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July 7, 2019

Quality scores for 32,000 genomes.

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). We have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences.Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes had quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes.The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.


July 7, 2019

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform.

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 7, 2019

Genomic analysis of the multi-drug-resistant clinical isolate Myroides odoratimimus PR63039.

Myroides odoratimimus (M. odoratimimus) has been gradually implicated as an important nosocomial pathogen that poses a serious health threat to immunocompromised patients owing to its multi-drug resistance. However, the resistance mechanism is currently unclear. To clarify the antibiotic resistance and infectivity mechanisms of M. odoratimimus, whole genome sequencing was performed on the multi-drug-resistant M. odoratimimus strain PR63039. The genome sequence was completed with single molecule real-time (SMRT) technologies. Then, annotation was performed using RAST and IMG-ER. A number of databases and software programs were used to analyze the genomic characteristics, including GC-Profile, ISfinder, CG viewer, ARDB, CARD, ResFinder, the VFDB database, PHAST and Progressive Mauve. The M. odoratimimus PR63039 genome consisted of a chromosome and a plasmid. The genome contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region named MY63039-RR was found. The subsystem features generated by RAST indicated that the annotated genome had 108 genes that were potentially involved in virulence, disease and defense, all of which had strong associations with resistance and pathogenicity. The prophage analysis showed two incomplete prophages in the genome. The genomic analysis of M. odoratimimus PR63039 partially clarified its antibiotic resistance mechanisms and virulence factors. Obtaining a clear understanding of its genomic characteristics will be conducive to the management of multidrug-resistant M. odoratimimus.


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.


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.


July 7, 2019

Coexistence of blaOXA-48 and truncated blaNDM-1 on different plasmids in a Klebsiella pneumoniae isolate in China.

Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both blaOXA-48 and blaNDM-1 on different plasmids from a Chinese hospital. Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring blaOXA-48 and blaNDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes. Plasmid DNA was sequenced using Pacbio RS II and annotated using RAST. Results:K. pneumoniae RJ119, carrying both blaOXA-48 and blaNDM-1, was resistant to almost all carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides and belonged to ST307. blaOXA-48 was located on a 61,748-bp IncL/M conjugative plasmid, which displayed overall nucleotide identity (99%) to pKPN-E1-Nr.7. blaNDM-1 was located on a 335,317-bp conjugative plasmid, which was a fusion of a blaNDM-1-harboring InA/C plasmid pNDM-US (140,825 bp, 99% identity) and an IncFIB plasmid pKPN-c22 (178,563 bp, 99% identity). The transconjugant RJ119-1 harboring blaNDM-1 was susceptible to carbapenem, and there was an insertion of IS10 into the blaNDM-1 gene. Conclusion: This is the first report of the coexistence of blaOXA-48 and blaNDM-1 in one K. pneumoniae clinical isolate in China. OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to the insertion of IS10. Our results provide new insight for the relationship between genetic diagnosis and clinical treatment. They also indicate that increased surveillance of blaOXA-48 is urgently needed in China.


July 7, 2019

Antibiotic discovery throughout the Small World Initiative: A molecular strategy to identify biosynthetic gene clusters involved in antagonistic activity.

The emergence of bacterial pathogens resistant to all known antibiotics is a global health crisis. Adding to this problem is that major pharmaceutical companies have shifted away from antibiotic discovery due to low profitability. As a result, the pipeline of new antibiotics is essentially dry and many bacteria now resist the effects of most commonly used drugs. To address this global health concern, citizen science through the Small World Initiative (SWI) was formed in 2012. As part of SWI, students isolate bacteria from their local environments, characterize the strains, and assay for antibiotic production. During the 2015 fall semester at Bowling Green State University, students isolated 77 soil-derived bacteria and genetically characterized strains using the 16S rRNA gene, identified strains exhibiting antagonistic activity, and performed an expanded SWI workflow using transposon mutagenesis to identify a biosynthetic gene cluster involved in toxigenic compound production. We identified one mutant with loss of antagonistic activity and through subsequent whole-genome sequencing and linker-mediated PCR identified a 24.9 kb biosynthetic gene locus likely involved in inhibitory activity in that mutant. Further assessment against human pathogens demonstrated the inhibition of Bacillus cereus, Listeria monocytogenes, and methicillin-resistant Staphylococcus aureus in the presence of this compound, thus supporting our molecular strategy as an effective research pipeline for SWI antibiotic discovery and genetic characterization.© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


July 7, 2019

Assessment of insertion sequence mobilization as an adaptive response to oxidative stress in Acinetobacter baumannii using IS-Seq.

Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. Few of the more than thirty IS elements described in Acinetobacter baumannii have been characterized for transposition activity or expression effects. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. Serial subculture in the presence of sub-inhibitory concentrations of hydrogen peroxide led to rapid selection of cells carrying an ISAba1 element upstream of the catalase/peroxidase gene katG Several additional sites for the elements ISAba1, ISAba13, ISAba25, ISAba26, and ISAba125 were found at low abundance after serial subculture, indicating that each element is active and contributes to genetic variation that may be subject to selection. Following hydrogen peroxide exposure, rapid changes in gene expression were observed in genes related to iron homeostasis. The IS insertions adjacent to katG resulted in more than 20-fold overexpression of the gene and increased hydrogen peroxide tolerance.Importance Insertion sequences (IS) are contribute to genomic and phenotypic variation in many bacterial species, but little is known about how transposition rates vary among elements or how selective pressure influences this process. A new method, termed “IS-seq” for identifying new insertion locations that arise under experimental growth conditions in the genome was developed and tested with cells grown in the presence of hydrogen peroxide, which causes oxidative stress. Gene expression changes in response to hydrogen peroxide exposure are similar to those observed in other species and include genes that control free iron concentrations. New IS insertions adjacent to a gene encoding a catalase enzyme confirm that IS elements can rapidly contribute to adaptive variation in the presence of selection. Copyright © 2017 Wright et al.


July 7, 2019

Genomic sequencing of a strain of Acinetobacter baumannii and potential mechanisms to antibiotics resistance.

Acinetobacter baumannii has been becoming a great challenge to clinicians due to their resistance to almost all available antibiotics. In this study, we sequenced the genome from a multiple antibiotics resistant Acinetobacter baumannii stain which was named A. baumannii-1isolated from China by SMRT sequencing technology to explore its potential mechanisms to antibiotic resistance. We found that several mechanisms might contribute to the antibiotic resistance of Acinetobacter baumannii. Specifically, we found that SNP in genes associated with nucleotide excision repair and ABC transporter might contribute to its resistance to multiple antibiotics; we also found that specific genes associated with bacterial DNA integration and recombination, DNA-mediated transposition and response to antibiotics might contribute to its resistance to multiple antibiotics; Furthermore, specific genes associated with penicillin and cephalosporin biosynthetic pathway and specific genes associated with CHDL and MBL ß-lactamase genes might contribute to its resistance to multiple antibiotics. Thus, the detailed mechanisms by which Acinetobacter baumannii show extensive resistance to multiple antibiotics are very complicated. Such a study might be helpful to develop new strategies to control Acinetobacter baumannii infection. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

Outbreak of KPC-2-producing Enterobacteriaceae caused by clonal dissemination of Klebsiella pneumoniae ST307 carrying an IncX3-type plasmid harboring a truncated Tn4401a.

Over a 5-month period between the end of June and the beginning of November in 2015, a KPC-producing Enterobacteriaceae outbreak occurred in a general hospital in Busan, South Korea, being associated with a total of 50 clinical isolates from 47 patients. Multilocus sequence typing and pulsed-field gel electrophoresis were carried out for strain typing and whole-genome sequencing was performed to characterize the plasmids. A clonal spread of K. pneumoniae sequence type 307 (ST307) carrying a self-transferable IncX3-type plasmid harboring blaKPC-2 was responsible for the outbreak. Sporadic emergence of K. pneumoniae ST697 carrying an IncFII-type plasmid and a ST11 isolate harboring a small plasmid devoid of any known origin of replication were observed to be associated with blaKPC-3, but no further dissemination of these strains was identified. The results indicated a healthcare-associated infection associated with a blaKPC-harboring plasmid dissemination and a clonal spread of KPC-producing Enterobacteriaceae. Copyright © 2016 Elsevier Inc. All rights reserved.


July 7, 2019

Multi-omics approach to study global changes in a triclosan-resistant mutant strain of Acinetobacter baumannii ATCC 17978.

Acinetobacter baumannii AB042, a triclosan-resistant mutant strain, was examined for modulated gene expression using whole-genome sequencing, transcriptomics and proteomics in order to understand the mechanism of triclosan resistance as well as its impact on A. baumannii. Data revealed modulated expression of the fatty acid metabolism pathway, co-factors known to play a role in the synthesis of fatty acids, as well as several transcriptional regulators. The membrane composition of the mutant revealed a decrease in C18 with a corresponding increase in C16 fatty acids compared with the parent strain A. baumannii ATCC 17978. These data indicate that A. baumannii responds to triclosan by altering the expression of genes involved in fatty acid metabolism, antibiotic resistance and amino acid metabolism. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.


July 7, 2019

Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ~30 to ~250 kb, among which there were conjugative plasmids of ~30 kb, ~60 kb, and ~250 kb and nonconjugative plasmids of ~140 kb, ~180 kb, and ~240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event. Copyright © 2017 American Society for Microbiology.


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