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September 22, 2019

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

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September 22, 2019

Comparative genomics of Campylobacter concisus: Analysis of clinical strains reveals genome diversity and pathogenic potential.

In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal (GI) diseases including gastroenteritis, inflammatory bowel disease, and colorectal cancer. Campylobacter concisus, an oral commensal historically linked to gingivitis and periodontitis, has been increasingly detected in the lower GI tract. In the present study, we generated robust genome sequence data from C. concisus strains and undertook a comprehensive pangenome assessment to identify C. concisus virulence properties and to explain potential adaptations acquired while residing in specific ecological niche(s) of the GI tract. Genomes of 53 new C. concisus strains were sequenced, assembled, and annotated including 36 strains from gastroenteritis patients, 13 strains from Crohn’s disease patients and four strains from colitis patients (three collagenous colitis and one lymphocytic colitis). When compared with previous published sequences, strains clustered into two main groups/genomospecies (GS) with phylogenetic clustering explained neither by disease phenotype nor sample location. Paired oral/faecal isolates, from the same patient, indicated that there are few genetic differences between oral and gut isolates which suggests that gut isolates most likely reflect oral strain relocation. Type IV and VI secretion systems genes, genes known to be important for pathogenicity in the Campylobacter genus, were present in the genomes assemblies, with 82% containing Type VI secretion system genes. Our findings indicate that C. concisus strains are genetically diverse, and the variability in bacterial secretion system content may play an important role in their virulence potential.

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September 22, 2019

Otitis in a cat associated with Corynebacterium provencense.

The role of corynebacteria in canine and feline otitis has not been investigated in detail; however, members of this genus are increasingly recognized as pathogens of otitis in both human and veterinary medicine.Here we report the first case of feline otitis associated with the recently described species Corynebacterium provencense. A seven-month old cat presented with a head tilt and ataxia was diagnosed with peripheral vestibular syndrome associated with an otitis media/interna. This took place 6 weeks after resection of a polyp, having initially shown a full recovery with topical ofloxacin and glucocorticoid treatment. Bacteriology of an ear swab yielded a pure culture of corynebacteria, which could not be identified at the species level using routine methods. However, the 16S rRNA gene sequence was 100% identical to the recently published novel corynebacterium species, Corynebacterium provencense. Whole genome sequencing of the cat isolate and calculation of average nucleotide identity (99.1%) confirmed this finding. The cat isolate was found to contain additional presumptive iron acquisition genes that are likely to encode virulence factors. Furthermore, the strain tested resistant to clindamycin, penicillin and ciprofloxacin. The cat was subsequently treated with chloramphenicol, which lead to clinical improvement.Corynebacteria from otitis cases are not routinely identified at the species level and not tested for antimicrobial susceptibility in veterinary laboratories, as they are not considered major pathogens. This may lead to underreporting of this genus or animals being treated with inappropriate antimicrobials since corynebacteria are often resistant to multiple drugs.

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September 22, 2019

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Adaptation of Pseudomonas aeruginosa to phage PaP1 predation via O-antigen polymerase mutation.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.

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September 22, 2019

Genome-wide DNA methylation and transcriptome changes in Mycobacterium tuberculosis with rifampicin and isoniazid resistance

We investigated the genome-wide DNA methylation and transcriptome changes in M. tuberculosis with rifampicin or isoniazid resistance. Single-molecule real-time (SMRT) sequencing and microarray technology were performed to expound DNA methylation profiles and differentially expressed genes in rifampicin or isoniazid resis- tant M. tuberculosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis and meth- ylated regulatory network analysis were conducted by online forecasting databases. Integrated analysis of DNA methylation and transcriptome revealed that 335 differentially methylated genes (175 hypermethylated and 160 hypomethylated) and 132 significant differentially expressed genes (68 up-regulated and 63 down-regulated) were found to be regulated by both rifampicin and isoniazid in M. tuberculosis H37Rv. Correlation analysis showed that differential methylated genes were negatively correlated with their transcriptional levels in rifampicin or isoniazid resistant strains. KEGG pathway analysis indicated that nitrogen metabolism pathway is closely related to differ- entially methylated genes induced by rifampicin and isoniazid. KEGG also suggested that differentially expressed genes in rifampicin or isoniazid-resistant strains may play different roles in regulating signal transduction events. Furthermore, five differentially methylated candidate genes (Rv0840c, Rv2243, Rv0644c, Rv2386c and Rv1130) in rifampicin resistant strains and three genes (Rv0405, Rv0252 and Rv0908) in isoniazid-resistant strains were verified the existence of protein-protein interaction in STRING database. Integrated DNA methylation and transcrip- tome analyses provide an epigenetic overview of rifampicin and isoniazid-induced antibiotic resistance in M. tuber- culosis H37Rv. Several interesting genes and regulatory pathways may provide valuable resources for epigenetic studies in M. tuberculosis antibiotic resistance.

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September 22, 2019

Whole genome sequencing of Klebsiella pneumoniae strain unravels a new model for the development of extensive drug resistance in Enterobacteriaceae.

Increased incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide. The WHO warns about the imminent risk to global health if the spread of resistant bacteria is not contained.Here, single molecule real time sequencing was used to analyse the whole genome and resistome of SKGH01, a strain of Klebsiella pneumoniae .The data showed that SKGH01 was resistant to all commercially available antibiotics. A complete account of extensively drug-resistant (XDR) CRE at a genomic level and the entire location map of all antibiotic resistance components are here presented. Additionally, this work proposes a model of XDR acquisition in Enterobacteriaceae.

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September 22, 2019

Co-culture of soil biofilm isolates enables the discovery of novel antibiotics

Bacterial natural products (NPs) are considered to be a promising source of drug discovery. However, the biosynthesis gene clusters (BGCs) of NP are not often expressed, making it difficult to identify them. Recently, the study of biofilm community showed bacteria may gain competitive advantages by the secretion of antibiotics, implying a possible way to screen antibiotic by evaluating the social behavior of bacteria. In this study, we have described an efficient workflow for novel antibiotic discovery by employing the bacterial social interaction strategy with biofilm cultivation, co-culture, transcriptomic and genomic methods. We showed that a biofilm dominant species, i.e. Pseudomonas sp. G7, which was isolated from cultivated soil biofilm community, was highly competitive in four-species biofilm communities, as the synergistic combinations preferred to exclude this strain while the antagonistic combinations did not. Through the analysis of transcriptomic changes in four-species co-culture and the complete genome of Pseudomonas sp. G7, we finally discovered two novel non-ribosomal polypeptide synthetic (NRPS) BGCs, whose products were predicted to have seven and six amino acid components, respectively. Furthermore, we provide evidence showing that only when Pseudomonas sp. G7 was co-cultivated with at least two or three other bacterial species can these BGC genes be induced, suggesting that the co-culture of the soil biofilm isolates is critical to the discovery of novel antibiotics. As a conclusion, we set a model of applying microbial interaction to the discovery of new antibiotics.

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September 22, 2019

Complete genome sequencing of exopolysaccharide-producing Lactobacillus plantarum K25 provides genetic evidence for the probiotic functionality and cold endurance capacity of the strain.

Lactobacillus plantarum (L. plantarum) K25 is a probiotic strain isolated from Tibetan kefir. Previous studies showed that this exopolysaccharide (EPS)-producing strain was antimicrobial active and cold tolerant. These functional traits were evidenced by complete genome sequencing of strain K25 with a circular 3,175,846-bp chromosome and six circular plasmids, encoding 3365 CDSs, 16 rRNA genes and 70 tRNA genes. Genomic analysis of L. plantarum K25 illustrates that this strain contains the previous reported mechanisms of probiotic functionality and cold tolerance, involving plantaricins, lysozyme, bile salt hydrolase, chaperone proteins, osmoprotectant, oxidoreductase, EPSs and terpenes. Interestingly, strain K25 harbors more genes that function in defense mechanisms, and lipid transport and metabolism, in comparison with other L. plantarum strains reported. The present study demonstrates the comprehensive analysis of genes related to probiotic functionalities of an EPS-producing L. plantarum strain based on whole genome sequencing.

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September 22, 2019

A molecular window into the biology and epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes of Mycobacterium tuberculosis.

Tuberculosis (TB) resulted in an estimated 1.7 million deaths in the year 2016. The disease is caused by the members of Mycobacterium tuberculosis complex, which includes Mycobacterium tuberculosis, Mycobacterium bovis and other closely related TB causing organisms. In order to understand the epidemiological dynamics of TB, national TB control programs often conduct standardized genotyping at 24 Mycobacterial-Interspersed-Repetitive-Units (MIRU)-Variable-Number-of-Tandem-Repeats (VNTR) loci. With the advent of next generation sequencing technology, whole-genome sequencing (WGS) has been widely used for studying TB transmission. However, an open-source software that can connect WGS and MIRU-VNTR typing is currently unavailable, which hinders interlaboratory communication. In this manuscript, we introduce the MIRU-profiler program which could be used for prediction of MIRU-VNTR profile from WGS of M. tuberculosis.The MIRU-profiler is implemented in shell scripting language and depends on EMBOSS software. The in-silico workflow of MIRU-profiler is similar to those described in the laboratory manuals for genotyping M. tuberculosis. Given an input genome sequence, the MIRU-profiler computes alleles at the standard 24-loci based on in-silico PCR amplicon lengths. The final output is a tab-delimited text file detailing the 24-loci MIRU-VNTR pattern of the input sequence.The MIRU-profiler was validated on four datasets: complete genomes from NCBI-GenBank (n = 11), complete genomes for locally isolated strains sequenced using PacBio (n = 4), complete genomes for BCG vaccine strains (n = 2) and draft genomes based on 250 bp paired-end Illumina reads (n = 106).The digital MIRU-VNTR results were identical to the experimental genotyping results for complete genomes of locally isolated strains, BCG vaccine strains and five out of 11 genomes from the NCBI-GenBank. For draft genomes based on short Illumina reads, 21 out of 24 loci were inferred with a high accuracy, while a number of inaccuracies were recorded for three specific loci (ETRA, QUB11b and QUB26). One of the unique features of the MIRU-profiler was its ability to process multiple genomes in a batch. This feature was tested on all complete M. tuberculosis genome (n = 157), for which results were successfully obtained in approximately 14 min.The MIRU-profiler is a rapid tool for inference of digital MIRU-VNTR profile from the assembled genome sequences. The tool can accurately infer repeat numbers at the standard 24 or 21/24 MIRU-VNTR loci from the complete or draft genomes respectively. Thus, the tool is expected to bridge the communication gap between the laboratories using WGS and those using the conventional MIRU-VNTR typing.

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September 22, 2019

Comparing two Mycobacterium tuberculosis genomes from Chinese immigrants with native genomes using mauve alignments.

The number of immigrants with tuberculosis (TB) increases each year in South Korea. Determining the transmission dynamics based on whole genome sequencing (WGS) to cluster the strains has been challenging.WGS, annotation refinement, and orthology assignment for the GenBank accession number acquisition were performed on two clinical isolates from Chinese immigrants. In addition, the genomes of the two isolates were compared with the genomes of Mycobacterium tuberculosis isolates, from two native Korean and five native Chinese individuals using a phylogenetic topology tree based on the Multiple Alignment of Conserved Genomic Sequence with Rearrangements (Mauve) package.The newly assigned accession numbers for two clinical isolates were CP020381.2 (a Korean-Chinese from Yanbian Province) and CP022014.1 (a Chinese from Shandong Province), respectively. Mauve alignment classified all nine TB isolates into a discriminative collinear set with matched regions. The phylogenetic analysis revealed a rooted phylogenetic tree grouping the nine strains into two lineages: strains from Chinese individuals and strains from Korean individuals.Phylogenetic trees based on the Mauve alignments were supposed to be useful in revealing the dynamics of TB transmission from immigrants in South Korea, which can provide valuable information for scaling up the TB screening policy for immigrants. Copyright©2018. The Korean Academy of Tuberculosis and Respiratory Diseases.

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September 22, 2019

Genomic characterization of extensively drug-resistant Acinetobacter baumannii strain, KAB03 belonging to ST451 from Korea.

Extensively drug-resistant (XDR) Acinetobacter baumannii strains have emerged rapidly worldwide. The antibiotic resistance characteristics of XDR A. baumannii strains show regional differences; therefore, it is necessary to analyze both genomic and proteomic characteristics of emerging XDR A. baumannii clinical strains isolated in Korea to elucidate their multidrug resistance. Here, we isolated new sequence type of XDR A. baumannii clinical strain (KAB03) from Korean hospitals and performed comprehensive genome analyses. The strain belongs to new sequence type, ST451. Single nucleotide polymorphism (SNP) analysis with other types of A. baumannii strains revealed that KAB03 has unique SNP pattern in the regions of gyrB and gpi of MLST profiles. A. baumannii KAB03 harbours three antibiotic resistance islands (AbGRI1, 2, and 3). AbGRI1 harbours two copies of Tn2006 containing blaOXA-23, which play an important role in antibiotic resistance. AbGRI2 possesses aminoglycoside resistant gene aph(3′)-Ic and class A ß-lactamase blaTEM. AbGIR3 has macrolide resistant genes and aminoglycoside resistant gene armA. A. baumannii KAB03 harbours mutations in pmrB and pmrC, which are believed to confer colistin resistance. In addition, proteomic and transcriptional analysis of KAB03 confirmed that ß-lactamases (ADC-73 and OXA-23), Ade efflux pumps (AdeIJK), outer membrane proteins (OmpA and OmpW), and colistin resistance genes (PmrCAB) were major proteins responsible for antibiotic resistance. Our proteogenomic results provide valuable information for multi-drug resistance in emerging XDR A. baumannii strains belonging to ST451. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Biosynthetic Baeyer-Villiger chemistry enables access to two anthracene scaffolds from a single gene cluster in Deep-Sea-derived Streptomyces olivaceus SCSIO T05.

Four known compounds, rishirilide B (1), rishirilide C (2), lupinacidin A (3), and galvaquinone B (4), representing two anthracene scaffolds typical of aromatic polyketides, were isolated from a culture of the deep-sea-derived Streptomyces olivaceus SCSIO T05. From the S. olivaceus producer was cloned and sequenced the rsd biosynthetic gene cluster (BGC) that drives rishirilide biosynthesis. The structural gene rsdK2 inactivation and heterologous expression of the rsd BGC confirmed the single rsd BGC encodes construction of 1-4 and, thus, accounts for two anthracene scaffolds. Precursor incubation experiments with 13C-labeled acetate revealed that a Baeyer-Villiger-type rearrangement plays a central role in construction of 1-4. Two luciferase monooxygenase components, along with a reductase component, are presumably involved in the Baeyer-Villiger-type rearrangement reaction enabling access to the two anthracene scaffold variants. Engineering of the rsd BGC unveiled three SARP family transcriptional regulators, enhancing anthracene production. Inactivation of rsdR4, a MarR family transcriptional regulator, failed to impact production of 1-4, although production of 3 was slightly improved; most importantly rsdR4 inactivation led to the new adduct 6 in high titer. Notably, inactivation of rsdH, a putative amidohydrolase, substantially improved the overall titers of 1-4 by more than 4-fold.

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September 22, 2019

PBHoover and CigarRoller: a method for confident haploid variant calling on Pacific Biosciences data and its application to heterogeneous population analysis

Motivation: Single Molecule Real-Time (SMRT) sequencing has important and underutilized advantages that amplification-based platforms lack. Lack of systematic error (e.g. GC-bias), complete de novo assembly (including large repetitive regions) without scaffolding, can be mentioned. SMRT sequencing, however suffers from high random error rate and low sequencing depth (older chemistries). Here, we introduce PBHoover, software that uses a heuristic calling algorithm in order to make base calls with high certainty in low coverage regions. This software is also capable of mixed population detection with high sensitivity. PBHoovertextquoterights CigarRoller attachment improves sequencing depth in low-coverage regions through CIGAR-string correction. Results: We tested both modules on 348 M.tuberculosis clinical isolates sequenced on C1 or C2 chemistries. On average, CigarRoller improved percentage of usable read count from 68.9% to 99.98% in C1 runs and from 50% to 99% in C2 runs. Using the greater depth provided by CigarRoller, PBHoover was able to make base and variant calls 99.95% concordant with Sanger calls (QV33). PBHoover also detected antibiotic-resistant subpopulations that went undetected by Sanger. Using C1 chemistry, subpopulations as small as 9% of the total colony can be detected by PBHoover. This provides the most sensitive amplification-free molecular method for heterogeneity analysis and is in line with phenotypic methodstextquoteright sensitivity. This sensitivity significantly improves with the greater depth and lower error rate of the newer chemistries. Availability and Implementation: Executables are freely available under GNU GPL v3+ at http://www.gitlab.com/LPCDRP/pbhoover and http://www.gitlab.com/LPCDRP/CigarRoller. PBHoover is also available on bioconda: https://anaconda.org/bioconda/pbhoover.

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