few strains from the six species have been sequenced. In this study, the type strain of E. asburiae 1497-78(T) (ATCC 35953) was sequenced. The genome sequence of the type strain of E. asburiae will help us to understand antibiotic resistance and evolution in Ecc. Copyright © 2017. Published by Elsevier Ltd.

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July 7, 2019

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

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July 7, 2019

Prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae recovered from bloodstream infections in China: a multicentre longitudinal study.

Polymyxin antibiotics are used as last-resort therapies to treat infections caused by multidrug-resistant Gram-negative bacteria. The plasmid-mediated colistin resistance determinant MCR-1 has been identified in Enterobacteriaceae in China. We did this study to investigate the prevalence of the mcr-1 gene in clinical isolates from patients with bloodstream infections in China.Clinical isolates of Escherichia coli and Klebsiella pneumoniae were collected from patients with bloodstream infections at 28 hospitals in China, then screened for colistin resistance by broth microdilution and for the presence of the mcr-1 gene by PCR amplification. We subjected mcr-1-positive isolates to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of mcr-1 with Southern blot hybridisation, and we analysed plasmids containing mcr-1 with filter mating, electroporation, and DNA sequencing.2066 isolates, consisting of 1495 E coli isolates and 571 K pneumoniae isolates were collected. Of the 1495 E coli isolates, 20 (1%) were mcr-1-positive, whereas we detected only one (<1%) mcr-1-positive isolate among the 571 K pneumoniae isolates. All mcr-1-positive E coli and K pneumoniae isolates were resistant to colistin, with minimum inhibitory concentrations values in the range of 4-32 mg/L, except for one E coli isolate that had a minimum inhibitory concentration less than or equal to 0·06 mg/L. All 21 mcr-1-positive isolates were susceptible to tigecycline and 20 isolates (95%) were susceptible to the carbapenem and ß-lactamase inhibitor combination piperacillin and tazobactam. One mcr-1-positive E coli isolate also produced NDM-5, which confers resistance to beta-lactam antibiotics. The 21 mcr-1-positive isolates were clonally diverse and carried mcr-1 on two types of plasmids, a 33 kb IncX4 plasmid and a 61 kb Inc12 plasmid. The 30 day mortality of the patients with bloodstream infections caused by mcr-1-positive isolates was zero.mcr-1-positive isolates from bloodstream infections were rare, sporadic, and remained susceptible to many antimicrobial agents. E coli, rather than K pneumoniae, was the main host of the mcr-1 gene. Further studies are needed to clarify the clinical impact of this novel resistance gene.National Natural Science Foundation of China. Copyright © 2017 Elsevier Ltd. All rights reserved.

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