Characterization and transmission of plasmid-mediated multidrug resistance in foodborne Vibrio parahaemolyticus.

Objectives: The purpose of this study was to determine the structural features and transferability of the multidrug-resistance (MDR) plasmid, and resistance phenotypes for the tested antimicrobials in foodborne Vibrio parahaemolyticus. Methods: Plasmids were isolated from a V. parahaemolyticus strain of seafood origin, then sequenced using the Illumina NovaSeq 6000 and PacBio Sequel II sequencing platforms to obtain the complete genome data. Characterization of the MDR plasmid pVP52-1, including determination of antimicrobial resistance genes (ARGs), plasmid incompatibility groups, and transferability, was carried out. Results: V. parahaemolyticus strain NJIFDCVp52 contained two circular chromosomes and two circular plasmids (pVP52-1 and pVP52-2). Plasmid typing indicated that pVP52-1 belonged to the incompatibility group IncA/C2 and the sequence type pST3. pVP52-1 carried 12 different ARGs, an IS110-composite transposon consisting of aac(6')-Ib-cr, qnrVC1, aac(6')-Ib, dfrA14, and the IS26-mphA-IS6100 unit flanked by inverted sequences of IS5075 and IS4321. pVP52-2 carried no ARGs. A plasmid elimination assay showed that only pVP52-1 and its ARGs were lost, the loss of resistance to several antimicrobials, causing a change from the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-trimethoprim/sulfamethoxazole resistance pattern to the ampicillin resistance pattern. In accordance, a conjugation transfer assay showed that only pVP52-1 and its ARGs were horizontally transferred, leading to increased antimicrobial resistance in Escherichia coli strain EC600, causing a change from the ampicillin-nalidixic acid resistance pattern to the ampicillin-ampicillin/sulbactam-cefazolin-cefoxitin-ceftazidime-cefotaxime-imipenem-nalidixic acid-chloramphenicol-tetracycline-trimethoprim/sulfamethoxazole-azithromycin resistance pattern. Further transferability experiments revealed that pVP52-1 could be transferred to other enterobacterial strains of E. coli and Salmonella. Discussion: This study emphasizes the urgent need for continued surveillance of resistance plasmids and changes in antimicrobial resistance profiles among the V. parahaemolyticus population.

A self-complementary AAV proviral plasmid that reduces cross-packaging and ITR promoter activity in AAV vector preparations.

Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences, (2) increases correctly packaged AAV payloads, and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.

Investigation of the impact of widely used pesticides on conjugative transfer of multidrug resistance plasmids.

Plasmid-mediated conjugative transfer has emerged as a major driver accounting for the dissemination of antibiotic resistance genes (ARGs). In addition to the use of antimicrobial agents, there is growing evidence that non-antibiotic factors also play an important role. Pesticides are widely used to protect crops against vectors of diseases, and are indispensable agents in agricultural production, whereas the impact of pesticide pollution on the transmission of antimicrobial resistance remains poorly understood. Here we reveal that the pesticides at environmentally relevant concentrations, especially cyromazine (Cyr) and kresoxim-methyl (Kre), greatly facilitate the conjugative transfer of antibiotic-resistance plasmids carrying clinically important ARGs. Mechanistic studies indicate that Cyr and Kre treatments trigger reactive oxygen species (ROS) production and SOS response, increase membrane permeability, upregulate bacterial proton motive force (PMF) and promote ATP supply. Further non-targeted metabolomics and biochemical analysis demonstrate that the addition of Cyr and Kre accelerates tricarboxylic acid (TCA) cycle and electron transport chain (ETC), thereby activating bacterial energy metabolism. In the constructed soil model, we prove that two pesticides contribute to the dissemination of resistance plasmids in the soil microbiota. 16S rRNA sequencing analyses indicate that pesticides alter transconjugant microbial communities, and enable more opportunistic pathogens, such as Pseudomonas and Enterobacter, to acquire the multidrug resistance plasmids. Collectively, our work indicates the potential risk in accelerating the spread of antimicrobial resistance owing to pesticide pollution, highlighting the importance of continuous surveillance of pesticide residues in complex environmental settings.

Identification and Characterization of an R-M System in P. denitrificans DYTN-1 to Improve the Plasmid Conjugation Transfer Efficiency.

Paracoccus denitrificans has been identified as a representative strain with heterotrophic nitrificationaerobic denitrification capabilities (HN-AD), and demonstrates strong denitrification proficiency. Previously, we isolated the DYTN-1 strain from activated sludge, and it has showcased remarkable nitrogen removal abilities and genetic editability, which positions P. denitrificans DYTN-1 as a promising chassis cell for synthetic biology engineering, with versatile pollutant degradation capabilities. However, the strain's low stability in plasmid conjugation transfer efficiency (PCTE) hampers gene editing efficacy, and is attributed to its restriction modification system (R-M system). To overcome this limitation, we characterized the R-M system in P. denitrificans DYTN-1 and identified a DNA endonuclease and 13 DNA methylases, with the DNA endonuclease identified as HNH endonuclease. Subsequently, we developed a plasmid artificial modification approach to enhance conjugation transfer efficiency, which resulted in a remarkable 44-fold improvement in single colony production. This was accompanied by an increase in the frequency of positive colonies from 33.3% to 100%. Simultaneously, we cloned, expressed, and characterized the speculative HNH endonuclease capable of degrading unmethylated DNA at 30°C without specific cutting site preference. Notably, the impact of DNA methylase M9 modification on the plasmid was discovered, significantly impeding the cutting efficiency of the HNH endonuclease. This revelation unveils a novel R-M system in P. denitrificans and sheds light on protective mechanisms employed against exogenous DNA invasion. These findings pave the way for future engineering endeavors aimed at enhancing the DNA editability of P. denitrificans.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Horizontal plasmid transfer promotes antibiotic resistance in selected bacteria in Chinese frog farms.

The emergence and dissemination of antibiotic resistance genes (ARGs) in the ecosystem are global public health concerns. One Health emphasizes the interconnectivity between different habitats and seeks to optimize animal, human, and environmental health. However, information on the dissemination of antibiotic resistance genes (ARGs) within complex microbiomes in natural habitats is scarce. We investigated the prevalence of antibiotic resistant bacteria (ARB) and the spread of ARGs in intensive bullfrog (Rana catesbeiana) farms in the Shantou area of China. Antibiotic susceptibilities of 361 strains, combined with microbiome analyses, revealed Escherichia coli, Edwardsiella tarda, Citrobacter and Klebsiella sp. as prevalent multidrug resistant bacteria on these farms. Whole genome sequencing of 95 ARB identified 250 large plasmids that harbored a wide range of ARGs. Plasmid sequences and sediment metagenomes revealed an abundance of tetA, sul1, and aph(3″)-Ib ARGs. Notably, antibiotic resistance (against 15 antibiotics) highly correlated with plasmid-borne rather than chromosome-borne ARGs. Based on sequence similarities, most plasmids (62%) fell into 32 distinct groups, indicating a potential for horizontal plasmid transfer (HPT) within the frog farm microbiome. HPT was confirmed in inter- and intra-species conjugation experiments. Furthermore, identical mobile ARGs, flanked by mobile genetic elements (MGEs), were found in different locations on the same plasmid, or on different plasmids residing in the same or different hosts. Our results suggest a synergy between MGEs and HPT to facilitate ARGs dissemination in frog farms. Mining public databases retrieved similar plasmids from different bacterial species found in other environmental niches globally. Our findings underscore the importance of HPT in mediating the spread of ARGs in frog farms and other microbiomes of the ecosystem.

An idea to explore: A systematic approach for solving plasmid double-digest puzzles.

A common exercise given to students early in a molecular biology course is the creation of a restriction map of a plasmid "digested" by two restriction enzymes (RE). Meanwhile, students have learned from an early age about the properties and analyses of circles in their mathematics courses. But it is rare for students to learn using puzzle-based assignments at the intersection of molecular biology and mathematics. Therefore, we should present students with a puzzle that allows them to combine knowledge and skills from these seemingly disconnected disciplines. Here, we present a method for analyzing RE digests of circular plasmids using basic geometric principles.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus.

BACKGROUND: To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process. RESULTS: Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R-M- without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained. CONCLUSIONS: The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.

Gemogenovatucel-T (Vigil): bi-shRNA plasmid-based targeted immunotherapy.

We describe in this review the historical evidence leading up to the concept and design of Vigil and subsequent clinical applications including safety and efficacy in a randomized, controlled Phase IIB trial. Vigil (gemogenovatucel-T) is a unique triple function targeted immunotherapy that demonstrates preclinical and clinical systemic anticancer activity. Construction of Vigil involves harvest of autologous malignant tissue for neoantigen targeting (ideally containing clonal neoantigens) followed by a two-day process involving transfection with a plasmid to provide a permissive 'training environment' for the patient's immune system. Transfected plasmid components contain an expressive human GMCSF DNA segment to enhance anticancer immune functional response and a second component expressing bi-shRNAfurin which reduces TGFß isomers (TGFß1 and TGFß2) thereby reducing cancer inhibition of the targeted immune response. Results generated to date justify advancement to confirmatory clinical trials supporting product regulatory approval.

Vigil is an anticancer treatment that employs three methods of enhancing the body's immune system to identify and kill cancer cells. The construction of Vigil involves cancer cells from the same person being treated (personalized therapy) in combination with added anticancer genetic signals to enhance the number and function anti-anticancer immune cells and to guide the immune cells to the cancer and not to normal organs of the body. In this manner, an army of immune cells are created that can move to attacking the cancer using blood vessels to get to the cancer anywhere it tries to grow in the body. One study (Phase I) performed with this product to determine safety and dose range demonstrated an optimal dose and schedule. Another study (Phase IIA) showed initial clinical benefit. A third more complex study (Phase IIB) in patients treated with Vigil compared with standard of care without Vigil demonstrated the ability to prolong the patients life and time without their cancer getting worse without any significant side effects associated with the treatment in a unique subset of ovarian cancer patients, those with the ability to repair their DNA. Based on the composite of these results, Vigil is an attractive targeted immunotherapy justified for late-stage clinical testing.

The virulence plasmid associated with AHPND in shrimp appears to have originated from Vibrio owensii through a process of homologous recombination of parental plasmids and the transposable insertion of two large fragments.

Acute hepatopancreatic necrosis disease (AHPND) is a highly contagious and lethal disease of shrimp caused by Vibrio strains carrying the virulence plasmid (pAHPND) containing the pirAB virulence genes. Through analysis of plasmid sequence similarity, clustering, and phylogeny, a horizontal transfer element similar to IS91 was discovered within the pAHPND plasmid. Additionally, two distinct clades of plasmids related to pAHPND (designated as pAHPND-r1 and pAHPND-r2) were identified, which may serve as potential parental plasmids for pAHPND. The available evidence, including the difference in G+C content between the plasmid and its host, codon usage preference, and plasmid recombination event prediction, suggests that the formation of the pAHPND plasmid in the Vibrio owensii strain was likely due to the synergistic effect of the recombinase RecA and the associated proteins RecBCD on the pAHPND-r1 and pAHPND-r2, resulting in the recombination and formation of the precursor plasmid for pAHPND (pre-pAHPND). The emergence of pAHPND was found to be a result of successive insertions of the horizontal transfer elements of pirAB-Tn903 and IS91-like segment, which led to the deletion of one third of the pre-pAHPND. This plasmid was then able to spread horizontally to other Vibrio strains, contributing to the epidemics of AHPND. These findings shed light on previously unknown mechanisms involved in the emergence of pAHPND and improve our understanding of the disease's spread.

NHEJ and HDR can occur simultaneously during gene integration into the genome of Aspergillus niger.

Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two mechanisms in filamentous fungi to repair DNA damages. NHEJ is the dominant response pathway to rapidly join DNA double-strand breaks, but often leads to insertions or deletions. On the other hand, HDR is more precise and utilizes a homologous DNA template to restore the damaged sequence. Both types are exploited in genetic engineering approaches ranging from knock-out mutations to precise sequence modifications.In this study, we evaluated the efficiency of an HDR based gene integration system designed for the pyrG locus of Aspergillus niger. While gene integration was achieved at a rate of 91.4%, we also discovered a mixed-type repair (MTR) mechanism with simultaneous repair of a Cas9-mediated double-strand break by both NHEJ and HDR. In 20.3% of the analyzed transformants the donor DNA was integrated by NHEJ at the 3' end and by HDR at the 5' end of the double-strand break. Furthermore, sequencing of the locus revealed different DNA repair mechanisms at the site of the NHEJ event.Together, the results support the applicability of the genome integration system and a novel DNA repair type with implication on the diversity of genetic modifications in filamentous fungi.

Small RNA GadY in Escherichia coli enhances conjugation system of IncP-1 by targeting SdiA.

Plasmid-mediated conjugation is a common mechanism for most bacteria to transfer antibiotic resistance genes (ARGs). The conjugative transfer of ARGs is emerging as a major threat to human beings. Although several transfer-related factors are known to regulate this process, small RNAs (sRNAs)-based regulatory roles remain to be clarified. Here, the Hfq-binding sRNA GadY in donor strain Escherichia coli (E. coli) SM10λπ was identified as a new regulator for bacterial conjugation. Two conjugation models established in our previous studies were used, which SM10λπ carrying a chromosomally integrated IncP-1α plasmid RP4 and a mobilizable plasmid pUCP24T served as donor cells, and P. aeruginosa PAO1 or E. coli EC600 as the recipients. GadY was found to promote SM10λπ-PAO1 conjugation by base-pairing with its target mRNA SdiA, an orphan LuxR-type receptor that responds to exogenous N-acylated homoserine lactones (AHLs). However, SM10λπ-EC600 conjugation was not affected due to EC600 lacking AHLs synthase. It indicates that the effects of GadY on conjugation depended on AHLs-SdiA signalling. Further study found GadY bound SdiA to negatively regulate the global RP4 repressors KorA and KorB. When under ciprofloxacin or levofloxacin treatment, GadY expression in donor strain was enhanced, and it positively regulated quinolone-induced SM10λπ-PAO1 conjugation. Thus, our study provides a novel role for sRNA GadY in regulating plasmid-mediated conjugation, which helps us better understand bacterial conjugation to counter antibiotic resistance.

Enterobacter adelaidei sp. nov. Isolation of an extensively drug resistant strain from hospital wastewater in Australia and the global distribution of the species.

BACKGROUND: Enterobacter species are included among the normal human gut microflora and persist in a diverse range of other environmental niches. They have become important opportunistic nosocomial pathogens known to harbour plasmid-mediated multi-class antimicrobial resistance (AMR) determinants. Global AMR surveillance of Enterobacterales isolates shows the genus is second to Klebsiella in terms of frequency of carbapenem resistance. Enterobacter taxonomy is confusing and standard species identification methods are largely inaccurate or insufficient. There are currently 27 named species and a total of 46 taxa in the genus distinguishable via average nucleotide identity (ANI) calculation between pairs of genomic sequences. Here we describe an Enterobacter strain, ECC3473, isolated from the wastewater of an Australian hospital whose species could not be determined by standard methods nor by ribosomal RNA gene multi-locus typing. AIM: To characterise ECC3473 in terms of phenotypic and genotypic antimicrobial resistance, biochemical characteristics and taxonomy as well as to determine the global distribution of the novel species to which it belongs. METHODS: Standard broth dilution and disk diffusion were used to determine phenotypic AMR. The strain's complete genome, including plasmids, was obtained following long- and short read sequencing and a novel long/short read hybrid assembly and polishing, and the genomic basis of AMR was determined. Phylogenomic analysis and quantitative measures of relatedness (ANI, digital DNA-DNA hybridisation, and difference in G+C content) were used to study the taxonomic relationship between ECC3473 and Enterobacter type-strains. NCBI and PubMLST databases and the literature were searched for additional members of the novel species to determine its global distribution. RESULTS: ECC3473 is one of 21 strains isolated globally belonging to a novel Enterobacter species for which the name, Enterobacter adelaidei sp. nov. is proposed. The novel species was found to be resilient in its capacity to persist in contaminated water and adaptable in its ability to accumulate multiple transmissible AMR determinants. CONCLUSION: E. adelaidei sp. nov. may become increasingly important to the dissemination of AMR.

An outbreak of severe or lethal infections by a multidrug-resistant Acinetobacter baumannii ST126 strain carrying a plasmid with blaNDM-1 and blaOXA-58 carbapenemases.

Acinetobacter baumannii poses a significant health threat because of its frequent implications in hospital outbreaks and multidrug resistance (MDR). Here, we studied four A. baumannii isolates recovered during a hospital outbreak of severe or fatal cases to elucidate their diversity and factors contributing to their increased virulence and antibiotic resistance. The isolates were identified using MALDI-ToF and characterized using comparative genomics, PCR, and antimicrobial susceptibility tests. They were classified as ST126 and exhibited fewer than five chromosomal single-nucleotide variants and the same extrachromosomal content, indicating that they are a single strain (A. baumannii AB01). A. baumannii AB01 showed an MDR phenotype that could be linked to the carriage of parC and gyrA mutations, efflux transporters, aminoglycoside resistance genes, a class C beta-lactamase, and three carbapenemases, some of which are encoded on a 72 kb plasmid. ST126 is infrequent and has not been reported in Latin America, and our genomic data indicate a plausible origin for A. baumannii AB01 within the Pan Pacific region.

Molecular Characterization of a KPC-2- and NDM-1-Producing Klebsiella michiganensis Clinical Isolate in Cerebrospinal Fluid.

Objective: Klebsiella michiganensis is an emerging pathogen. In this context, we characterised a strain fxq isolated from a cerebrospinal fluid specimen of a patient with tentorial meningioma, and the K. michiganensis isolate produced carbapenemases of KPC and NDM types. Methods: The Phoenix 100 Automated Microbiology System, MALDI-TOF and whole-genome sequencing were used to identify the species. Anti-microbial susceptibility testing was also conducted with the Phoenix 100. The plasmid locations of the bla KPC-2 and bla NDM-1 genes were determined by S1-nuclease pulsed-field gel electrophoresis and Southern blot. The transfer capacity of plasmids carrying bla KPC-2 and bla NDM-1 was investigated by conjugation experiments, and the resistance plasmid stability was evaluated by culture and subculture. K. michiganensis subtypes were identified by multi-locus sequence typing. We performed whole-genome sequencing to confirm species, characterise plasmids and analyse core genes. Results: fxq was originally identified as Klebsiella oxytoca and showed resistance to imipenem and meropenem, but whole-genome sequencing identified it to be K. michiganensis. The strain fxq belonged to the novel sequence type 202 (ST202) and carried the bla KPC-2 and bla NDM-1 genes located on the pB_KPC InFIA and pE_NDM IncU plasmids, respectively. The bla KPC-2-carrying plasmid was successfully transferred to Escherichia coli EC600 by conjugation, whereas the bla NDM-1 gene on the pE_NDM plasmid was not. The pB_KPC and pE_NDM plasmids demonstrated high stability. Conclusion: This work is the first report on a carbapenem-resistant clinical isolate K. michiganensis ST202 harbouring the bla KPC-2 and bla NDM-1 genes encoded by the IncFIA and IncU plasmids, respectively.

Extraction of high-molecular-weight DNA from Streptococcus spp. for nanopore sequencing in resource-limited settings.

The long-read sequencing platform MinION, developed by Oxford Nanopore Technologies, enables the sequencing of bacterial genomes in resource-limited settings, such as field conditions or low- and middle-income countries. For this purpose, protocols for extracting high-molecular-weight DNA using nonhazardous, inexpensive reagents and equipment are needed, and some methods have been developed for gram-negative bacteria. However, we found that without modification, these protocols are unsuitable for gram-positive Streptococcus spp., a major threat to fish farming and food security in low- and middle-income countries. Multiple approaches were evaluated, and the most effective was an extraction method using lysozyme, sodium dodecyl sulfate, and proteinase K for lysis of bacterial cells and magnetic beads for DNA recovery. We optimized the method to consistently achieve sufficient yields of pure high-molecular-weight DNA with minimal reagents and time and developed a version of the protocol which can be performed without a centrifuge or electrical power. The suitability of the method was verified by MinION sequencing and assembly of 12 genomes of epidemiologically diverse fish-pathogenic Streptococcus iniae and Streptococcus agalactiae isolates. The combination of effective high-molecular-weight DNA extraction and MinION sequencing enabled the discovery of a naturally occurring 15 kb low-copy number mobilizable plasmid in S. iniae, which we name pSI1. We expect that our resource-limited settings-adapted protocol for high-molecular-weight DNA extraction could be implemented successfully for similarly recalcitrant-to-lysis gram-positive bacteria, and it represents a method of choice for MinION-based disease diagnostics in low- and middle-income countries.

DNA Electrotransfer Regulates Molecular Functions in Skeletal Muscle.

Background: Tissues, such as skeletal muscle, have been targeted for the delivery of plasmid DNA (pDNA) encoding vaccines and therapeutics. The application of electric pulses (electroporation or electrotransfer) increases cell membrane permeability to enhance plasmid delivery and expression. However, the molecular effects of DNA electrotransfer on the muscle tissue are poorly characterized. Materials and Methods: Four hours after intramuscular plasmid electrotransfer, we evaluated gene expression changes by RNA sequencing. Differentially expressed genes were analyzed by gene ontology (GO) pathway enrichment analysis. Results: GO analysis highlighted many enriched molecular functions. The terms regulated by pulse application were related to muscle stress, the cytoskeleton and inflammation. The terms regulated by pDNA injection were related to a DNA-directed response and its control. Several terms regulated by pDNA electrotransfer were similar to those regulated by pulse application. However, the terms related to pDNA injection differed, focusing on entry of the plasmid into the cells and intracellular trafficking. Conclusion: Each muscle stimulus resulted in specific regulated molecular functions. Identifying the unique intrinsic molecular changes driven by intramuscular DNA electrotransfer will aid in the design of preventative and therapeutic gene therapies.

Beta-blocker drives the conjugative transfer of multidrug resistance genes in pure and complex biological systems.

Drug resistance poses a high risk to human health. Extensive use of non-antibiotic drugs contributes to antibiotic resistance genes (ARGs) transfer. However, how they affect the spread of broad-host plasmids in complex biological systems remains unknown. This study investigated the effect of metoprolol on the transfer frequency and host range of ARGs in both intrageneric and intergeneric pure culture systems, as well as in anammox microbiome. The results showed that environmental concentrations of metoprolol significantly promoted the intrageneric and intergeneric conjugative transfer. Initially, metoprolol induced excessive oxidative stress, resulting in high cell membrane permeability and bacterial SOS response. Meanwhile, more pili formation increased the adhesion and contact between bacteria, and the abundance of conjugation-related genes also increased significantly. Activation of the electron transport chain provided more ATP for this energy-consuming process. The underlying mechanism was further verified in the complex anammox conjugative system. Metoprolol induced the enrichment of ARGs and mobile genetic elements. The enhanced bacterial interaction and energy generation facilitated the high conjugative transfer frequency of ARGs. In addition, plasmid-borne ARGs tended to transfer to opportunistic pathogens. This work raises public concerns about the health and ecological risks of non-antibiotic drugs.

Complete genome sequences of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs.

We report the complete genomes of Geobacillus stearothermophilus strains EF60045 and SJEF4-2 from Korean hot springs, with 3,769 and 3,625 thermophilic genes, respectively. G. stearothermophilus EF60045 shows four methylation patterns. G. stearothermophilus SJEF4-2 harbors three plasmids. These findings enhance understanding of Geobacillus strains, aiding in their development as microbial platform hosts.