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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.


September 22, 2019  |  

Genomic insights into nematicidal activity of a bacterial endophyte, Raoultella ornithinolytica MG against pine wilt nematode.

Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is one of the most devastating conifer diseases decimating several species of pine trees on a global scale. Here, we report the draft genome of Raoultella ornithinolytica MG, which is isolated from mountain-cultivated ginseng plant as an bacterial endophyte and shows nematicidal activity against B. xylophilus. Our analysis of R. ornithinolytica MG genome showed that it possesses many genes encoding potential nematicidal factors in addition to some secondary metabolite biosynthetic gene clusters that may contribute to the observed nematicidal activity of the strain. Furthermore, the genome was lacking key components of avermectin gene cluster, suggesting that nematicidal activity of the bacterium is not likely due to the famous anthelmintic agent of wide-spread use, avermectin. This genomic information of R. ornithinolytica will provide basis for identification and engineering of genes and their products toward control of pine wilt disease.


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.


September 22, 2019  |  

Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata.

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.


September 22, 2019  |  

Mosaic structure as the main feature of Mycobacterium bovis BCG genomes

Background: The genome stability of attenuated live BCG vaccine preventing the acute forms of childhood tuberculosis is an important aspect of vaccine production. The pur- pose of our study was a whole genome comparative analysis of BCG sub-strains and identification of potential triggers of sub-strains’ transition. Results: Genomes of three BCG Russia seed lots (1963, 1982, 2006 years) have been sequenced, and the stability of vaccine sub-strain genomes has been confirmed. A com- parative genome analysis of nine Mycobacterium bovis BCG and three M. bovis strains revealed their specific genome features associated with prophage profiles. A number of prophage-coded homologs to Caudovirales ORFs were common to all BCG genomes. Prophage profiles of BCG Tice and BCG Montreal genomes were unique and coded homologs to herpes viruses ORFs. The data of phylogenetic analysis of BCG sub-strain groups based on whole genome sequences and genome restriction maps were in con- gruence with prophage profiles. The only fragmentary similarity of specific prophage sequences of BCG Tice, BCG Montreal, and BCG Russia 368 in pair-wise alignments was observed, suggesting the impact of prophages on mosaic structure of genomes. Conclusions: The whole genome sequencing approach is essential for genomes with mosaic structure, harboring numerous prophage sequences. Tools for prophage search are effective instruments in this analysis.


September 22, 2019  |  

Characterization of a novel multidrug resistance plasmid pSGB23 isolated from Salmonella enterica subspecies enterica serovar Saintpaul.

Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken.Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance.We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study.


September 22, 2019  |  

Stendomycin and pantomycin are identical natural products: Preparation of a functionalized bioactive analogue.

The natural products pantomycin and stendomycin were both reported as antimicrobial agents. We demonstrate by gene cluster analysis, LC-MS analysis, and isolation that these polypeptides are identical, and we identify previously unknown congeners. We show that stendomycin can be chemically modified at its electrophilic dehydrobutyrine moiety yielding the first bioactive analogue of this natural product which can undergo additional functionalization. This compound may be a valuable starting point for molecular probe development, and we invite its distribution to the scientific community.


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.


September 22, 2019  |  

Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae.

The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene blaNDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.


September 22, 2019  |  

Heterogeneous and flexible transmission of mcr-1 in hospital-associated Escherichia coli.

The recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route of mcr-1 among Enterobacteriaceae species in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis of Escherichia coli isolates collected in a hospital in Hangzhou, China. We found that mcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread of mcr-1 The mcr-1 gene was found on either chromosomes or plasmids, but in most of the E. coli isolates, mcr-1 was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission of mcr-1 and the coexistence of mcr-1 with other genes encoding ß-lactamases and fluoroquinolone resistance in the E. coli isolates. These findings indicate that mcr-1 is heterogeneously disseminated in both commensal and pathogenic strains of E. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology of mcr-1 among hospital-associated E. coli strains. IMPORTANCE Colistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergent mcr-1 colistin resistance gene threatens the clinical utility of colistin and has gained global attention. How mcr-1 spreads in hospital settings remains unknown and was investigated by whole-genome sequencing of mcr-1-carrying Escherichia coli in this study. The findings revealed extraordinary flexibility of mcr-1 in its spread among genetically diverse E. coli hosts and plasmids, nosocomial transmission of mcr-1-carrying E. coli, and the continuous emergence of novel Inc types of plasmids carrying mcr-1 and new mcr-1 variants. Additionally, mcr-1 was found to be frequently associated with other genes encoding ß-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology of mcr-1 and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat. Copyright © 2018 Shen et al.


September 22, 2019  |  

Genomic comparison of highly virulent, moderately virulent, and avirulent strains from a genetically closely-related MRSA ST239 sub-lineage provides insights into pathogenesis.

The genomic comparison of virulent (TW20), moderately virulent (CMRSA6/CMRSA3), and avirulent (M92) strains from a genetically closely-related MRSA ST239 sub-lineage revealed striking similarities in their genomes and antibiotic resistance profiles, despite differences in virulence and pathogenicity. The main differences were in the spa gene (coding for staphylococcal protein A), lpl genes (coding for lipoprotein-like membrane proteins), cta genes (genes involved in heme synthesis), and the dfrG gene (coding for a trimethoprim-resistant dihydrofolate reductase), as well as variations in the presence or content of some prophages and plasmids, which could explain the virulence differences of these strains. TW20 was positive for all genetic traits tested, compared to CMRSA6, CMRSA3, and M92. The major components differing among these strains included spa and lpl with TW20 carrying both whereas CMRSA6/CMRSA3 carry spa identical to TW20 but have a disrupted lpl. M92 is devoid of both these traits. Considering the role played by these components in innate immunity and virulence, it is predicted that since TW20 has both the components intact and functional, these traits contribute to its pathogenesis. However, CMRSA6/CMRSA3 are missing one of these components, hence their intermediately virulent nature. On the contrary, M92 is completely devoid of both the spa and lpl genes and is avirulent. Mobile genetic elements play a potential role in virulence. TW20 carries three prophages (?Sa6, ?Sa3, and ?SPß-like), a pathogenicity island and two plasmids. CMRSA6, CMRSA3, and M92 contain variations in one or more of these components. The virulence associated genes in these components include staphylokinase, entertoxins, antibiotic/antiseptic/heavy metal resistance and bacterial persistence. Additionally, there are many hypothetical proteins (present with variations among strains) with unknown function in these mobile elements which could be making an important contribution in the virulence of these strains. The above mentioned repertoire of virulence components in TW20 likely contributes to its increased virulence, while the absence and/or modification of one or more of these components in CMRSA6/CMRSA3 and M92 likely affects the virulence of the strains.


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.


September 22, 2019  |  

The Chara genome: Secondary complexity and implications for plant terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote. Copyright © 2018 Elsevier Inc. All rights reserved.


September 22, 2019  |  

An outbreak of a rare Shiga-toxin-producing Escherichia coli serotype (O117:H7) among men who have sex with men.

Sexually transmissible enteric infections (STEIs) are commonly associated with transmission among men who have sex with men (MSM). In the past decade, the UK has experienced multiple parallel STEI emergences in MSM caused by a range of bacterial species of the genus Shigella, and an outbreak of an uncommon serotype (O117?:?H7) of Shiga-toxin-producing Escherichia coli (STEC). Here, we used microbial genomics on 6 outbreak and 30 sporadic STEC O117?:?H7 isolates to explore the origins and pathogenic drivers of the STEC O117?:?H7 emergence in MSM. Using genomic epidemiology, we found that the STEC O117?:?H7 outbreak lineage was potentially imported from Latin America and likely continues to circulate both in the UK MSM population and in Latin America. We found genomic relationships consistent with existing symptomatic evidence for chronic infection with this STEC serotype. Comparative genomic analysis indicated the existence of a novel Shiga toxin 1-encoding prophage in the outbreak isolates, and evidence of horizontal gene exchange among the STEC O117?:?H7 outbreak lineage and other enteric pathogens. There was no evidence of increased virulence in the outbreak strains relative to contextual isolates, but the outbreak lineage was associated with azithromycin resistance. Comparing these findings with similar genomic investigations of emerging MSM-associated Shigella in the UK highlighted many parallels, the most striking of which was the importance of the azithromycin phenotype for STEI emergence in this patient group.


September 22, 2019  |  

Potential survival and pathogenesis of a novel strain, Vibrio parahaemolyticus FORC_022, isolated from a soy sauce marinated crab by genome and transcriptome analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.


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