Search

De novo assembly of short DNA reads remains an essential technology, especially for large-scale projects and high-resolution variant analyses in epidemiology. However, the existing tools often lack sufficient accuracy required to compare closely related strains. To facilitate such studies on bacterial genomes, we developed Platanus_B, a de novo assembler that employs iterations of multiple error-removal algorithms. The benchmarks demonstrated the superior accuracy and high contiguity of Platanus_B, in addition to its ability to enhance the hybrid assembly of both short and nanopore long reads. Although the hybrid strategies for short and long reads were effective in achieving near full-length genomes, we found that short-read-only assemblies generated with Platanus_B were sufficient to obtain ≥90% of exact coding sequences in most cases. In addition, while nanopore long-read-only assemblies lacked fine-scale accuracies, inclusion of short reads was effective in improving the accuracies. Platanus_B can, therefore, be used for comprehensive genomic surveillances of bacterial pathogens and high-resolution phylogenomic analyses of a wide range of bacteria. [ABSTRACT FROM AUTHOR]

PHAST (PHAge Search Tool) and its successor PHASTER (PHAge Search Tool – Enhanced Release) have become two of the most widely used web servers for identifying putative prophages in bacterial genomes. Here we review the main capabilities of these web resources, provide some practical guidance regarding their use and discuss possible future improvements. PHAST, which was first described in 2011, made its debut just as whole bacterial genome sequencing and was becoming inexpensive and relatively routine. PHAST quickly gained popularity among bacterial genome researchers because of its web accessibility, its ease of use along with its enhanced accuracy and rapid processing times. PHASTER, which appeared in 2016, provided a number of much-needed enhancements to the PHAST server, including greater processing speed (to cope with very large submission volumes), increased database sizes, a more modern user interface, improved graphical displays and support for metagenomic submissions. Continuing developments in the field, along with increased interest in automated phage and prophage finding, have already led to several improvements to the PHASTER server and will soon lead to the development of a successor to PHASTER (to be called PHASTEST). [ABSTRACT FROM AUTHOR]

Background: Persistence of bacterial pathogens in the airways has profound consequences on the course and pathogenesis of chronic obstructive pulmonary disease (COPD). Patients with COPD continuously acquire and clear strains of Moraxella catarrhalis, a major pathogen in COPD. Some strains are cleared quickly and some persist for months to years. The mechanism of the variability in duration of persistence is unknown.Methods: Guided by genome sequences of selected strains, we studied the expression of Hag/MID, hag/mid gene sequences, adherence to human cells, and autoaggregation in longitudinally collected strains of M. catarrhalis from adults with COPD.Results: Twenty-eight of 30 cleared strains of M. catarrhalis expressed Hag/MID whereas 17 of 30 persistent strains expressed Hag/MID upon acquisition by patients. All persistent strains ceased expression of Hag/MID during persistence. Expression of Hag/MID in human airways was regulated by slipped-strand mispairing. Virulence-associated phenotypes (adherence to human respiratory epithelial cells and autoaggregation) paralleled Hag/MID expression in airway isolates.Conclusions: Most strains of M. catarrhalis express Hag/MID upon acquisition by adults with COPD and all persistent strains shut off expression during persistence. These observations suggest that Hag/MID is important for initial colonization by M. catarrhalis and that cessation of expression facilitates persistence in COPD airways. [ABSTRACT FROM AUTHOR]

Background:  Many countries worldwide have reported increasing numbers of emm89 group A Streptococcus (GAS) infections during last decade. Pathogen genetic factors linked to this increase need assessment.Methods:  We investigated epidemiological characteristics of emm89 GAS bacteremic infections, including 7-day and 30-day case-fatality rates, in Finland during 2004-2014 and linked them to whole-genome sequencing data obtained from corresponding strains. The Fisher exact test and exact logistic regression were used to compare differences between bacteremic infections due to emm89 GAS belonging to different genetic clades and subclades.Results:  Out of 1928 cases of GAS bacteremic infection, 278 were caused by emm89 GAS. We identified 2 genetically distinct clades, arbitrarily designated clade 2 and clade 3. Both clades were present during 2004-2008, but clade 3 increased rapidly from 2009 onward. Six subclades (designated subclades A-F) were identified within clade 3, based on phylogenetic core genome analysis. The case-fatality rate differed significantly between subclades (P < .05), with subclade D having the highest 30-day estimated case-fatality rate (19% vs 3%-14%).Conclusions:  A new emm89 clone, clade 3, emerged in 2009 and spread rapidly in Finland. Patients infected with certain subclades of clade 3 were significantly more likely to die. A specific polymerase chain reaction assay was developed to follow the spread of subclade D in 2015. [ABSTRACT FROM AUTHOR]

Background. Listeriosis is a serious foodborne infection. Outbreaks of listeriosis occur rarely, but have often proved difficult to solve. In June 2014, we detected and investigated a listeriosis outbreak in Denmark using patient interviews and whole-genome sequencing (WGS). Methods. We performed WGS on Listeria monocytogenes isolates from patients and available isolates from ready-to-eat foods and compared them using single-nucleotide polymorphism (SNP) analysis. Case patients had L. monocytogenes with ≤3 SNPs (the outbreak strain) isolated in September 2013-December 2014. Through interviews, we established case patients' food and clinical histories. Food production facilities were inspected and sampled, and we performed trace-back/trace-forward of food delivery chains. Results. In total, 41 cases were identified; 17 deaths occurred (41%). An isolate from a delicatessen meat (spiced meat roll) from company A was identical to the outbreak strain. Half of the patients were infected while hospitalized/institutionalized; institutions were supplied food by company A. The outbreak strain was repeatedly isolated from further samples taken within this company and within companies in its distribution chain. Products from company Awere traced and recalled from >6000 food establishments, after which the outbreak ended. Conclusions. Ready-to-eat spiced meat roll from a single production facility caused this outbreak. The product, served sliced and cold, is popular among the elderly; serving it at hospitals probably contributed to the high case-fatality rate. WGS used for patient isolates and isolates from food control inspections, coupled with routine epidemiological follow-up, was instrumental in swiftly locating the source of infections, preventing further illnesses and deaths. [ABSTRACT FROM AUTHOR]

Mutation and selection are both thought to impact significantly the nucleotide composition of bacterial genomes. Earlier studies have compared closely related strains to obtain mutation patterns based on the hypothesis that these bacterial strains had diverged so recently that selection will not have had enough time to play its role. In this study, we used a SOLiD autosequencer that was based on a dual-base encoding scheme to sequence the genome of Staphylococcus aureus with a mapping coverage of over 5,000×. By directly counting the variation obtained from these ultradeep sequencing reads, we found that A → G was the predominant single-base substitution and 1 bp deletions were the major small indel. These patterns are completely different from those obtained by comparison of closely related S. aureus strains, where C → T accounted for a larger proportion of mutations and deletions were shown to occur at an almost equal frequency to insertion. These findings suggest that the genomic differences between closely related bacterial strains have already undergone selection and are therefore not representative of spontaneous mutation. [ABSTRACT FROM AUTHOR]

Aims To perform a systematic evaluation of the applicability, validity and reliability of the long PCR- RFLP of 16 S- ITS-23 S r RNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures. Methods and Results 3301 Full-length sequences of 16 S- ITS-23 S r RNA genes were retrieved from 885 published bacterial genomes. Copy numbers of the whole set of 16 S- ITS-23 S r RNA genes per genome ranged from 1 ( n = 161) to 14 ( n = 4) with an average of 3·71. Their length varied greatly, from 4319 to 6568 bp with an average of 4952 bp. Computer-simulated RFLP analyses of the 16 S- ITS-23 S fragments flanked by the conserved primers 27 F and 2241 R suggested Msp I, Rsa I, Hha I and Taq I as the most appropriate enzymes for long PCR- RFLP analysis of the 16 S- ITS-23 S sequence. Msp I was used to screen over 900 bacterial cultures isolated from the Huguangyan Maar Lake in southern China. An experimental sequencing of 16 S r RNA genes of the isolates possessing a unique RFLP band pattern proved the broad applicability and high resolution of this approach. Conclusions These results indicate that long PCR- RFLP of 16 S- ITS-23 S r RNA genes is a potentially universal and reliable bacterial genotyping tool with a high resolution. Significance and Impact of the Study The methodology of long PCR- RFLP of 16 S- ITS-23 S r RNA genes will facilitate the exploration and tracing of cultivable microbial diversity in natural environments. [ABSTRACT FROM AUTHOR]

Next-generation sequencing technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads. In this context, an important issue is the need of a careful assessment of the accuracy of the assembly process. Here, we review the efficiency of a panel of assemblers, specifically designed to handle data from GS FLX 454 platform, on three bacterial data sets with different characteristics in terms of reads coverage and repeats content. Our aim is to investigate their strengths and weaknesses in the reconstruction of the reference genomes. In our benchmarking, we assess assemblers’ performance, quantifying and characterizing assembly gaps and errors, and evaluating their ability to solve complex genomic regions containing repeats. The final goal of this analysis is to highlight pros and cons of each method, in order to provide the final user with general criteria for the right choice of the appropriate assembly strategy, depending on the specific needs. A further aspect we have explored is the relationship between coverage of a sequencing project and quality of the obtained results. The final outcome suggests that, for a good tradeoff between costs and results, the planned genome coverage of an experiment should not exceed 20–30 ×. [ABSTRACT FROM PUBLISHER]

A large number of complete microorganism genomes has been sequenced and submitted to the public database and then incorporated into our complete genome database, Genome Information Broker (GIB, ). However, when comparative genomics is carried out, researchers must be aware that there are protein-coding genes not confirmed by homology or motif search and that reliable protein-coding genes are missing. Therefore, we developed a protocol (Gene Trek in Prokaryote Space, GTPS) for finding possible protein-coding genes in bacterial genomes. GTPS assigns a degree of reliability to predicted protein-coding genes. We first systematically applied the protocol to the complete genomes of all 123 bacterial species and strains that were publicly available as of July 2003, and then to those of 183 species and strains available as of September 2004. We found a number of incorrect genes and several new ones in the genome data in question. We also found a way to estimate the total number of orthologous genes in the bacterial world. [ABSTRACT FROM PUBLISHER]

We present a simple method to detect pathogenicity islands and anomalous gene clusters in bacterial genomes. The method uses iterative discriminant analysis to define genomic regions that deviate most from the rest of the genome in three compositional criteria: G+C content, dinucleotide frequency and codon usage. Using this method, we identify many virulence-related gene islands, e.g. encoding protein secretion systems, adhesins, toxins, and other anomalous gene clusters, such as prophages. The program and the whole dataset, including the catalogs of genes in the detected anomalous segments, are publicly available at http://compbio.sibsnet.org/projects/pai-ida/. This program can be used in searching for virulence-related factors in newly sequenced bacterial genomes. [Copyright &y& Elsevier]

Summary: Administration of appropriate therapeutic regimes for infections arising from pathogenic species of Burkholderia is critically dependent upon rapid and accurate diagnoses. The purpose of this work is to establish a bioinformatic pipeline to assess protein sequences for their potential as diagnostic targets for the detection of Burkholderia species. Data are presented showing both a bioinformatic methodology for prediction of surface-associated and secreted proteins and its application to a test dataset of proteins from the pathogen B. pseudomallei. A subset of proteins, known to be produced by the organism, is identified which represents potential targets for development of new diagnostic reagents. In addition, a ‘reverse diagnostics’ bioinformatics approach has been established which can now be extended to whole genome analyses. [Copyright &y& Elsevier]