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April 21, 2020  |  

Comparative analysis of KPC-2-encoding chimera plasmids with multi-replicon IncR:IncpA1763-KPC:IncN1 or IncFIIpHN7A8:IncpA1763-KPC:IncN1.

IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described.Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1).The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary Inc-FIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as blaKPC, tetA, dfrA, and qnr).In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number.

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April 21, 2020  |  

Genetic characterization and potential molecular dissemination mechanism of tet(31) gene in Aeromonas caviae from an oxytetracycline wastewater treatment system.

Recently, the rarely reported tet(31) tetracycline resistance determinant was commonly found in Aeromonas salmonicida, Gallibacterium anatis, and Oblitimonas alkaliphila isolated from farming animals and related environment. However, its distribution in other bacteria and potential molecular dissemination mechanism in environment are still unknown. The purpose of this study was to investigate the potential mechanism underlying dissemination of tet(31) by analysing the tet(31)-carrying fragments in A. caviae strains isolated from an aerobic biofilm reactor treating oxytetracycline bearing wastewater. Twenty-three A. caviae strains were screened for the tet(31) gene by polymerase chain reaction (PCR). Three strains (two harbouring tet(31), one not) were subjected to whole genome sequencing using the PacBio RSII platform. Seventeen A. caviae strains carried the tet(31) gene and exhibited high resistance levels to oxytetracycline with minimum inhibitory concentrations (MICs) ranging from 256 to 512?mg/L. tet(31) was comprised of the transposon Tn6432 on the chromosome of A. caviae, and Tn6432 was also found in 15 additional tet(31)-positive A. caviae isolates by PCR. More important, Tn6432 was located on an integrative conjugative element (ICE)-like element, which could mediate the dissemination of the tet(31)-carrying transposon Tn6432 between bacteria. Comparative analysis demonstrated that Tn6432 homologs with the structure ISCR2-?phzF-tetR(31)-tet(31)-?glmM-sul2 were also carried by A. salmonicida, G. anatis, and O. alkaliphila, suggesting that this transposon can be transferred between species and even genera. This work provides the first report on the identification of the tet(31) gene in A. caviae, and will be helpful in exploring the dissemination mechanisms of tet(31) in water environment.Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020  |  

Analysis of a poxtA- and optrA-co-carrying conjugative multiresistance plasmid from Enterococcus faecalis.

To investigate the presence and transferability of the poxtA gene and identify the genetic context of poxtA in two enterococcal plasmids from swine.MICs were determined by broth microdilution. A total of 114 porcine enterococci with florfenicol MICs of =16?mg/L were screened for the presence of the poxtA gene by PCR. Transferability of poxtA was investigated by conjugation and transformation. The poxtA-carrying plasmids were completely sequenced using the Illumina Miseq and PacBio RSII platform. The presence of circular intermediates was examined by inverse PCR.The poxtA gene was present in 57.9% (66/114) of the florfenicol-resistant porcine enterococci. Two poxtA-carrying plasmids, pE035 and pE076, were identified. The conjugative 121524?bp plasmid pE035 carried poxtA and optrA along with the resistance genes erm(A), erm(B), aac(A)-aph(D), lnu(G), fexB, dfrG and bcrABDR. Three mobile elements, comprising a mobile dfrG locus, a mobile bcrABDR locus and an unconventional circularizable structure containing aac(A)-aph(D), were located on this plasmid and all proved to be active by inverse PCR. The non-conjugative 19832?bp plasmid pE076 only carried poxtA and fexB. After transfer, both the transconjugant and the transformant displayed elevated MICs of the respective antimicrobial agents.To the best of our knowledge, this is the first report of the co-location of the oxazolidinone resistance genes poxtA and optrA on a conjugative multiresistance plasmid from a porcine enterococcal strain. In addition, the presence of three mobile elements in such a plasmid will aid in the persistence and dissemination of poxtA and optrA among enterococci. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020  |  

Plastid genomes from diverse glaucophyte genera reveal a largely conserved gene content and limited architectural diversity.

Plastid genome (ptDNA) data of Glaucophyta have been limited for many years to the genus Cyanophora. Here, we sequenced the ptDNAs of Gloeochaete wittrockiana, Cyanoptyche gloeocystis, Glaucocystis incrassata, and Glaucocystis sp. BBH. The reported sequences are the first genome-scale plastid data available for these three poorly studied glaucophyte genera. Although the Glaucophyta plastids appear morphologically “ancestral,” they actually bear derived genomes not radically different from those of red algae or viridiplants. The glaucophyte plastid coding capacity is highly conserved (112 genes shared) and the architecture of the plastid chromosomes is relatively simple. Phylogenomic analyses recovered Glaucophyta as the earliest diverging Archaeplastida lineage, but the position of viridiplants as the first branching group was not rejected by the approximately unbiased test. Pairwise distances estimated from 19 different plastid genes revealed that the highest sequence divergence between glaucophyte genera is frequently higher than distances between species of different classes within red algae or viridiplants. Gene synteny and sequence similarity in the ptDNAs of the two Glaucocystis species analyzed is conserved. However, the ptDNA of Gla. incrassata contains a 7.9-kb insertion not detected in Glaucocystis sp. BBH. The insertion contains ten open reading frames that include four coding regions similar to bacterial serine recombinases (two open reading frames), DNA primases, and peptidoglycan aminohydrolases. These three enzymes, often encoded in bacterial plasmids and bacteriophage genomes, are known to participate in the mobilization and replication of DNA mobile elements. It is therefore plausible that the insertion in Gla. incrassata ptDNA is derived from a DNA mobile element.

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April 21, 2020  |  

Genome Comparisons of Wild Isolates of Caulobacter crescentus Reveal Rates of Inversion and Horizontal Gene Transfer.

Since previous interspecies comparisons of Caulobacter genomes have revealed extensive genome rearrangements, we decided to compare the nucleotide sequences of four C. crescentus genomes, NA1000, CB1, CB2, and CB13. To accomplish this goal, we used PacBio sequencing technology to determine the nucleotide sequence of the CB1, CB2, and CB13 genomes, and obtained each genome sequence as a single contig. To correct for possible sequencing errors, each genome was sequenced twice. The only differences we observed between the two sets of independently determined sequences were random omissions of a single base in a small percentage of the homopolymer regions where a single base is repeated multiple times. Comparisons of these four genomes indicated that horizontal gene transfer events that included small numbers of genes occurred at frequencies in the range of 10-3 to 10-4 insertions per generation. Large insertions were about 100 times less frequent. Also, in contrast to previous interspecies comparisons, we found no genome rearrangements when the closely related NA1000, CB1, and CB2 genomes were compared, and only eight inversions and one translocation when the more distantly related CB13 genome was compared to the other genomes. Thus, we estimate that inversions occur at a rate of one per 10 to 12 million generations in Caulobacter genomes. The inversions seem to be complex events that include the simultaneous creation of indels.

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April 21, 2020  |  

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected.To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms.The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing.A novel IncZ-7 plasmid pEC013 (~118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013.The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid’s heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020  |  

Characterization of vanM carrying clinical Enterococcus isolates and diversity of the suppressed vanM gene cluster.

Here we report the prevalence of the suppressed vanM gene cluster as a reservoir of vancomycin resistance genes. Among 1284 clinical isolates of enterococci from four hospitals in Hangzhou, China, 55 isolates of Enterococcus faecium and one isolate of Enterococcus faecalis were screened positive for the vanM genotype. Antimicrobial susceptibility testing showed that 55 of the 56 vanM-positive isolates were susceptible to vancomycin and teicoplanin. Most of them (54/56) belonged to the main epidemic lineage CC17, mostly the ST78 type. The vanM gene clusters in the 55 vancomycin-susceptible isolates showed sequence diversity owing to different insertion locations of IS1216E. The vanM transposons could be classified into five types and they all carried two or more IS1216E elements, leading to complete or partial deletions of vanR, vanS, or vanX. Quantitative reverse transcription polymerase chain reaction showed that the expression level of vanM was significantly lower in the vancomycin-susceptible isolates than in the vancomycin-resistant isolate. Considering the prevalence of the vanM genotype and the potential for conversion to a resistant phenotype, vanM might act as an important determinant of glycopeptide resistance in the future. It is essential to strengthen the surveillance of vanM-containing enterococci to control the dissemination of vancomycin resistance. Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020  |  

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel’s original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

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April 21, 2020  |  

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

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April 21, 2020  |  

Large Enriched Fragment Targeted Sequencing (LEFT-SEQ) Applied to Capture of Wolbachia Genomes.

Symbiosis is a major force of evolutionary change, influencing virtually all aspects of biology, from population ecology and evolution to genomics and molecular/biochemical mechanisms of development and reproduction. A remarkable example is Wolbachia endobacteria, present in some parasitic nematodes and many arthropod species. Acquisition of genomic data from diverse Wolbachia clades will aid in the elucidation of the different symbiotic mechanisms(s). However, challenges of de novo assembly of Wolbachia genomes include the presence in the sample of host DNA: nematode/vertebrate or insect. We designed biotinylated probes to capture large fragments of Wolbachia DNA for sequencing using PacBio technology (LEFT-SEQ: Large Enriched Fragment Targeted Sequencing). LEFT-SEQ was used to capture and sequence four Wolbachia genomes: the filarial nematode Brugia malayi, wBm, (21-fold enrichment), Drosophila mauritiana flies (2 isolates), wMau (11-fold enrichment), and Aedes albopictus mosquitoes, wAlbB (200-fold enrichment). LEFT-SEQ resulted in complete genomes for wBm and for wMau. For wBm, 18 single-nucleotide polymorphisms (SNPs), relative to the wBm reference, were identified and confirmed by PCR. A limit of LEFT-SEQ is illustrated by the wAlbB genome, characterized by a very high level of insertion sequences elements (ISs) and DNA repeats, for which only a 20-contig draft assembly was achieved.

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April 21, 2020  |  

Sequencing and Genomic Diversity Analysis of IncHI5 Plasmids.

IncHI plasmids could be divided into five different subgroups IncHI1-5. In this study, the complete nucleotide sequences of seven blaIMP- or blaVIM-carrying IncHI5 plasmids from Klebsiella pneumoniae, K. quasipneumoniae, and K. variicola were determined and compared in detail with all the other four available sequenced IncHI5 plasmids. These plasmids carried conserved IncHI5 backbones composed of repHI5B and a repFIB-like gene (replication), parABC (partition), and tra1 (conjugal transfer). Integration of a number of accessory modules, through horizontal gene transfer, at various sites of IncHI5 backbones resulted in various deletions of surrounding backbone regions and thus considerable diversification of IncHI5 backbones. Among the accessory modules were three kinds of resistance accessory modules, namely Tn10 and two antibiotic resistance islands designated ARI-A and ARI-B. These two islands, inserted at two different fixed sites (one island was at one site and the other was at a different site) of IncHI5 backbones, were derived from the prototype Tn3-family transposons Tn1696 and Tn6535, respectively, and could be further discriminated as various intact transposons and transposon-like structures. The ARI-A or ARI-B islands from different IncHI5 plasmids carried distinct profiles of antimicrobial resistance markers and associated mobile elements, and complex events of transposition and homologous recombination accounted for assembly of these islands. The carbapenemase genes blaIMP-4, blaIMP-38 and blaVIM-1 were identified within various class 1 integrons from ARI-A or ARI-B of the seven plasmids sequenced in this study. Data presented here would provide a deeper insight into diversification and evolution history of IncHI5 plasmids.

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April 21, 2020  |  

Genome analysis of the rice coral Montipora capitata.

Corals comprise a biomineralizing cnidarian, dinoflagellate algal symbionts, and associated microbiome of prokaryotes and viruses. Ongoing efforts to conserve coral reefs by identifying the major stress response pathways and thereby laying the foundation to select resistant genotypes rely on a robust genomic foundation. Here we generated and analyzed a high quality long-read based ~886 Mbp nuclear genome assembly and transcriptome data from the dominant rice coral, Montipora capitata from Hawai’i. Our work provides insights into the architecture of coral genomes and shows how they differ in size and gene inventory, putatively due to population size variation. We describe a recent example of foreign gene acquisition via a bacterial gene transfer agent and illustrate the major pathways of stress response that can be used to predict regulatory components of the transcriptional networks in M. capitata. These genomic resources provide insights into the adaptive potential of these sessile, long-lived species in both natural and human influenced environments and facilitate functional and population genomic studies aimed at Hawaiian reef restoration and conservation.

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April 21, 2020  |  

Mobilome of Brevibacterium aurantiacum Sheds Light on Its Genetic Diversity and Its Adaptation to Smear-Ripened Cheeses.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.

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April 21, 2020  |  

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is =95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.Comparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

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