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.


April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.


April 21, 2020  |  

Analysis of two pheromone-responsive conjugative multiresistance plasmids carrying the novel mobile optrA locus from Enterococcus faecalis

Background: The acquired optrA gene, which encodes a ribosomal protection protein of the ABC-F family, can confer cross-resistance to linezolid and florfenicol, posing a serious therapeutic challenge to both human and veterinary medicine. Purpose: The objective of this study was to investigate the two Enterococcus faecalis (E. faecalis) plasmids for their fine structure, their transferability and the presence of mobile antimicrobial resistance loci. Methods: To elucidate their fine structure, the two plasmids were completely sequenced and the sequences analysed. Besides conjugation experiments, inverse PCR assays were conducted to see whether minicircles are produced from the mobile antimicrobial resistance loci. Results: Two pheromone-responsive conjugative optrA-carrying plasmids from E. faecalis, pE211 and pE508 were identified, which can transfer with frequencies of 2.6 ×10-2 and 3.7 ×10-2 (transconjugant per donor), respectively. In both plasmids, optrA was located on the novel mobile optrA locus with different sizes (12,834 bp in pE211 and 7,561 bp in pE508, respectively), flanked by two copies of IS1216 genes in the same orientation. Inverse PCR revealed that circular forms can be generated, consisting of optrA and one copy of IS1216, indicating they are all active. The 77,562 bp plasmid pE211 also carried Tn558 and a mobile bcrABDR locus, and the 84,468 bp plasmid pE508 also harbored the genes fexA, tet(L), tet(O/W/32/O) and a mobile aac(A)-aph(D) locus. Conclusion: The presence of mobile genetic elements in these plasmids renders them flexible and these elements will aid to the persistence and dissemination of these plasmids among enterococci and potentially also other gram-positive bacteria.


April 21, 2020  |  

Dissemination of multiple carbapenem resistance genes in an in vitro gut model simulating the human colon.

Carbapenemase-producing Enterobacteriaceae (CPE) pose a major global health risk. Mobile genetic elements account for much of the increasing CPE burden.To investigate CPE colonization and the impact of antibiotic exposure on subsequent resistance gene dissemination within the gut microbiota using a model to simulate the human colon.Gut models seeded with CPE-negative human faeces [screened with BioMérieux chromID® CARBA-SMART (Carba-Smart), Cepheid Xpert® Carba-R assay (XCR)] were inoculated with distinct carbapenemase-producing Klebsiella pneumoniae strains (KPC, NDM) and challenged with imipenem or piperacillin/tazobactam then meropenem. Resistant populations were enumerated daily on selective agars (Carba-Smart); CPE genes were confirmed by PCR (XCR, Check-Direct CPE Screen for BD MAX™). CPE gene dissemination was tracked using PacBio long-read sequencing.CPE populations increased during inoculation, plateauing at ~105?log10?cfu/mL in both models and persisting throughout the experiments (>65?days), with no evidence of CPE ‘washout’. After antibiotic administration, there was evidence of interspecies plasmid transfer of blaKPC-2 (111742?bp IncFII/IncR plasmid, 99% identity to pKpQIL-D2) and blaNDM-1 (~170?kb IncFIB/IncFII plasmid), and CPE populations rose from <0.01% to >45% of the total lactose-fermenting populations in the KPC model. Isolation of a blaNDM-1K. pneumoniae with one chromosomal single-nucleotide variant compared with the inoculated strain indicated clonal expansion within the model. Antibiotic administration exposed a previously undetected K. pneumoniae encoding blaOXA-232 (KPC model).CPE exposure can lead to colonization, clonal expansion and resistance gene transfer within intact human colonic microbiota. Furthermore, under antibiotic selective pressure, new resistant populations emerge, emphasizing the need to control exposure to antimicrobials. © 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.


April 21, 2020  |  

WGS of 1058 Enterococcus faecium from Copenhagen, Denmark, reveals rapid clonal expansion of vancomycin-resistant clone ST80 combined with widespread dissemination of a vanA-containing plasmid and acquisition of a heterogeneous accessory genome.

From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized.WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems.Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in =99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ~29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes.Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges. © 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.


April 21, 2020  |  

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.


April 21, 2020  |  

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.


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.


April 21, 2020  |  

A novel plasmid carrying carbapenem-resistant gene blaKPC-2 in Pseudomonas aeruginosa.

A carbapenem-resistant Pseudomonas aeruginosa strain PA1011 (ST463) was isolated from a patient in a surgical intensive care unit. PCR detection showed that PA1011 carried the blaKPC-2 gene. A plasmid was isolated and sequenced using the Illumina NextSeq 500 and PacBio RSII sequencing platforms. The plasmid was named pPA1011 and carried the carbapenem-resistant gene blaKPC-2. pPA1011 was a 62,793 bp in length with an average G+C content of 58.8%. It was identified as a novel plasmid and encoded a novel genetic environment of blaKPC-2 gene (?IS6-Tn3-ISKpn8-blaKPC-2-ISKpn6-IS26).


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.


April 21, 2020  |  

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race.

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.


April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.


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.


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.


April 21, 2020  |  

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ~53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ~4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.


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