Plasmid Fragment Research Articles

High-throughput single-cell sequencing of activated sludge microbiome

Wastewater treatment plants (WWTPs) represent one of biotechnology's largest and most critical applications, playing a pivotal role in environmental protection and public health. In WWTPs, activated sludge (AS) plays a major role in removing contaminants and pathogens from wastewater. While metagenomics has advanced our understanding of microbial communities, it still faces challenges in revealing the genomic heterogeneity of cells, uncovering the microbial dark matter, and establishing precise links between genetic elements and their host cells as a bulk method. These issues could be largely resolved by single-cell sequencing, which can offer unprecedented resolution to show the unique genetic information. Here we show the high-throughput single-cell sequencing to the AS microbiome. The single-amplified genomes (SAGs) of 15,110 individual cells were clustered into 2,454 SAG bins. We find that 27.5% of the genomes in the AS microbial community represent potential novel species, highlighting the presence of microbial dark matter. Furthermore, we identified 1,137 antibiotic resistance genes (ARGs), 10,450 plasmid fragments, and 1,343 phage contigs, with shared plasmid and phage groups broadly distributed among hosts, indicating a high frequency of horizontal gene transfer (HGT) within the AS microbiome. Complementary analysis using 1,529 metagenome-assembled genomes from the AS samples allowed for the taxonomic classification of 98 SAG bins, which were previously unclassified. Our study establishes the feasibility of single-cell sequencing in characterizing the AS microbiome, providing novel insights into its ecological dynamics, and deepening our understanding of HGT processes, particularly those involving ARGs. Additionally, this valuable tool could monitor the distribution, spread, and pathogenic hosts of ARGs both within AS environments and between AS and other environments, which will ultimately contribute to developing a health risk evaluation system for diverse environments within a One Health framework.

Metagenomic assembly is the main bottleneck in the identification of mobile genetic elements.

Antimicrobial resistance genes (ARG) are commonly found on acquired mobile genetic elements (MGEs) such as plasmids or transposons. Understanding the spread of resistance genes associated with mobile elements (mARGs) across different hosts and environments requires linking ARGs to the existing mobile reservoir within bacterial communities. However, reconstructing mARGs in metagenomic data from diverse ecosystems poses computational challenges, including genome fragment reconstruction (assembly), high-throughput annotation of MGEs, and identification of their association with ARGs. Recently, several bioinformatics tools have been developed to identify assembled fragments of plasmids, phages, and insertion sequence (IS) elements in metagenomic data. These methods can help in understanding the dissemination of mARGs. To streamline the process of identifying mARGs in multiple samples, we combined these tools in an automated high-throughput open-source pipeline, MetaMobilePicker, that identifies ARGs associated with plasmids, IS elements and phages, starting from short metagenomic sequencing reads. This pipeline was used to identify these three elements on a simplified simulated metagenome dataset, comprising whole genome sequences from seven clinically relevant bacterial species containing 55 ARGs, nine plasmids and five phages. The results demonstrated moderate precision for the identification of plasmids (0.57) and phages (0.71), and moderate sensitivity of identification of IS elements (0.58) and ARGs (0.70). In this study, we aim to assess the main causes of this moderate performance of the MGE prediction tools in a comprehensive manner. We conducted a systematic benchmark, considering metagenomic read coverage, contig length cutoffs and investigating the performance of the classification algorithms. Our analysis revealed that the metagenomic assembly process is the primary bottleneck when linking ARGs to identified MGEs in short-read metagenomics sequencing experiments rather than ARGs and MGEs identification by the different tools.

MOBFinder: a tool for mobilization typing of plasmid metagenomic fragments based on a language model.

Mobilization typing (MOB) is a classification scheme for plasmid genomes based on their relaxase gene. The host ranges of plasmids of different MOB categories are diverse, and MOB is crucial for investigating plasmid mobilization, especially the transmission of resistance genes and virulence factors. However, MOB typing of plasmid metagenomic data is challenging due to the highly fragmented characteristics of metagenomic contigs. We developed MOBFinder, an 11-class classifier, for categorizing plasmid fragments into 10 MOB types and a nonmobilizable category. We first performed MOB typing to classify complete plasmid genomes according to relaxase information and then constructed an artificial benchmark dataset of plasmid metagenomic fragments (PMFs) from those complete plasmid genomes whose MOB types are well annotated. Next, based on natural language models, we used word vectors to characterize the PMFs. Several random forest classification models were trained and integrated to predict fragments of different lengths. Evaluating the tool using the benchmark dataset, we found that MOBFinder outperforms previous tools such as MOBscan and MOB-suite, with an overall accuracy approximately 59% higher than that of MOB-suite. Moreover, the balanced accuracy, harmonic mean, and F1-score reached up to 99% for some MOB types. When applied to a cohort of patients with type 2 diabetes (T2D), MOBFinder offered insights suggesting that the MOBF type plasmid, which is widely present in Escherichia and Klebsiella, and the MOBQ type plasmid might accelerate antibiotic resistance transmission in patients with T2D. To the best of our knowledge, MOBFinder is the first tool for MOB typing of PMFs. The tool is freely available at https://github.com/FengTaoSMU/MOBFinder.

Exploring the genetic diversity of Edwardsiella ictaluri in Vietnamese striped catfish (Pangasianodon hypophthalmus) farms over a 20-year period

Since first being identified in 1999, disease outbreaks from Edwardsiella ictaluri remain a significant health challenge for the farmed catfish sector in Vietnam. To better understand the population structure of E. ictaluri circulating in Vietnamese striped catfish (Pangasianodon hypophthalmus) farms, this study aimed to investigate the genetic diversity of 83 E. ictaluri isolates recovered from natural disease outbreaks occurring in the Mekong Delta region between 2001 and 2021. Pulsed-field gel electrophoresis resolved isolates into 15 pulsotypes following restriction digest with speI, with a Simpson’s diversity index of 0.8548. The genetic fingerprints of isolates recovered from recent outbreaks across different provinces were highly shared (100% similarity), suggesting clonal expansion within the production systems situated in the Mekong Delta region. Findings from this study also showed that E. ictaluri populations circulating this region have changed over time, as isolates recovered between 2001 and 2011 were genetically distinct from those recovered after 2017. Furthermore, isolates recovered after 2004 and 2010 lacked the virulence gene traD and a 5.8 Kb plasmid DNA fragment, respectively. Findings from this study illustrate the need for continued epidemiological monitoring of E. ictaluri to ensure prevention and treatment strategies in the Vietnamese striped catfish sector remain robust and effective.

Plasmid DNA extraction by double magnetic beads method and its application

Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.

The origin and evolution of IncF33 plasmids based on large-scale data sets.

To comprehensively understand the formation and evolution of IncF33 plasmids, a global collection of whole-genome sequences of 863 strains positive for IncF33 plasmid replicons was analyzed. The results showed that the IncF33 plasmids were mainly identified in Enterobacterales, of which Escherichia coli (86.44%) was the dominant host, followed by Salmonella (8.57%) and Klebsiella pneumoniae (3.48%). Salmonella ST11 and K. pneumoniae ST11 were common and mostly from humans. IncF33 plasmids were found worldwide but prevalent in Chinese farm animals, predominantly carrying antibiotic resistance genes such as blaCTX-M-55, fosA3, blaTEM-1B, rmtB, and floR. Comparative genomics analysis of 103 complete IncF33 plasmid sequences showed highly similar backbones except for 16 lacking partial backbone fragments. Variable regions were diverse, containing various antibiotic resistance genes, insertion sequences, or other plasmid fragments. They can be roughly divided into two sublines based on the production of different CTX-M enzymes. Similar IncF33 plasmids from different countries were identified. Some early IncF33 plasmids lacked part of their leader regions, which showed over 99% homology with early F2:A-:B- plasmids, indicating that leader regions of IncF33 likely came from F2:A-:B- plasmids. In addition, IncF33 plasmids cointegrating with other types of plasmids to form new cointegrate plasmids are increasing, making them more efficient in their dissemination and persistence in Enterobacterales, which could pose a significant threat to global public health. IMPORTANCE Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.

A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.

Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.

Emergence of transferable ceftazidime–avibactam resistance in KPC-producing Klebsiella pneumoniae due to a novel CMY AmpC β-lactamase in China

ObjectivesTo evaluate the molecular mechanisms of ceftazidime/avibactam (CAZ/AVI) resistance in six Klebsiella pneumoniae strains that co-produce K. pneumoniae carbapenemase (KPC)-2 and a novel variant of CMY cephalosporinase in a Chinese hospital. MethodsAntimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing (WGS) was performed to investigate potential resistance determinants. Plasmid conjugation, electroporation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) hybridization and cloning experiment were carried out to investigate the resistance plasmids and genes. ResultsA high level of CAZ/AVI resistance was observed in six KPC-Kp strains (MIC 128 mg/L). Five strains were isolated in 2015 and one in 2016, before the approval of CAZ/AVI in China. Sequence analysis indicated that all the strains belonged to sequence type (ST) 11 and uniformly carried a novel CMY AmpC β-lactamase gene, designated blaCMY-172. When compared with CMY-2, CMY-172 has a deletion of three consecutive amino acids (K290, V291 and A292) in the R2-loop region and a non-synonymous amino acid substitution at position 346 (N346I). The blaCMY-172-bearing plasmid, pKPCZA02_4, was 93.3 Kb, IncI1-I type, and conjugative; blaCMY-172 was located in an IS1294-mediated transposon. Plasmid conjugation and DNA fragment cloning proved that blaCMY-172 was responsible for CAZ/AVI resistance. ConclusionsOur study identified conjugative plasmid-mediated blaCMY-172 as a new mechanism for CAZ/AVI resistance in clinical KPC-Kp strains. Careful monitoring of CAZ/AVI susceptibility is imperative for preventing the spread of the resistance gene.

Population genomics study of Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacillus that causes vibriosis to human and aquatic products, including fish, shrimp and shellfish. It poses a threat to public health and causes enormous economic losses to the aquaculture industry. However, research on genetic diversity and pathogenicity-related genetic elements based on whole genome is still lacking. In this study, sixty-eight strains of V. alginolyticus were collected from four provinces of China and the whole genome sequences were obtained. Combined with 113 publicly available genome sequences downloaded from NCBI, we inferred the population structure of V. alginolyticus by using fineSTRUCTURE software, and identified the virulence and antibiotic resistance factors using the VFDB, CARD and ResFinder database. The results indicated that V. alginolyticus included two main lineages, named Lineage 1 and Lineage 2. Both lineages distributed in America and Asia, but all the European genomes were classified into Lineage 1. A single cross-ocean transmission event was inferred from one of the 12 identified clonal groups in our dataset. V. alginolyticus genome contains a variety of virulence factors, such as tlh, OmpU, and IlpA, etc. The distribution of virulence factors revealed no lineage-specificity, but some of which revealed differences in their geographical distribution. A lower frequency of VP1611, vcrD, vopD, fleR/flrC and a higher frequency of IlpA were observed in genomes of Europe than other continents. In China, a lower frequency of fleR/flrC, and no IlpA were observed in genomes from Guangxi province. Among the identified antibiotic resistance genes, TxR and fos are significantly enriched in Lineage 2. In addition, TxR is more common in genomes from Asia, compared with the American and European genomes. But in China, the frequency of TxR in Sichuan genomes is much lower than in other provinces. We also found that large fragments of plasmids or ICEs that carried multiple drug resistance genes were present in five V. alginolyticus genomes (VA24, VA28, 2014V-1011, ZJ-T and Vb1833). Based on population genomics analysis, our study delineated the population structure, distribution of virulence and antibiotic resistance related factors of V. alginolyticus, which lays a foundation for future study of genetic characters and pathogenesis mechanism of this pathogen and will improve the works on monitoring, prevention and control of this pathogen.

T-DNA regions from 350 Agrobacterium genomes: maps and phylogeny.

Analysis of 350 Agrobacterium wgs sequences reveals complex evolutionary history of T-DNA regions Virulent Agrobacterium strains transfer one or more plasmid DNA fragments to plant cells during a well-characterized transformation process. The transferred DNA sequences (T-DNA regions) are delimited by 25 nucleotide long conserved border sequences. Until recently, relatively few T-DNA regions were known. However, due to increased whole genome sequencing efforts, about 400 Agrobacterium sequences have now become available, 350 of which contain T-DNA regions. Detailed analysis identified 92 different T-DNA regions and several new T-DNA genes. T-DNA regions can be divided into three groups. I. Typical Agrobacterium rhizogenes T-DNA regions with rol genes. II. A large group of T-DNA regions with iaa and ipt genes, which can be further subdivided into seven subgroups. III. A small group of unusual T-DNA regions. The evolutionary relation between the T-DNA regions could not be completely elucidated, because of the lack of evolutionary intermediates. Several clusters of highly related structures suggest that evolution of T-DNA regions proceeds by slow, progressive evolution of gene sequences, accompanied by rapid changes in overall structure, due to recombination between T-DNA regions of different origins, and insertion of bacterial insertion sequences (IS). Divergence values for T-DNA genes suggest that they were recruited at different times in evolution. An attempt was made to link T-DNA region evolution to plasmid evolution. The present study provides a solid basis for further studies on T-DNA region diversity and evolution.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

Development of a Simple Method for the Specific Detection of Anthrax Antibody in Sera by Colored Slide Agglutination Test

This study was aimed to identify humoral immune response against anthrax vaccine in mice model by using colored slide agglutination test and detection of field infectivity of anthrax. The field isolates of B. anthracis (n=05) and F34 stern strain vaccine was isolated on agar plates in order to carry out the slide agglutination test. The field isolates of B. anthracis and vaccine bacteria grew on PLET agar medium produced roughly circular and creamy white colonies with ground glass appearance. The bacteria on sheep blood agar media produced rough, sticky, white-gray non hemolytic colonies. Colony polymerase chain reaction (PCR) protocol was adapted to detect fragments of pX01 (596bp) and pX02 (777bp) plasmid of virulent field isolates of B. anthracis. The fragment of pX01 plasmid was only detected in vaccine bacteria. Growth of a field isolate and a vaccine bacteria were colored with crystal violet and used in slide agglutination test to detect anthrax antibodies. The anti-anthrax antibody was prepared by immunizing female mice with 100ül anthrax vaccine through subcutaneous route. Tail bleed were collected on day 0, 30, 60, 120 and 180 of immunization. Cardiac bleed was collected on day 180 of immunization for extensive study. 25μl of diluted (1:10, 1:20, 1:50 and 1:100) antisera and 25μl colored antigen was mixed together onto a clean slide at room temperature and the results was red following 5min, 10mins, 15mins and 20mins of reaction. Unstained antigens and non-immunized sera from the mice were used as control. Results of slide agglutination test showed that the colored vaccine bacteria and field isolates clumped the mice anti-antisera (day 30, 60, and 120) at 1:20 dilution as seen in naked eye but the reaction was seen only at 1:10 dilution while colorless antigens were used. Under microscopic investigation of slide agglutination test, the reaction was read up to 1:100 dilutions with the sera collected at day 30, 60, 120 and 180 of immunization. The Anthrax Sterne strain vaccine induced anti-anthrax immunity in mice that was detected until day 180 of immunization. The clumping reaction was distinct while colored anthrax antigen was used in slide agglutination tests. The colored slide agglutination tests protocol developed in this study can be used to detect anti-anthrax immune response and anthrax bacteria in the field condition with minimum laboratory facilities.
 Res. Agric., Livest. Fish.7(3): 497-506, December 2020

Identification of the conjugative and mobilizable plasmid fragments in the plasmidome using sequence signatures.

Plasmids are the key element in horizontal gene transfer in the microbial community. Recently, a large number of experimental and computational methods have been developed to obtain the plasmidomes of microbial communities. Distinguishing transmissible plasmid sequences, which are derived from conjugative or at least mobilizable plasmids, from non-transmissible plasmid sequences in the plasmidome is essential for understanding the diversity of plasmids and how they regulate the microbial community. Unfortunately, due to the highly fragmented characteristics of DNA sequences in the plasmidome, effective identification methods are lacking. In this work, we used information entropy from information theory to assess the randomness of synonymous codon usage over 4424 plasmid genomes. The results showed that for all amino acids, the choice of a synonymous codon in conjugative and mobilizable plasmids is more random than that in non-transmissible plasmids, indicating that transmissible plasmids have different sequence signatures from non-transmissible plasmids. Inspired by this phenomenon, we further developed a novel algorithm named PlasTrans. PlasTrans takes the triplet code sequences and base sequences of plasmid DNA fragments as input and uses the convolutional neural network of the deep learning technique to further extract the more complex signatures of the plasmid sequences and identify the conjugative and mobilizable DNA fragments. Tests showed that PlasTrans could achieve an AUC of as high as 84–91%, even though the fragments only contained hundreds of base pairs. To the best of our knowledge, this is the first quantitative analysis of the difference in sequence signatures between transmissible and non-transmissible plasmids, and we developed the first tool to perform transferability annotation for DNA fragments in the plasmidome. We expect that PlasTrans will be a useful tool for researchers who analyse the properties of novel plasmids in the microbial community and horizontal gene transfer, especially the spread of resistance genes and virulence factors associated with plasmids. PlasTrans is freely available via https://github.com/zhenchengfang/PlasTrans

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.

IncN3 and IncHI2 plasmids with an In1763 integron carrying bla IMP-1 in carbapenem-resistant Enterobacterales clinical isolates from the UK.

Introduction. Imipenemase (IMP) carbapenemase genes are relatively rare among Enterobacterales in the UK. Emergence in multiple hospitals, in different strains and species, prompted an investigation into their genetic context.Aim. Our goal was to identify and describe the elements carrying bla IMP genes in a variety of Enterobacterales from five hospitals in the UK.Methodology. Long-read nanopore sequencing was carried out on 18 IMP-positive isolates belonging to 6 species. The locations of the bla IMP genes and other associated genetic elements were identified.Results. Ten out of 18 isolates carried bla IMP-1 on an IncN3 plasmid (52-57 kb) in an In1763 class 1 integron. These plasmids also contained genes encoding type IV secretion and conjugal transfer proteins. Five out of 18 isolates carried bla IMP-1 in the same In1763 integron in much larger IncHI2 plasmids. A further isolate carried the In1763 integron in a chromosomally located plasmid fragment. Two isolates carried bla IMP-4 in IncHI2 plasmids. The isolates included three representatives of sequence type 20 of Klebsiella pneumoniae, with one carrying a distinct plasmid from the other two.Conclusion. Highly similar IncN3 plasmids were found in a range of Enterobacterales, mostly K. pneumoniae and the Enterobacter cloacae complex, from three of four London hospitals, with the same In1763 integron carrying bla IMP-1 also being found in IncHI2 plasmids and chromosomally. These plasmids carried multiple elements facilitating self-transmission. Strain typing alone was not sufficient to investigate cross-infection among this set of isolates, many of which appeared to be unrelated until plasmid analysis was undertaken, and vice versa.

Development of a multiplex RT-PCR assay for the routine detection of seven RNA viruses in Apis mellifera

Colony losses in apiaries are frequently one of the most important problems in beekeeping. Colony loss is multifactorial with many reported disorders, Colony Collapse Disorder (CCD), is an increasingly recognised phenomenon which is thought to be caused by many pathogens, including viruses. The aim of this study was to develop a multiplex RT-PCR (mRT-PCR) test to obtain faster results in routine diagnostic laboratories for seven crucial bee viruses. Specific primers for seven RNA viruses, including Israeli acute bee paralysis virus (IAPV), deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), black queen cell virus (BQCV), kashmir bee virus (KBV) and chronic paralysis virus (CBPV), were used for testing procedure. The mRT-PCR assay can amplify seven plasmid DNA fragments from the pooled viral genomes and it was shown to be sensitive because virus copy numbers were detected to be 104 copies/μl when log10 serial dilutions were performed for the optimized mRT- PCR method. It is concluded that, mRT-PCR test can be used in routine analysis because this assay can perform specific, sensitive and reliable results also achieves economic gains and time due to detecting seven viral agents simultaneously.

The <i>rolC</i> gene of agrobacteria: towards the understanding of its functions

Agrobacterium rhizogenesConn is a soil bacterium, which can transform plants by inserting a plasmid fragment into the plant genome. This fragment contains a “root locus”: four genes that cause root overgrowth of the transformed plant, the so-called “hairy root syndrome”. The most studied gene of the root locus isrolC. For more than 30 years of research on this gene, data have been obtained on its expression, protein localization and putative functions of the protein as well as on its effect on plant morphology and biochemistry. TherolCtransformation leads to multiple morphological effects, most common among which are dwarfism, bushiness, and a change in the shape of the leaf blade. Such specific plant reactions are associated with changes in hormone balance under the influence ofrolC. The levels of auxins, cytokinins, and abscisic acid do change in transformed plants, but no regularities have been revealed. Also, the signaling pathways ofrolCaffecting the hormonal system of plants are not established. Morphogenetic effects can occur in varying degrees depending on the promoter under which therolCworks. A constitutive promoter usually leads to a more pronounced effect when compared to a gene that operates under a native promoter. Secondary plant metabolism is also affected byrolC. The synthesis of various metabolites is amplified in transformants, and, in contrast to morphological effects, this biochemical effect does not depend on the promoter. Some secondary metabolites are associated with the plant defense system; thus,rolCis able to indirectly influence this aspect of plant physiology. This review summarizes the results of therolCgene studies in plants. The authors formulate the main hypotheses regarding the mechanisms of the gene in order to promote our understanding of its function in plants.

Genomic epidemiology of emerging ESBL-producing Salmonella Kentucky bla CTX-M-14b in Europe

ABSTRACT Global dissemination of ciprofloxacin-resistant Salmonella Kentucky has been observed over the past decades. In recent years, there have been reports of extended-spectrum β-lactamase (ESBL) producing S. Kentucky. Routine surveillance at the European Centre for Disease Prevention and Control (ECDC) detected cases with a ciprofloxacin-resistant S. Kentucky with the ESBL-gene bla CTX-M-14b. Ensuing research identified 78 cases in 2013–2018 in eight European countries. Compared to other S. Kentucky and non-typhoidal Salmonella infections, reported to the European Surveillance System, these cases were more likely to be elderly and to present urinary-tract infections. Bayesian time-scaled phylogeny on whole genome sequences of isolates from these cases and supplementary isolates from public sequence databases was used to infer the origin and spread of this clone. We dated the origin of the bla CTX-M-14b clone to approximately 2005 in Northern Africa, most likely in Egypt. The geographic origin predicted by the phylogenetic analysis is consistent with the patients’ travel history. Next to multiple introductions of the clone to Europe from Egypt, our analysis suggests that in some parts of Europe the clone might have formed a stable population, from which further spread has occurred. Comparative genomics indicated that the bla CTX-M-14b gene is present on the bacterial chromosome, within the type VI secretion system region. The bla CTX-M-14b gene is integrated downstream of the hcp1 gene, on a 2854 bp plasmid fragment containing also ISEcp1. This is the first report of a chromosomally integrated CTX-M gene in Salmonella spp. in Europe, previous studies having identified similar genes only on plasmids.

High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach.

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR. Unfortunately, this approach is very often coupled with PCR artifacts which compromise overall efficiency of site-directed mutagenesis. To reduce the failure rate due to the PCR artifacts, we have set up a high-throughput mutagenesis protocol based on a two-fragment PCR approach. To this end, each mutation is introduced in two separate PCRs resulting in two linear fragments of the mutated plasmid. In the next steps, the PCR template is digested and the two matching plasmid fragments are joined together using Gibson assembly. Separating the corresponding mutagenic primers into two different PCRs decreases a number of artifacts and thus increases overall cloning efficiency. Furthermore, free software that we developed facilitates both high-throughput primer design and analysis of sequencing results. Overall, this protocol enabled us to efficiently produce several alanine-scanning libraries of 400 single-point mutations with complete coverage of the protein sequence.

High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.