Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

BACKGROUND: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. METHODS: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). RESULTS: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. CONCLUSIONS: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Genomic and functional divergence of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals: insights from global and local scales.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease worldwide and is characterized by a complex interplay with skin microbiota, with Staphylococcus aureus often abnormally more abundant in AD patients than in healthy individuals (HE). S. aureus harbors diverse strains with varied genetic compositions and functionalities, which exhibit differential connections with the severity of AD. However, the differences in S. aureus strains between AD and HE remain unclear, with most variations seen at a specific geographic level, implying spontaneous adaptations rather than systematic distinctions. This study presents genomic and functional differences between these S. aureus strains from AD and HE on both global and local levels. We observed reduced gene content diversity but increased functional variation in the global AD-associated strains. Two additional AD-dominant clusters emerged, with Cluster 1 enriched in transposases and Cluster 2 showcasing genes linked to adaptability and antibiotic resistance. Particularly, robust evidence illustrates that the lantibiotic operon of S. aureus, involved in the biosynthesis of lantibiotics, was acquired via horizontal gene transfer from environmental bacteria. Comparisons of the gene abundance profiles in functional categories also indicate limited zoonotic potential between human and animal isolates. Local analysis mirrored global gene diversity but showed distinct functional variations between AD and HE strains. Overall, this research provides foundational insights into the genomic evolution, adaptability, and antibiotic resistance of S. aureus, with significant implications for clinical microbiology.IMPORTANCEOur study uncovers significant genomic variations in Staphylococcus aureus strains associated with atopic dermatitis. We observed adaptive evolution tailored to the disease microenvironment, characterized by a smaller pan-genome than strains from healthy skin both on the global and local levels. Key functional categories driving strain diversification include "replication and repair" and "transporters," with transposases being pivotal. Interestingly, the local strains predominantly featured metal-related genes, whereas global ones emphasized antimicrobial resistances, signifying scale-dependent diversification nuances. We also pinpointed horizontal gene transfer events, indicating interactions between human-associated and environmental bacteria. These insights expand our comprehension of S. aureus's genetic adaptation in atopic dermatitis, yielding valuable implications for clinical approaches.

CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (CRISPR)-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a One Health approach.IMPORTANCEAntibiotic resistance is a growing global health crisis in human and veterinary medicine. Previous work has shown technologies based on CRISPR-Cas-a bacterial defense system-to be effective in tackling antibiotic resistance. Here we test if CRISPR-Cas is present and effective in agricultural niches, specifically in the ubiquitously present bacterium, Enterococcus faecalis. We show that CRISPR-Cas is both prevalent and functional in manure and has the potential to be used to specifically kill bacteria carrying antibiotic resistance genes. This study demonstrates the utility of CRISPR-Cas-based strategies for control of antibiotic resistance in agricultural settings.

Introducing Casbah, Kronus, and MmasiCarm, Members of the Mycobacteriophage Subcluster B3.

Background: As part of a large science education effort, bacteriophages that lyse Mycobacterium smegmatis mc2155 continue to be discovered. Materials and Methods: Phages were isolated from soil samples from urban sites in the Northeastern United States. Their genomes were sequenced, assembled, and bioinformatically compared. Results: Three lytic siphoviruses belonging to subcluster B3 with high similarity to each other and other B3 mycobacteriophages were isolated. These phages contain double-stranded DNA genomes (68,754 to 69,495 bp) with high GC content (67.4-67.5%) and 102-104 putative protein coding genes. Notable features include a HicA-like toxin and 33 genes exclusive to subcluster B3. One phage had an intein in its terminase sequence. Conclusions: Genomic analyses of these phages provide insights into genome evolution and horizontal gene transfer (HGT). The networks for HGT are apparently vast and gene specific. Interestingly, a number of genes are found in both B3 and Gordonia DR phages.

Cardiomyocytes Permeabilization and Electrotransfection by Unipolar and Bipolar Asymmetric Electric Field Pulses.

Short electric field pulses represent a novel potential approach for achieving uniform electroporation within tissue containing elongated cells oriented in various directions, such as electroporation-based cardiac ablation procedures. In this study, we investigated how electroporation with nanosecond pulses with respect to different pulse shapes (unipolar, bipolar, and asymmetric) influences cardiomyocyte permeabilization and gene transfer. For this purpose, rat cardiomyocytes (H9c2) were used. The efficacy of the pulsed electric field protocols was assessed by flow cytometry and electrogene transfer by fluorescent and holotomographic microscopy. The response of the cells was assessed by the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay), F-actin distribution in cells by confocal microscopy, and muscle atrophy F-box (MAFbx) marker. We show nano- and microsecond pulse protocols, which are not cytotoxic for cardiac muscle cells and can be efficiently used for gene electrotransfection. Asymmetric nanosecond pulsed electric fields were similarly efficient in plasmid delivery as microsecond and millisecond protocols. However, the millisecond protocol induced a higher MAFbx expression in H9c2 cells.

Antibiotic-resistant characteristics and horizontal gene transfer ability analysis of extended-spectrum ß-lactamase-producing Escherichia coli isolated from giant pandas.

Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) is regarded as one of the most important priority pathogens within the One Health interface. However, few studies have investigated the occurrence of ESBL-EC in giant pandas, along with their antibiotic-resistant characteristics and horizontal gene transfer abilities. In this study, we successfully identified 12 ESBL-EC strains (8.33%, 12/144) out of 144 E. coli strains which isolated from giant pandas. We further detected antibiotic resistance genes (ARGs), virulence-associated genes (VAGs) and mobile genetic elements (MGEs) among the 12 ESBL-EC strains, and the results showed that 13 ARGs and 11 VAGs were detected, of which bla CTX-M (100.00%, 12/12, with 5 variants observed) and papA (83.33%, 10/12) were the most prevalent, respectively. And ISEcp1 (66.67%, 8/12) and IS26 (66.67%, 8/12) were the predominant MGEs. Furthermore, horizontal gene transfer ability analysis of the 12 ESBL-EC showed that all bla CTX-M genes could be transferred by conjugative plasmids, indicating high horizontal gene transfer ability. In addition, ARGs of rmtB and sul2, VAGs of papA, fimC and ompT, MGEs of ISEcp1 and IS26 were all found to be co-transferred with bla CTX-M. Phylogenetic analysis clustered these ESBL-EC strains into group B2 (75.00%, 9/12), D (16.67%, 2/12), and B1 (8.33%, 1/12), and 10 sequence types (STs) were identified among 12 ESBL-EC (including ST48, ST127, ST206, ST354, ST648, ST1706, and four new STs). Our present study showed that ESBL-EC strains from captive giant pandas are reservoirs of ARGs, VAGs and MGEs that can co-transfer with bla CTX-M via plasmids. Transmissible ESBL-EC strains with high diversity of resistance and virulence elements are a potential threat to humans, animals and surrounding environment.

Analysing carbapenemases in hospital wastewater: Insights from intracellular and extracellular DNA using qPCR and digital PCR.

The widespread dissemination of carbapenem-resistant bacteria in wastewater systems, particularly from clinical sources, poses a significant public health risk. This study assessed the concentrations and distributions of extracellular DNA (exDNA) and intracellular DNA (iDNA) harboring carbapenemase genes in wastewater from six tertiary care hospitals in Germany. We collected a total of 36 samples, comprising six biological replicates from each hospital, and analysed them using quantitative real-time PCR (qPCR) and digital PCR (dPCR). The analysis targeted seven carbapenemase genes: blaNDM, blaKPC, blaIMP, blaVIM, blaOXA-23-like, blaOXA-48-like, and blaOXA-58-like across both DNA fractions. Our results revealed significant variability in the concentrations of exDNA and iDNA across the sampling sites, with iDNA typically present at higher concentrations. Using NanoDrop One spectrophotometry and the Qubit dsDNA kit, exDNA concentrations ranged from 2.7 to 7.7 ng/mL, while Qubit recorded lower values between 1.1 and 4.0 ng/mL. Conversely, iDNA concentrations were markedly higher, spanning from 42.3 to 191.7 ng/mL with NanoDrop and 12.0 to 46.5 ng/mL with Qubit, highlighting the variability between DNA types and quantification methods. Despite its lower concentrations, exDNA comprised up to 18.2 % of total DNA, highlighting its potential role in the horizontal transfer of antimicrobial resistance genes (ARGs). The study detected target ARGs in both DNA fractions at all sites, with notable differences in their concentrations; iDNA consistently exhibited higher levels of ARGs, with the highest concentrations reaching 10.57 ±â€¯0.20 log gene copies per liter (GC/L) for blaVIM in iDNA and 6.96 ±â€¯0.72 log GC/L for blaIMP in exDNA. dPCR demonstrated greater sensitivity than qPCR, especially effective for detecting low-abundance targets like blaOXA-23-like in the exDNA fraction. Additionally, qPCR's susceptibility to inhibition and contamination emphasizes the superior robustness of dPCR. This research highlights the need for improved monitoring and the implementation of advanced treatment technologies to mitigate ARG dissemination in wastewater.

Precision Genetic Therapies: Balancing Risk and Benefit in Patients with Heart Failure.

PURPOSE OF REVIEW: Precision genetic medicine is evolving at a rapid pace and bears significant implications for clinical cardiology. Herein, we discuss the latest advancements and emerging strategies in gene therapy for cardiomyopathy and heart failure. RECENT FINDINGS: Elucidating the genetic architecture of heart failure has paved the way for precision therapies in cardiovascular medicine. Recent preclinical studies and early-phase clinical trials have demonstrated encouraging results that support the development of gene therapies for heart failure arising from a variety of etiologies. In addition to the discovery of new therapeutic targets, innovative delivery platforms are being leveraged to improve the safety and efficacy of cardiac gene therapies. Precision genetic therapy represents a potentially safe and effective approach for improving outcomes in patients with heart failure. It holds promise for radically transforming the treatment paradigm for heart failure by directly targeting the underlying etiology. As this new generation of cardiovascular medicines progress to the clinic, it is especially important to carefully evaluate the benefits and risks for patients.

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.

The interkingdom horizontal gene transfer in 44 early diverging fungi boosted their metabolic, adaptive, and immune capabilities.

Numerous studies have been devoted to individual cases of horizontally acquired genes in fungi. It has been shown that such genes expand the hosts' metabolic capabilities and contribute to their adaptations as parasites or symbionts. Some studies have provided an extensive characterization of the horizontal gene transfer (HGT) in Dikarya. However, in the early diverging fungi (EDF), a similar characterization is still missing. In order to fill this gap, we have designed a computational pipeline to obtain a statistical sample of reliable HGT events with a low false discovery rate. We have analyzed 44 EDF proteomes and identified 829 xenologs in fungi ranging from Chytridiomycota to Mucoromycota. We have identified several patterns and statistical properties of EDF HGT. We show that HGT is driven by bursts of gene exchange and duplication, resulting in highly divergent numbers and molecular properties of xenologs between fungal lineages. Ancestrally aquatic fungi are generally more likely to acquire foreign genetic material than terrestrial ones. Endosymbiotic bacteria can be a source of useful xenologs, as exemplified by NOD-like receptors transferred to Mortierellomycota. Closely related fungi have similar rates of intronization of xenologs. Posttransfer gene fusions and losses of protein domains are common and may influence the encoded proteins' functions. We argue that there is no universal approach for HGT identification and inter- and intra-kingdom transfers require tailored identification methods. Our results help to better understand how and to what extent HGT has shaped the metabolic, adaptive, and immune capabilities of fungi.

Exploration of Alicyclobacillus spp. Genome in Search of Antibiotic Resistance.

The study investigates the antibiotic resistance (AR) profiles and genetic determinants in three strains of guaiacol-producing Alicyclobacillus spp. isolated from orchard soil and pears. Their phenotypic characteristics, such as spore formation; resistance to different factors, including drugs or disinfectants; or production of off-flavor compounds, can affect the taste and aroma of spoiled products. Food and beverages are potential vectors for the transfer of antibiotic resistance genes, which is a growing health concern; thus, microorganisms in food and beverages should not be a potential source of drug resistance to consumers. Whole-genome sequencing (WGS) was utilized to identify antibiotic resistance genes, metabolic pathways, and elements associated with guaiacol and halophenol production. Minimum inhibitory concentration (MIC) testing revealed that all strains were susceptible to eight out of nine tested antibiotics (ampicillin, gentamycin, kanamycin, streptomycin, clindamycin, tetracycline, chloramphenicol, and vancomycin) but exhibited high resistance to erythromycin. Analysis indicated that the erythromycin resistance gene, ribosomal RNA small subunit methyltransferase A (RsmA), was intrinsic and likely acquired through horizontal gene transfer (HGT). The comprehensive genomic analysis provides insights into the molecular mechanisms of antibiotic resistance in Alicyclobacillus spp., highlighting the potential risk of these bacteria as vectors for antibiotic resistance genes in the food chain. This study expands the understanding of the genetic makeup of these spoilage bacteria and their role in antimicrobial resistance dissemination.

Frequent Acquisition of Glycoside Hydrolase Family 32 (GH32) Genes from Bacteria via Horizontal Gene Transfer Drives Adaptation of Invertebrates to Diverse Sources of Food and Living Habitats.

Glycoside hydrolases (GHs, also called glycosidases) catalyze the hydrolysis of glycosidic bonds in polysaccharides. Numerous GH genes have been identified from various organisms and are classified into 188 families, abbreviated GH1 to GH188. Enzymes in the GH32 family hydrolyze fructans, which are present in approximately 15% of flowering plants and are widespread across microorganisms. GH32 genes are rarely found in animals, as fructans are not a typical carbohydrate source utilized in animals. Here, we report the discovery of 242 GH32 genes identified in 84 animal species, ranging from nematodes to crabs. Genetic analyses of these genes indicated that the GH32 genes in various animals were derived from different bacteria via multiple, independent horizontal gene transfer events. The GH32 genes in animals appear functional based on the highly conserved catalytic blades and triads in the active center despite the overall low (35-60%) sequence similarities among the predicted proteins. The acquisition of GH32 genes by animals may have a profound impact on sugar metabolism for the recipient organisms. Our results together with previous reports suggest that the acquired GH32 enzymes may not only serve as digestive enzymes, but also may serve as effectors for manipulating host plants, and as metabolic enzymes in the non-digestive tissues of certain animals. Our results provide a foundation for future studies on the significance of horizontally transferred GH32 genes in animals. The information reported here enriches our knowledge of horizontal gene transfer, GH32 functions, and animal-plant interactions, which may result in practical applications. For example, developing crops via targeted engineering that inhibits GH32 enzymes could aid in the plant's resistance to animal pests.

Antioxidant Defense in the Toughest Animals on the Earth: Its Contribution to the Extreme Resistance of Tardigrades.

Tardigrades are unique among animals in their resistance to dehydration, mainly due to anhydrobiosis and tun formation. They are also very resistant to high-energy radiation, low and high temperatures, low and high pressure, and various chemical agents, Interestingly, they are resistant to ionizing radiation both in the hydrated and dehydrated states to a similar extent. They are able to survive in the cosmic space. Apparently, many mechanisms contribute to the resistance of tardigrades to harmful factors, including the presence of trehalose (though not common to all tardigrades), heat shock proteins, late embryogenesis-abundant proteins, tardigrade-unique proteins, DNA repair proteins, proteins directly protecting DNA (Dsup and TDR1), and efficient antioxidant system. Antioxidant enzymes and small-molecular-weight antioxidants are an important element in the tardigrade resistance. The levels and activities of many antioxidant proteins is elevated by anhydrobiosis and UV radiation; one explanation for their induction during dehydration is provided by the theory of "preparation for oxidative stress", which occurs during rehydration. Genes coding for some antioxidant proteins are expanded in tardigrades; some genes (especially those coding for catalases) were hypothesized to be of bacterial origin, acquired by horizontal gene transfer. An interesting antioxidant protein found in tardigrades is the new Mn-dependent peroxidase.

The evolution of autonomy from two cooperative specialists in fluctuating environments.

From microbes to humans, organisms perform numerous tasks for their survival, including food acquisition, migration, and reproduction. A complex biological task can be performed by either an autonomous organism or by cooperation among several specialized organisms. However, it remains unclear how autonomy and cooperation evolutionarily switch. Specifically, it remains unclear whether and how cooperative specialists can repair deleted genes through direct genetic exchange, thereby regaining metabolic autonomy. Here, we address this question by experimentally evolving a mutualistic microbial consortium composed of two specialists that cooperatively degrade naphthalene. We observed that autonomous genotypes capable of performing the entire naphthalene degradation pathway evolved from two cooperative specialists and dominated the community. This evolutionary transition was driven by the horizontal gene transfer (HGT) between the two specialists. However, this evolution was exclusively observed in the fluctuating environment alternately supplied with naphthalene and pyruvate, where mutualism and competition between the two specialists alternated. The naphthalene-supplied environment exerted selective pressure that favors the expansion of autonomous genotypes. The pyruvate-supplied environment promoted the coexistence and cell density of the cooperative specialists, thereby increasing the likelihood of HGT. Using a mathematical model, we quantitatively demonstrate that environmental fluctuations facilitate the evolution of autonomy through HGT when the relative growth rate and carrying capacity of the cooperative specialists allow enhanced coexistence and higher cell density in the competitive environment. Together, our results demonstrate that cooperative specialists can repair deleted genes through a direct genetic exchange under specific conditions, thereby regaining metabolic autonomy.

Outer membrane vesicles of Acinetobacter baumannii DS002 carry circular DNA similar to bovine meat and milk factors (BMMFs) and SPHINX 2.36 and probably play a role in interdomain lateral gene transfer.

The discovery of Replication Competent Circular DNA molecules in mammalian cells and tissues is being linked to debilitating diseases, such as multiple sclerosis (MS), bovine spongiform encephalopathy (BSE), and colorectal cancer (CRC). These circular DNA molecules, otherwise known as bovine meat and milk factors (BMMFs) and Slow Progressive Hidden INfections of variable (X) latency (SPHINX), bear significant (80%) sequence similarity with the plasmids of Acinetobacter baumannii strains. Nanostructures, such as bacterial outer membrane vesicles (OMVs) serve as vehicles for transporting biomolecular cargo and have the potential to facilitate interkingdom lateral mobility of DNA. Strengthening the proposed hypothesis, this study demonstrates that OMVs derived from A. baumannii DS002 carrying four plasmids and genome (pTS236) of phage, AbDs1, successfully reached different parts of the body, including the central nervous system, following the injection of fluorescein isothiocyanate (FITC)-labeled OMVs into experimental mice. Out of the four OMV-associated plasmids, three (pTS4586, pTS9900, and pTS134338) were identified within the lumen, and the fourth one (pTS11291) was found on the surface of OMVs. In addition to the indigenous plasmids, the phage-encoded protein, Orf96, anchored on the surface of the OMVs by establishing a strong interaction with the OMV-associated porin, OmpA. Intriguingly, a subset of labeled OMVs, when incubated with Neuro2A cells, translocated across the membrane and reached to the cytoplasmic space of the cells. Collectively, the experimental evidence presented herein underscores the promising potential of OMVs as vehicles for delivering molecular cargo containing plasmids and phage genomes to diverse mammalian tissues and cells. IMPORTANCE: Several independent studies have demonstrated the existence of replication competent circular DNA molecules of bacterial and viral origin in mammalian cells and tissues. However, studies about their origin and lateral mobility to mammalian cells are scarce. Our work describes the existence of circular DNA, similar to that of DNA molecules identified in mammalian cells, OMVs derived from soil isolate of A. baumannii DS002. Furthermore, the work also provides visual evidence that demonstrates the passage of labeled OMVs to different organs of experimental mice within hours after intravenously administering OMVs into experimental mice. Some of the labeled OMVs have even crossed the membrane of Neuro2A, suggesting the existence of interkingdom horizontal mobility between bacteria and mammals.

Understanding the Evolution and Transmission Dynamics of Antibiotic Resistance Genes: A Comprehensive Review.

Antibiotic resistance poses a formidable challenge to global public health, necessitating comprehensive understanding and strategic interventions. This review explores the evolution and transmission dynamics of antibiotic resistance genes, with a focus on Bangladesh. The indiscriminate use of antibiotics, compounded by substandard formulations and clinical misdiagnosis, fuels the emergence and spread of resistance in the country. Studies reveal high resistance rates among common pathogens, emphasizing the urgent need for targeted interventions and rational antibiotic use. Molecular assessments uncover a diverse array of antibiotic resistance genes in environmental reservoirs, highlighting the complex interplay between human activities and resistance dissemination. Horizontal gene transfer mechanisms, particularly plasmid-mediated conjugation, facilitate the exchange of resistance determinants among bacterial populations, driving the evolution of multidrug-resistant strains. The review discusses clinical implications, emphasizing the interconnectedness of environmental and clinical settings in resistance dynamics. Furthermore, bioinformatic and experimental evidence elucidates novel mechanisms of resistance gene transfer, underscoring the dynamic nature of resistance evolution. In conclusion, combating antibiotic resistance requires a multifaceted approach, integrating surveillance, stewardship, and innovative research to preserve the efficacy of antimicrobial agents and safeguard public health.

Ribozyme-Mediated Gene-Fragment Complementation for Nondestructive Reporting of DNA Transfer within Soil.

Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the mht gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht-ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli, coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht-ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht-ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

Exploring the antibiotic resistance genes removal dynamics in chicken manure by composting.

Prolonged antibiotic usage in livestock farming leads to the accumulation of antibiotic resistance genes in animal manure. Composting has been shown as an effective way of removing antibiotic resistance from manures, but the specific mechanisms remain unclear. This study used time-series sampling and metagenomics to analyse the resistome types and their bacterial hosts in chicken manures. Composting significantly altered the physicochemical properties and microbiome composition, reduced antibiotic resistance genes by 65.71 %, mobile genetic elements by 68.15 % and horizontal gene transfer frequency. Source tracking revealed that Firmicutes, Actinobacteria, and Proteobacteria are the major bacterial hosts involved in the resistome and gene transfer events. Composting reduces the resistome risk by targeting pathogens such as Staphylococcus aureus. Structural equation modelling confirmed that composting reduces resistome risk by changing pH and pathogen abundance. This study demonstrates that composting is an effective strategy for mitigating resistome risk in chicken manure, thereby supporting the One Health initiative.

Gene Transfer Among Viruses Substantially Contributes to Gene Gain of Giant Viruses.

The phylum Nucleocytoviricota comprises a diverse group of double-stranded DNA viruses that display a wide range of gene repertoires. Although these gene repertoires determine the characteristics of individual viruses, the evolutionary processes that have shaped the gene repertoires of extant viruses since their common ancestor are poorly characterized. In this study, we aimed to address this gap in knowledge by using amalgamated likelihood estimation, a probabilistic tree reconciliation method that infers evolutionary scenarios by distinguishing origination, gene duplications, virus-to-virus horizontal gene transfer (vHGT), and gene losses. We analyzed over 4,700 gene families from 195 genomes spanning all known viral orders. The evolutionary reconstruction suggests a history of extensive gene gains and losses during the evolution of these viruses, notably with vHGT contributing to gene gains at a comparable level to duplications and originations. The vHGT frequently occurred between phylogenetically closely related viruses, as well as between distantly related viruses with an overlapping host range. We observed a pattern of massive gene duplications that followed vHGTs for gene families that was potentially related to host range control and virus-host arms race. These results suggest that vHGT represents a previously overlooked, yet important, evolutionary force that integrates the evolutionary paths of multiple viruses and affects shaping of Nucleocytoviricota virus gene repertoires.

Escherichia coli persisters in biofilm can perform horizontal gene transfer by transformation.

Persisters represent a subset of cells that exhibit transient tolerance to antimicrobials. These persisters can withstand sudden exposure to antimicrobials, even as the majority of normal cells perish. In this study, we have demonstrated the capacity of ampicillin-tolerant and alkali-tolerant persisters to execute horizontal gene transfer via in situ transformation within biofilms. Air-solid biofilms, comprising two Escherichia coli populations each with a distinct plasmid, were formed on agar media. They were treated with lethal doses of ampicillin or NaOH for 24 h, followed by a 1-min glass-ball roll. This process led to a high frequency of horizontal plasmid transfer (10-7-10-6 per cell) from dead cells to surviving persisters within the biofilms. Plasmid transfer was DNase-sensitive and also occurred by adding purified plasmid DNA to plasmid-free biofilms, demonstrating a transformation mechanism. This marks the first evidence of persisters' novel ability for horizontal gene transfer, via transformation.