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The hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a substantial challenge to the global health care. However, the mechanism behind its evolution and transmission remain elusive. Here, four virulence plasmid types were identified from 310 hv-CRKP isolates collected nationwide during 2017-2018, based on their aerobactin (iuc locus) lineage and IncFIB replicons. Notably, pIUC1-IncFIB(K)37 and pIUC1-IncFIB(Mar), representing two epidemic virulence plasmids in Asia and Europe, respectively, accounted for >90% of the hv-CRKP episodes. Analysis of 494 K. pneumoniae isolates (376 from 2010-2013; 118 from 2017-2018) and 2578 public K. pneumoniae genomes indicated the notable role of IncFIB(Mar) plasmids in the hv-CRKP emergence and spread. Conjugation assays showed the helper IncFIB(Mar) plasmid could efficiently transfer into a hypervirulent strain and uniquely retromobilize with pIUC1-IncFIB(K)37 back into CRKP. Thereafter, the IncFIB(Mar) plasmid either lost rapidly or recombined with pIUC1-IncFIB(K)37, generating the hybrid pIUC1-IncFIB(Mar) plasmid. Our findings elucidated formation, evolution, and dissemination trajectories of the two major hv-CRKP strains in different regions. [ABSTRACT FROM AUTHOR]

Virus assembly is a crucial step for the completion of the viral replication cycle. In addition to ensuring efficient incorporation of viral genomes into nascent virions, high specificity is required to prevent incorporation of host nucleic acids. For picornaviruses, including FMDV, the mechanisms required to fulfil these requirements are not well understood. However, recent evidence has suggested that specific RNA sequences dispersed throughout picornavirus genomes are involved in packaging. Here, we have shown that such sequences are essential for FMDV RNA packaging and have demonstrated roles for both the pseudoknot (PK) region and the poly-(C) tract in this process, where the length of the poly-(C) tract was found to influence the efficiency of RNA encapsidation. Sub-genomic replicons containing longer poly-(C) tracts were packaged with greater efficiency in trans, and viruses recovered from transcripts containing short poly-(C) tracts were found to have greatly extended poly-(C) tracts after only a single passage in cells, suggesting that maintaining a long poly-(C) tract provides a selective advantage. We also demonstrated a critical role for a packaging signal (PS) located in the pseudoknot (PK) region, adjacent to the poly-(C) tract, as well as several other non-essential but beneficial PSs elsewhere in the genome. Collectively, these PSs greatly enhanced encapsidation efficiency, with the poly-(C) tract possibly facilitating nearby PSs to adopt the correct conformation. Using these data, we have proposed a model where interactions with capsid precursors control a transition between two RNA conformations, directing the fate of nascent genomes to either be packaged or alternatively to act as templates for replication and/or for protein translation. Author summary: Genome packaging, whereby viral RNA is incorporated into protective protein capsids to produce more virus particles, is a crucial step in RNA virus life cycles. It is a stringent process as only viral RNA is encapsidated, while cellular RNA is excluded. This study reveals the essential role of packaging signals in FMDV RNA encapsidation, specifically those in the pseudoknot region and in a region that can contain >100 cytosines, termed the poly-(C) tract. We demonstrate that the length of the poly-(C) tract significantly affects packaging efficiency; genomes containing longer poly-(C) tracts are favoured. This is the first role that has been identified for the poly-(C) tract in FMDV. We have also found an essential packaging signal in the pseudoknot region, which is assisted by other packaging signals located throughout the genome, that together facilitate encapsidation of FMDV RNA. These results provide compelling evidence for the involvement of RNA packaging signals in FMDV assembly. Based on this, we propose a simple model for FMDV RNA packaging, which involves a transition from genome replication to genome packaging and is controlled by packaging signals. This knowledge could pave the way for future research and development of novel antiviral strategies targeting FMDV and other picornaviruses. [ABSTRACT FROM AUTHOR]

The fork protection complex (FPC), composed of Mrc1, Tof1, and Csm3, supports rapid and stable DNA replication. Here, we show that FPC activity also introduces DNA damage by increasing DNA topological stress during replication. Mrc1 action increases DNA topological stress during plasmid replication, while Mrc1 or Tof1 activity causes replication stress and DNA damage within topologically constrained regions. We show that the recruitment of Top1 to the fork by Tof1 suppresses the DNA damage generated in these loci. While FPC activity introduces some DNA damage due to increased topological stress, the FPC is also necessary to prevent DNA damage in long replicons across the genome, indicating that the FPC is required for complete and faithful genome duplication. We conclude that FPC regulation must balance ensuring full genome duplication through rapid replication with minimizing the consequential DNA topological stress-induced DNA damage caused by rapid replication through constrained regions. [ABSTRACT FROM AUTHOR]

Topoisomerases are the main enzymes capable of resolving the topological constraints imposed by DNA transactions such as transcription or replication. All bacteria possess topoisomerases of different types. Although bacteria with circular replicons should encounter similar DNA topology issues, the distribution of topoisomerases varies from one bacterium to another, suggesting polymorphic functioning. Recently, several proteins restricting, enhancing or modifying the activity of topoisomerases were discovered, opening the way to a new area of understanding DNA topology management during the bacterial cell cycle. In this review, we discuss the distribution of topoisomerases across the bacterial phylum and current knowledge on the interplay among the different topoisomerases to maintain topological homeostasis. [ABSTRACT FROM AUTHOR]

Multipartite genomes are thought to confer evolutionary advantages to bacteria by providing greater metabolic flexibility in fluctuating environments and enabling rapid adaptation to new ecological niches and stress conditions. This genome architecture is commonly found in plant symbionts, including nitrogen-fixing rhizobia, such as Rhizobium leguminosarum bv. trifolii TA1 (RtTA1), whose genome comprises a chromosome and four extrachromosomal replicons (ECRs). In this study, the transcriptomic responses of RtTA1 to partial nutrient limitation and low acidic pH were analyzed using high-throughput RNA sequencing. RtTA1 growth under these conditions resulted in the differential expression of 1035 to 1700 genes (DEGs), which were assigned to functional categories primarily related to amino acid and carbohydrate metabolism, ribosome and cell envelope biogenesis, signal transduction, and transcription. These results highlight the complexity of the bacterial response to stress. Notably, the distribution of DEGs among the replicons indicated that ECRs played a significant role in the stress response. The transcriptomic data align with the Rhizobium pangenome analysis, which revealed an over-representation of functional categories related to transport, metabolism, and regulatory functions on ECRs. These findings confirm that ECRs contribute substantially to the ability of rhizobia to adapt to challenging environmental conditions. [ABSTRACT FROM AUTHOR]

Robbsia andropogonis is one of the most destructive leaf spot disease pathogens of numerous host plants and causes heavy economic damage. In the present study, the complete genome of R. andropogonis strain BLB1, causing the leaf spot disease of areca palm, was generated using a hybrid method combining ONT PromethION long reads and BGISEQ-500 short reads. The resulting genome consists of seven replicons totaling 6,828,120 bp, and 5,808 genes were annotated, including 788 virulence-related genes. Function analysis showed that genes involved in metabolism were the most abundant group. Impressively, the bacteria were well-equipped with four, two, and four sets of type three, four, and six secretion systems, respectively, highlighting the virulence features of R. andropogonis BLB1. As the first complete genome sequence of the species of genus Robbsia, the BLB1 genome provides a solid foundation for investigation of mechanisms underlying the pathogen virulence and disease control, and will promote further discovery and characterization of the genus Robbsia. [ABSTRACT FROM AUTHOR]

Statins, such as lovastatin, have been known to inhibit 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. Statins were reported to moderately suppress hepatitis C virus (HCV) replication in cultured cells harboring HCV RNA replicons. We report here using an HCV cell culture (HCVcc) system that high concentrations of lovastatin (5–20 μg/mL) markedly enhanced the release of HCV infectious particles (virion) in the culture supernatants by up to 40 times, without enhancing HCV RNA replication, HCV protein synthesis, or HCV virion assembly in the cells. We also found that lovastatin increased the phosphorylation (activation) level of extracellular‐signal‐regulated kinase 5 (ERK5) in both the infected and uninfected cells in a dose‐dependent manner. The lovastatin‐mediated increase of HCV virion release was partially reversed by selective ERK5 inhibitors, BIX02189 and XMD8‐92, or by ERK5 knockdown using small interfering RNA (siRNA). Moreover, we demonstrated that other cholesterol‐lowering statins, but not dehydrolovastatin that is incapable of inhibiting HMG‐CoA reductase and activating ERK5, enhanced HCV virion release to the same extent as observed with lovastatin. These results collectively suggest that statins markedly enhance HCV virion release from infected cells through HMG‐CoA reductase inhibition and ERK5 activation. [ABSTRACT FROM AUTHOR]

Abstract Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo.

Multiple drug resistance (MDR) has gained pronounced attention among Enterobacterales. The transfer of multiple antimicrobial resistance genes, frequently carried on conjugative incompatibility F (IncF) plasmids and facilitating interspecies resistance transmission, has been linked to Salmonella spp. and E. coli in broilers. In Egypt, the growing resistance is exacerbated by the limited clinical efficacy of many antimicrobials. In this study, IncF groups were screened and characterized in drug-resistant Salmonella spp. and E. coli isolated from broilers. The antimicrobial resistance profile, PCR-based replicon typing of bacterial isolates pre- and post-plasmid curing, and IncF replicon allele sequence typing were investigated. Five isolates of E. coli (5/31; 16.13%) and Salmonella spp. (5/36; 13.89%) were pan-susceptible to the examined antimicrobial agents, and 85.07% of tested isolates were MDR and extensively drug-resistant (XDR). Twelve MDR and XDR E. coli and Salmonella spp. isolates were examined for the existence of IncF replicons (FII, FIA, and FIB). They shared resistance to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanate, doxycycline, cefotaxime, and colistin. All isolates carried from one to two IncF replicons. The FII-FIA-FIB+ and FII-FIA+FIB- were the predominant replicon patterns. FIB was the most frequently detected replicon after plasmid curing. Three XDR E. coli isolates that were resistant to 12–14 antimicrobials carried a newly FIB replicon allele with four nucleotide substitutions: C99→A, G112→T, C113→T, and G114→A. These findings suggest that broilers are a significant reservoir of IncF replicons with highly divergent IncF-FIB plasmid incompatibility groups circulating among XDR Enterobacterales. Supporting these data with additional comprehensive epidemiological studies involving replicons other than the IncF can provide insights for implementing efficient policies to prevent the spreading of new replicons to humans. [ABSTRACT FROM AUTHOR]

Self-amplifying RNAs (saRNAs) are versatile vaccine platforms that take advantage of a viral RNA-dependent RNA polymerase (RdRp) to amplify the messenger RNA (mRNA) of an antigen of interest encoded within the backbone of the viral genome once inside the target cell. In recent years, more saRNA vaccines have been clinically tested with the hope of reducing the vaccination dose compared to the conventional mRNA approach. The use of N1-methyl-pseudouridine (1mΨ), which enhances RNA stability and reduces the innate immune response triggered by RNAs, is among the improvements included in the current mRNA vaccines. In the present study, we evaluated the effects of this modified nucleoside on various saRNA platforms based on different viruses. The results showed that different stages of the replication process were affected depending on the backbone virus. For TNCL, an insect virus of the Alphanodavirus genus, replication was impaired by poor recognition of viral RNA by RdRp. In contrast, the translation step was severely abrogated in coxsackievirus B3 (CVB3), a member of the Picornaviridae family. Finally, the effects of 1mΨ on Semliki forest virus (SFV), were not detrimental in in vitro studies, but no advantages were observed when immunogenicity was tested in vivo. [ABSTRACT FROM AUTHOR]

ABSTRACT Messenger (m)RNA has taken center stage in vaccine development, gene therapy, and cancer immunotherapy. A next-generation of mRNA is the self-amplifying (sa)mRNA, which induces broad and long-lasting immunity at a lower dose which provides better clinical outcomes in conjunction with fewer adverse effects. SamRNA, also known as “replicon” RNA, encodes the replication machinery of an alphavirus together with an antigen. Efficient delivery of replicon RNA to target tissues can be accomplished by packaging the replicon RNA in virus-like replicon particles (VRPs) via co-transfection of producer cells with defective helper RNA(s) encoding the alphavirus structural proteins. During the manufacture of VRPs, however, there is a potential risk of RNA recombination, which may lead to the formation of replication-competent virus (RCV). To investigate the factors influencing the unwanted RCV formation, we evaluated how sequence homology orchestrates alphavirus RNA recombination. Several combinations of complementing alphavirus replicon and helper RNAs varying in length of sequences overlap were co-transfected in mammalian cells. The culture fluid was serially passaged to detect RCV. Nanopore sequencing of cells after the first passage in combination with amplicon-based Sanger sequencing of RCV in the culture fluid after four passages led to the detection of RNA recombination. RCV was generated between replicon and helper RNAs with sequence homology in either the non-structural or structural genes, whereas RNAs without overlapping gene regions did not generate RCV. Remarkably, no sequence overlap was detected at the recombination junction sites in the RCV genome, suggesting a mechanism of “aberrant homologous RNA recombination.” Accordingly, we conclude that the alphavirus RNA recombination process leading to the formation of RCV is homology-assisted and can be prevented by avoiding sequence homology between replicon and helper RNAs.IMPORTANCEThere is a growing interest in the use of self-amplifying (sa)mRNA vectors for next-generation vaccine development, gene therapy, and cancer immunotherapy. The delivery of samRNA in the form of virus-like replicon particles (VRPs) enables efficient delivery of samRNA to target tissue. The production of these VRPs, however, suffers from contamination with replication-competent virus (RCV) that is thought to arise from recombination events between samRNA and helper RNAs for VRP packaging. The presence of RCV in samRNA in the clinical product is undesirable as alphaviruses may cause serious disease in humans. However, the underlying recombination mechanism leading to RCV is currently unknown. In our work, we demonstrate a detailed evaluation of the recombination sites, which indicates that RCV is formed through an unusual mechanism of “aberrant homologous RNA recombination.” The results are useful for researchers in the field of RNA vaccine manufacture and delivery.

Background: Escherichia. coli is the most frequent host for New Delhi metallo-β-lactamase (NDM) which hydrolyzes almost all β-lactams except aztreonam. The worldwide spread of blaNDM-carrying E. coli heavily threatens public health. Objective: This study aimed to explore the global genomic epidemiology of blaNDM- carrying E. coli isolates, providing information for preventing the dissemination of such strains. Methods: Global E. coli genomes were downloaded from NCBI database and blaNDM was detected using BLASTP. Per software was used to extract meta information on hosts, resources, collection data, and countries of origin from GenBank. The sequence types (STs) and distribution of antimicrobial resistance gene (ARG) were analyzed by CLC Workbench; Plasmid replicons, serotypes and virulence genes (VFs) were analyzed by submitting the genomes to the websites. Statistical analyses were performed to access the relationships among ARGs and plasmid replicons. Results: Until March 2023, 1,774 out of 33,055 isolates collected during 2003–2022 were found to contain blaNDM in total. Among them, 15 blaNDM variants were found with blaNDM-5 (74.1%) being most frequent, followed by blaNDM-1 (16.6%) and blaNDM-9 (4.6%). Among the 213 ARGs identified, 27 blaCTX-M and 39 blaTEM variants were found with blaCTX-M-15 (n = 438, 24.7%) and blaTEM-1B (n = 1092, 61.6%) being the most frequent ones, respectively. In addition, 546 (30.8%) plasmids mediated ampC genes, 508 (28.6%) exogenously acquired 16 S rRNA methyltransferase encoding genes and 262 (14.8%) mcr were also detected. Among the 232 distinct STs, ST167 (17.2%) were the most prevalent. As for plasmids, more than half of isolates contained IncFII, IncFIB and IncX3. The VF terC, gad, traT and iss as well as the serotypes O101:H9 (n = 231, 13.0%), O8:H9 (n = 115, 6.5%) and O9:H30 (n = 99, 5.6%) were frequently observed. Conclusions: The study delves into the intricate relationship between plasmid types, virulence factors, and ARGs, which provides valuable insights for clinical treatment and public health interventions, and serves as a critical resource for guiding future research, surveillance, and implementation of effective strategies to address the challenges posed by blaNDM-carrying E. coli. The findings underscore the urgent need for sustained global collaboration, surveillance efforts, and antimicrobial stewardship to mitigate the impact of these highly resistant strains on public health. [ABSTRACT FROM AUTHOR]

This study proposes a landscape-based scenario for the origin of viruses and cells, focusing on the adaptability of preexisting replicons from the RNP (ribonucleoprotein) world. The scenario postulates that life emerged in a subterranean "warm little pond" where organic matter accumulated, resulting in a prebiotic soup rich in nucleotides, amino acids, and lipids, which served as nutrients for the first self-replicating entities. Over time, the RNA world, followed by the RNP world, came into existence. Replicators/replicons, along with the nutritious soup from the pond, were washed out into the river and diluted. Lipid bubbles, enclosing organic matter, provided the last suitable environment for replicons to replicate. Two survival strategies emerged under these conditions: cell-like structures that obtained nutrients by merging with new bubbles, and virus-like entities that developed various techniques to transmit themselves to fresh bubbles. The presented hypothesis provides the possibility for the common origin of cells and viruses on rocky worlds hosting liquid water, like Earth. [ABSTRACT FROM AUTHOR]

To investigate the clinical and molecular characteristics of Salmonella spp. causing bloodstream infections (BSIs) in our hospital. Methods: We studied 22 clinical Salmonella isolates from BSIs and 16 from non-BSIs, performing antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS). The analysis included serovars, antibiotic resistance genes (ARGs), virulence factors (VFs), sequence types (STs), plasmid replicons, and genetic relationships. We also assessed pathogenicity of the isolates causing BSIs through growth, biofilm formation, and anti-serum killing assays. Results: WGS analysis identified 13 Salmonella serovars, with four responsible for BSIs. S. Enteritidis was the most prevalent serovar, involved in 19 (50.0%) cases. BSIs were caused by 17S. Enteritidis, two S. Typhimurium, two S. Munster and one S. Diguel. Of the 38 isolates, 27 (71.1%) exhibited high resistance to ampicillin, and 24 (63.2%) to ampicillin/sulbactam. Thirty-six types of ARGs were identified, with blaTEM-1B (n = 25, 65.8%) being the most frequent. Ten plasmid replicons were found; the combination of IncFIB(S)-IncFII(S)-IncX1 was the most common in S. Enteritidis (94.7%). Fifteen STs were identified, among which ST11 was the most prevalent and clonally disseminated, primarily responsible for BSIs. A total of 333 different VFs were detected, 177 of which were common across all strains. No significant differences were observed between the BSI and non-BSI isolates in terms of resistance rates, ARGs, plasmid replicons, and VFs, except for seven VFs. No strong pathogenicity was observed in the BSI-causing isolates. Conclusion: BSIs were predominantly caused by clonally disseminated S. Enteritidis ST11, the majority of which carried multiple ARGs, VFs and plasmid replicons. This study provides the first data on clonally disseminated S. Enteritidis ST11 causing BSIs, highlighting the urgent need for enhanced infection control measures. [ABSTRACT FROM AUTHOR]

Objectives While an increase in the levels of MDR in Salmonella enterica sevorar Choleraesuis has been reported in Europe, little is known about the situation in Spain. Therefore, we first aimed to assess the phenotypic resistance profile and to determine the presence of genetic determinants of resistance of S. Choleraesuis isolates collected in animal and human. Our second objective was to identify and characterize clusters of highly related isolates. Methods We analysed 50 human and 45 animal isolates retrieved from 2006 to 2021 using the disc diffusion method and performed WGS followed by analyses of genetic determinants and phylogenetic analysis. Results All isolates were of ST145 and corresponded to the variant Kunzendorf. Swine isolates harboured a significantly higher number of antimicrobial resistance genes than human isolates, and often carried plasmid replicons of the IncHI2/IncHI2A type (42% of all animal isolates). In addition, we identified several MDR S. Choleraesuis strains circulating in humans and swine between 2006 and 2021. The phylogenetic analyses identified four clades associated with specific patterns of resistance genes and plasmid replicons. The clades also included isolates that differed in terms of year and region of isolation as well as host of origin. Conclusions This One Health approach highlights that reducing human MDR S. Choleraesuis infections may require the adoption of strategies that not only seek to prevent cases in humans but also to characterize and reduce the infection burden in swine. [ABSTRACT FROM AUTHOR]

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is a pathogenic bacterium that causes Pullorum disease (PD). PD is an acute systemic disease that affects young chickens, causing white diarrhea and high mortality. Although many sanitary programs have been carried out to eradicate S. Pullorum, PD outbreaks have been reported in different types of birds (layers, broilers, breeders) worldwide. This study aimed to evaluate the evolution and genetic characteristics of S. Pullorum isolated from PD in Brazil. Phylogenetic analysis of S. Pullorum genomes sequenced in this study and available genomic databases demonstrated that all isolates from Brazil are from sequence type 92 (ST92) and cluster into two lineages (III and IV). ColpVC, IncFIC(FII), and IncFII(S) were plasmid replicons frequently found in the Brazilian lineages. Two resistance genes (aac(6')-Iaa, conferring resistance to aminoglycoside, disinfecting agents, and antiseptics (mdf(A)) and tetracycline (mdf(A)) were detected frequently. Altogether, these results are important to understand the circulation of S. Pullorum and, consequently, to develop strategies to reduce losses due to PD. Evolución y perfil genómico de aislados de Salmonella enterica serovar Gallinarum biovar Pullorum de Brasil. Salmonella enterica subespecie enterica serovar Gallinarum biovar Pullorum (S. Pullorum) es una bacteria patógena que causa la enfermedad de Pullorum (EP). La EP es una enfermedad sistémica aguda que afecta a los pollos jóvenes causando diarrea blanca y alta mortalidad. Aunque se han llevado a cabo muchos programas sanitarios para erradicar S. Pullorum, se han informado brotes de EP en diferentes tipos de aves (ponedoras, pollos de engorde, reproductoras) en todo el mundo. Este estudio tuvo como objetivo evaluar la evolución y las características genéticas de S. Pullorum aislado de EP en Brasil. El análisis filogenético de los genomas de S. Pullorum secuenciados en este estudio y las bases de datos genómicas disponibles demostraron que todos los aislamientos de Brasil son del tipo de secuencia 92 (ST92) y se agrupan en dos linajes (III y IV). ColpVC, IncFIC (FII) e IncFII(S) fueron replicones de plásmidos frecuentemente encontrados en los linajes brasileños. Dos genes de resistencia (aac(6′)-Iaa, que confiere resistencia a aminoglucósidos, desinfectantes y antisépticos (mdf(A)), y tetraciclina (mdf(A)) fueron detectados con frecuencia. En conjunto, estos resultados son importantes para comprender la circulación de S. Pullorum y, en consecuencia, desarrollar estrategias para reducir las pérdidas por EP. [ABSTRACT FROM AUTHOR]

Background: Multi-drug-resistant organisms (MDROs) in gram-negative bacteria have caused a global epidemic, especially the bacterial resistance to carbapenem agents. Plasmid is the common vehicle for carrying antimicrobial resistance genes (ARGs), and the transmission of plasmids is also one of the important reasons for the emergence of MDROs. Different incompatibility group plasmid replicons are highly correlated with the acquisition, dissemination, and evolution of resistance genes. Based on this, the study aims to identify relevant characteristics of various plasmids and provide a theoretical foundation for clinical anti-infection treatment. Methods: 330 gram-negative strains with different antimicrobial phenotypes from a tertiary hospital in Henan Province were included in this study to clarify the difference in incompatibility group plasmid replicons. Additionally, we combined the information from the PLSDB database to elaborate on the potential association between different plasmid replicons and ARGs. The VITEK mass spectrometer was used for species identification, and the VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). PCR-based replicon typing (PBRT) detected the plasmid profiles, and thirty-three different plasmid replicons were determined. All the carbapenem-resistant organisms (CROs) were tested for the carbapenemase genes. Results: 21 plasmid replicon types were detected in this experiment, with the highest prevalence of IncFII, IncFIB, IncR, and IncFIA. Notably, the detection rate of IncX3 plasmids in CROs is higher, which is different in strains with other antimicrobial phenotypes. The number of plasmid replicons they carried increased with the strain resistance increase. Enterobacterales took a higher number of plasmid replicons than other gram-negative bacteria. The same strain tends to have more than one plasmid replicon type. IncF-type plasmids tend to be associated with MDROs. Combined with PLSDB database analysis, IncFII and IncX3 are critical platforms for taking blaKPC−2 and blaNDM. Conclusions: MDROs tend to carry more complex plasmid replicons compared with non-MDROs. The plasmid replicons that are predominantly prevalent and associated with ARGs differ in various species. The wide distribution of IncF-type plasmids and their close association with MDROs should deserve our attention. Further investigation into the critical role of plasmids in the carriage, evolution, and transmission of ARGs is needed. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis has the potential to be an optimal chassis for the production of bulk chemicals derived from pyruvate. However, a lack of available standardized and characterized genetic tools hinders both efficient engineering of Z. mobilis and progress in basic research on this organism. In this study, a series of different shuttle vectors were constructed based on the replication mechanisms of the native Z. mobilis plasmids pZMO1, pZMOB04, pZMOB05, pZMOB06, pZMO7 and p29191_2 and on the broad host range replication origin of pBBR1. These plasmids as well as genomic integration sites were characterized for efficiency of heterologous gene expression, stability without selection and compatibility. We were able to show that a wide range of expression levels could be achieved by using different plasmid replicons. The expression levels of the constructs were consistent with the relative copy numbers, as determined by quantitative PCR. In addition, most plasmids are compatible and could be combined. To avoid plasmid loss, antibiotic selection is required for all plasmids except the pZMO7‐based plasmid, which is stable also without selection pressure. Stable expression of reporter genes without the need for selection was also achieved by genomic integration. All modules were adapted to the modular cloning toolbox Zymo‐Parts, allowing easy reuse and combination of elements. This work provides an overview of heterologous gene expression in Z. mobilis and adds a rich set of standardized genetic elements to an efficient cloning system, laying the foundation for future engineering and research in this area. [ABSTRACT FROM AUTHOR]

Alphaviruses have been used as vectors for the expression of heterologous genetic information for a few decades. Their genomes contain two open reading frames. One of these encodes viral nonstructural proteins (nsPs), which mediate the replication of viral genome (G RNA) and synthesis of subgenomic (SG) RNA. Viral structural proteins encoded by SG RNA are dispensable for G RNA replication and transcription of the SG RNA. Their genes can be replaced by heterologous genetic materials. Such defective viral genomes, replicons, can efficiently express the encoded heterologous genes of interest. Within recent years, alphavirus replicons, also termed self-amplifying RNAs (saRNAs), attracted additional attention as a tool to increase the expression level of mRNA vaccines. The results of this study demonstrate that replicons derived from both New World and Old World alphaviruses can efficiently express heterologous genetic information. However, the levels of expression are cell-specific and are strongly determined by the origin of the replicons. Moreover, replication of alphavirus replicons mimics many aspects of alphavirus infections in terms of virus-host interactions and induction of the innate immune response. They also express viral nsPs that may induce vector immunity. These characteristics need to be carefully examined before the wide application of saRNAs. On the other hand, the data show that alphavirus replicons are very flexible expression systems. The efficiencies of protein expression and interactions with host cells can be manipulated by introducing mutations into nsPs and other modifications in replicon genomes. [ABSTRACT FROM AUTHOR]

The next step in mRNA vaccine design is the application of viral-based self-amplifying mRNAs (replicons) that provide long-lasting humoral and cellular immune responses upon single, low-dose immunization. Replicons encode their own replication machinery to boost their copy numbers directly after administration in target cells, which dramatically lowers the required initial mRNA dose and may consequently reduce adverse effects in individuals. Recent advances in mRNA formulation using lipid or solid nanoparticles create opportunities for novel applications for replicons such as mucosal delivery. Replicon vaccines hold potential as a platform technology when safety aspects are properly addressed. A diverse spectrum of replicons have been developed for innovative applications such as multivalent and therapeutic cancer vaccines. mRNA vaccines have won the race for early COVID-19 vaccine approval, yet improvements are necessary to retain this leading role in combating infectious diseases. A next generation of self-amplifying mRNAs, also known as replicons, form an ideal vaccine platform. Replicons induce potent humoral and cellular responses with few adverse effects upon a minimal, single-dose immunization. Delivery of replicons is achieved with virus-like replicon particles (VRPs), or in nonviral vehicles such as liposomes or lipid nanoparticles. Here, we discuss innovative advances, including multivalent, mucosal, and therapeutic replicon vaccines, and highlight novelties in replicon design. As soon as essential safety evaluations have been resolved, this promising vaccine concept can transform into a widely applied clinical platform technology taking center stage in pandemic preparedness. [ABSTRACT FROM AUTHOR]

Background: Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT's recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. Results: Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. Conclusion: The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads. [ABSTRACT FROM AUTHOR]

Rhodococcus bacteria are a fast-growing platform for biocatalysis, biodegradation, and biosynthesis, but not a platform for molecular biology. That is, Rhodococcus are not convenient for genetic engineering. One major issue for the engineering of Rhodococcus is the absence of a publicly available, curated, and commented collection of sequences of genetic parts that are functional in biotechnologically relevant species of Rhodococcus (R. erythropolis, R. rhodochrous, R. ruber, and R. jostii). Here, we present a collection of genetic parts for Rhodococcus (vector replicons, promoter regions, regulators, markers, and reporters) supported by a thorough analysis of their functionality. We also highlight and discuss the gaps in Rhodococcus-related genetic parts and techniques, which should be filled in order to make these bacteria a full-fledged molecular biology platform independent of Escherichia coli. We conclude that all major types of required genetic parts for Rhodococcus are available now, except multicopy replicons. As for model Rhodococcus strains, there is a particular shortage of strains with high electrocompetence levels and strains designed for solving specific genetic engineering tasks. We suggest that these obstacles are surmountable in the near future due to an intensification of research work in the field of genetic techniques for non-conventional bacteria. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic resistance under treatment depends on the availability of resistance alleles and their establishment in the population. Novel resistance alleles are encoded either in chromosomal or extrachromosomal genetic elements; both types may be present in multiple copies within the cell. However, the effect of polyploidy on the emergence of antibiotic resistance remains understudied. Here we show that the establishment of resistance alleles in microbial populations depends on the ploidy level. Evolving bacterial populations under selection for antibiotic resistance, we demonstrate that resistance alleles in polyploid elements are lost frequently in comparison to alleles in monoploid elements due to segregational drift. Integrating the experiments with a mathematical model, we find a remarkable agreement between the theoretical and empirical results, confirming our understanding of the allele segregation process. Using the mathematical model, we further show that the effect of polyploidy on the establishment probability of beneficial alleles is strongest for low replicon copy numbers and plateaus for high replicon copy numbers. Our results suggest that the distribution of fitness effects for mutations that are eventually fixed in a population depends on the replicon ploidy level. Our study indicates that the emergence of antibiotic resistance in bacterial pathogens depends on the pathogen ploidy level. Author summary: Prokaryotic replicons—chromosomes and plasmids—may be present in the cell in multiple copies, a phenomenon termed polyploidy. Here we studied the effect of replicon copy number, i.e., polyploidy level, on the emergence of beneficial traits in prokaryotes, with a focus on the evolution of antibiotic resistance. We discovered that beneficial mutations encoded in multicopy replicons are bound to be frequently lost due to stochasticity during cell division that ultimately increases genetic drift. Our study presents empirical evidence for the effect of polyploidy on the rate of adaptation in prokaryotes. A comparison to simulations of a mathematical model confirms our understanding of the allele dynamics. We conclude that drug treatment strategies against pathogenic bacteria should consider the ploidy level of the organism. [ABSTRACT FROM AUTHOR]

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation. Omicron BA.1 is attenuated in infection models though the precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging. Here the authors present pGLUE, a plasmid-based viral genome assembly and rescue strategy, to systematically characterize Omicron mutations and show that Omicron NSP6 has weakened lipid flow to replication organelles and reduced viral RNA replication. [ABSTRACT FROM AUTHOR]

Background: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). Methods: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. Results: The liposome–replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. Conclusion: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines. [ABSTRACT FROM AUTHOR]

Salmonella serovars Heidelberg and Minnesota encoding antimicrobial resistance to third‐generation cephalosporins and fluoroquinolones are often detected in poultry/poultry meat. We analysed the genomes of 10 Salmonella Heidelberg (SH) and 4 Salmonella Minnesota (SM) from faecal isolates of Brazilian poultry. These featured virulent and multidrug‐resistant characteristics, with AmpC beta‐lactamase (blaCMY‐2) predominance (9/14), for all SM (4/4) and some SH (3/10) located on IncC plasmid replicons. IncC carrying blaCTX‐M‐2 was only detected among SH (3/10). Mutation in the gyrA/parC genes was present in all SH, whereas SM harboured parC mutation plus qnrB19 on ColRNAI plasmids (3/4). In silico resistance overall corroborated with phenotypic results. Core genome phylogenies showed close clustering and high similarities between the Brazilian and poultry meat/food isolates from Europe, and to human isolates from European countries with documented import of Brazilian poultry meat. Conjugation assays with SM successfully transferred blaCMY‐2, and qnrB19 to an Escherichia coli recipient. The findings reinforce the ongoing antimicrobial resistance acquisition of SH and Minnesota and the risks for disseminating resistant strains and/or mobile elements which may increasingly affect importing countries and the need for controlling AMR in major poultry‐exporting countries like Brazil. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) has received relatively little attention for decades although it is now considered as one of the most frequent causes of acute hepatitis worldwide. Our knowledge of this enterically-transmitted, positive-strand RNA virus and its life cycle remains scarce but research on HEV has gained momentum more recently. Indeed, advances in the molecular virology of hepatitis E, including the establishment of subgenomic replicons and infectious molecular clones, now allow study of the entire viral life cycle and to explore host factors required for productive infection. Here, we provide an overview on currently available systems, with an emphasis on selectable replicons and recombinant reporter genomes. Furthermore, we discuss the challenges in developing new systems which should enable to further investigate this widely distributed and important pathogen. [ABSTRACT FROM AUTHOR]

We found a carbapenem-resistant Enterobacter clinical strain which was susceptible to cefotaxime and ceftazidime. This unusual susceptibility profile promoted the investigation. This strain had blaFRI-11, a rare carbapenemase-encoding gene, on a 93,864-bp plasmid containing two replicons of IncFII(pECLA) and IncFIA(HI1). FRI-11, FRI-2, FRI-3, FRI-4, FRI-6, FRI-7, and FRI-9 belong to the same group of FRI β-lactamases based on the amino acid sequence similarity and their encoding genes are carried by plasmids containing an IncFII(pECLA) replicon. Awareness should be raised towards FRI carbapenemases that are plasmid-encoded and confer an unusual carbapenem-resistant but 3rd-generation-cephalosporin-susceptible resistance profile. [ABSTRACT FROM AUTHOR]

BackgroundThe genomes of halophilic archaea (haloarchaea) often comprise multiple replicons. Genomic variation in haloarchaea has been linked to viral infection pressure and, in the case of Antarctic communities, can be caused by intergenera gene exchange. To expand understanding of genome variation and biogeography of Antarctic haloarchaea, here we assessed genomic variation between two strains of Halorubrum lacusprofundi that were isolated from Antarctic hypersaline lakes from different regions (Vestfold Hills and Rauer Islands). To assess variation in haloarchaeal populations, including the presence of genomic islands, metagenomes from six hypersaline Antarctic lakes were characterised.ResultsThe sequence of the largest replicon of each Hrr. lacusprofundi strain (primary replicon) was highly conserved, while each of the strains' two smaller replicons (secondary replicons) were highly variable. Intergenera gene exchange was identified, including the sharing of a type I-B CRISPR system. Evaluation of infectivity of an Antarctic halovirus provided experimental evidence for the differential susceptibility of the strains, bolstering inferences that strain variation is important for modulating interactions with viruses. A relationship was found between genomic structuring and the location of variation within replicons and genomic islands, demonstrating that the way in which haloarchaea accommodate genomic variability relates to replicon structuring. Metagenome read and contig mapping and clustering and scaling analyses demonstrated biogeographical patterning of variation consistent with environment and distance effects. The metagenome data also demonstrated that specific haloarchaeal species dominated the hypersaline systems indicating they are endemic to Antarctica.ConclusionThe study describes how genomic variation manifests in Antarctic-lake haloarchaeal communities and provides the basis for future assessments of Antarctic regional and global biogeography of haloarchaea.

DNA-based gene therapy and vaccine development has received plenty of attention lately. DNA replicons based on self-replicating RNA viruses such as alphaviruses and flaviviruses have been of particular interest due to the amplification of RNA transcripts leading to enhanced transgene expression in transfected host cells. Moreover, significantly reduced doses of DNA replicons compared to conventional DNA plasmids can elicit equivalent immune responses. DNA replicons have been evaluated in preclinical animal models for cancer immunotherapy and for vaccines against infectious diseases and various cancers. Strong immune responses and tumor regression have been obtained in rodent tumor models. Immunization with DNA replicons has provided robust immune responses and protection against challenges with pathogens and tumor cells. DNA replicon-based COVID-19 vaccines have shown positive results in preclinical animal models. [ABSTRACT FROM AUTHOR]

Background: VIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, blaVIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM- 1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of blaVIM genes. Materials and methods: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. Results: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the blaVIM-1 allele while six isolates carried the blaVIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The blaVIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the blaVIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two blaVIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. Conclusion: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution. [ABSTRACT FROM AUTHOR]

How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, 'RNA replicons', in which RNAs produce proteins to replicate their coding RNA, and 'DNA replicons', in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in Escherichia coli. This mini-RNA replicon encoded the β subunit, one of the subunits of the Qβ replicase derived from the positive-sense single-stranded Qβ RNA phage and is replicated by the replicase in E. coli. To maintain the mini-RNA replicon persistently in E. coli cells, we employed a system of α complementation of LacZ that was dependent on the Qβ replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (E. coli genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA–protein world to the DNA world, which will contribute to progress in the research field of the origin of life. [ABSTRACT FROM AUTHOR]

Background: Phospholipase D (PLD) is highly valuable in the food and medicine industries, where it is used to convert low-cost phosphatidylcholine into high-value phospholipids (PLs). Despite being overexpressed in Streptomyces, PLD production requires expensive thiostrepton feeding during fermentation, limiting its industrialization. To address this issue, we propose a new thiostrepton-free system. Results: We developed a system using a combinatorial strategy containing the constitutive promoter kasOp* and PLD G215S mutation fused to a signal peptide sigcin of Streptoverticillium cinnamoneum pld. To find a candidate vector, we first expressed PLD using the integrative vector pSET152 and then built three autonomously replicating vectors by substituting Streptomyces replicons to increase PLD expression. According to our findings, replicon 3 with stability gene (sta) inserted had an ideal result. The retention rate of the plasmid pOJ260-rep3-pld* was 99% after five passages under non-resistance conditions. In addition, the strain SK-3 harboring plasmid pOJ260-rep3-pld* produced 62 U/mL (3.48 mg/g) of PLD, which further improved to 86.8 U/mL (7.51 mg/g) at 32 °C in the optimized medium, which is the highest activity achieved in the PLD secretory expression to date. Conclusions: This is the first time that a thiostrepton-free PLD production system has been reported in Streptomyces. The new system produced stable PLD secretion and lays the groundwork for the production of PLs from fermentation stock. Meanwhile, in the Streptomyces expression system, we present a highly promising solution for producing other complex proteins. [ABSTRACT FROM AUTHOR]

Background: pTE15 is a ~ 15-kb narrow-host-range indigenous plasmid from Lactobacillus reuteri N16 that does not replicate in selected Bacillus spp., Staphylococcus spp., and other Lactobacillus spp. Methods: Combined deletion analysis the minireplicon essential of pTE15 with replicon-probe vector pUE80 (−) to confirmed sufficient for replication and from the ssDNA intermediate detection, plasmid amplification tested by chloramphenicol treatment, and replication origin sequence analysis to delineated the novel theta-type replication of pTE15. Results: Single-stranded intermediate of pTE15 DNA was not detected in L. reuteri, indicating that this plasmid does not replicate via a rolling circle mechanism. The replicon of pTE15 did not display the structural organization typical of rolling-circle plasmids, nor were they similar to known rolling-circle plasmids. We further provided evidence that this plasmid applied a new mode of theta-type replication mechanism: (1) the size of this plasmid was > 10-kb; (2) the minireplicon consisted of AT-rich (directed repeat, iteron) and DnaA sequences; (3) the minireplicon did not contain double-strand origin (DSO) and essential rep genes, and it also showed no single-strand origin (SSO) structure; (4) the intermediate single-stranded DNA products were not observed for pTE15 replication; (5) the minireplicon did not contain a typical essential replication protein, Rep, (6) its copy number was decreased by chloramphenicol treatment, and (7) genes in pTE15 replication region encoded truncated RepA (TRepA), RepB and RepC, which were replication-associated proteins, but they were not essential for pTE15 replication. Conclusions: Collectively, our results strongly suggested that the indigenous plasmid pTE15 of L. reuteri N16 belongs to a new class of theta replicons. [ABSTRACT FROM AUTHOR]

Objective: To analyze the characteristics of global β-lactamase-producing Enterobacter cloacae including the distribution of β-lactamase, sequence types (STs) as well as plasmid replicons. Methods: All the genomes of the E. cloacae were downloaded from GenBank. The distribution of β-lactamase encoding genes were investigated by genome annotation after the genome quality was checked. The STs of these strains were analyzed by multi-locus sequence typing (MLST). The distribution of plasmid replicons was further explored by submitting these genomes to the genome epidemiology center. The isolation information of these strains was extracted by Per program from GenBank. Results: A total of 272 out of 276 strains were found to carry β-lactamase encoding genes. Among them, 23 varieties of β-lactamase were identified, blaCMH (n = 130, 47.8%) and blaACT (n = 126, 46.3%) were the most predominant ones, 9 genotypes of carbapenem-hydrolyzing β-lactamase (CHβLs) were identified with blaVIM (n = 29, 10.7%) and blaKPC (n = 24, 8.9%) being the most dominant ones. In addition, 115 distinct STs for the 272 ß-lactamase-carrying E. cloacae and 48 different STs for 106 CHβLs-producing E. cloacae were detected. ST873 (n = 27, 9.9%) was the most common ST. Furthermore, 25 different plasmid replicons were identified, IncHI2 (n = 65, 23.9%), IncHI2A (n = 64, 23.5%) and IncFII (n = 62, 22.8%) were the most common ones. Notably, the distribution of plasmid replicons IncHI2 and IncHI2A among CHβLs-producing strains were significantly higher than theat among non-CHβLs-producing strains (p < 0.05). Conclusion: Almost all the E. cloacae contained β-lactamase encoding gene. Among the global E. cloacae, blaCMH and blaACT were main blaAmpC genes. BlaTEM and blaCTX-M were the predominant ESBLs. BlaKPC, blaVIM and blaNDM were the major CHβLs. Additionally, diversely distinct STs and different replicons were identified. [ABSTRACT FROM AUTHOR]

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1pVEF1, rep1pIP501, rep2pRE25, rep9pAD1/pTEF2/pCF10 and rep6pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18ap200B and repUS1pVEF1 genes and located on ~ 20 kb and 150–200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals. [ABSTRACT FROM AUTHOR]

The RA3 plasmid, the archetype of IncU incompatibility group, represents a mosaic-modular genome of 45.9 kb. The replication module encompasses repA and repB (initiator) surrounded by two long repetitive sequences DR1 and DR2 of unknown function. Here, we mapped the origin of replication oriV to the 3′ end of repB and showed that oriV was activated by the transcription coming from orf02revp in the adjacent stability module. Using various in vivo and in vitro methods we demonstrated that the repB expression proceeded either from repBp located in the intergenic repA-repB region or from the upstream strong repAp that was autoregulated by RepA. Additionally, the repBp activity was modulated by the transcription from the overlapping, divergently oriented repXp. Both repXmRNA (antisense for repAmRNA) and its small polypeptide product, RepX, were strong incompatibility determinants. Hence, we showed that the sophisticated RA3 copy number control combined the multivalent regulation of repB expression, RepB titration by DR1, and transcriptional activation of oriV, dependent on the RA3 global regulatory network. Similarly organized replicons have been found in diverse bacterial species confirming the significance of these mechanisms in establishing the IncU plasmids in a broad spectrum of hosts. [ABSTRACT FROM AUTHOR]

The acceleration of climate change has been associated with an alarming increase in the prevalence and geographic range of tick-borne diseases (TBD), many of which have severe and long-lasting effects—particularly when treatment is delayed principally due to inadequate diagnostics and lack of physician suspicion. Moreover, there is a paucity of treatment options for many TBDs that are complicated by diagnostic limitations for correctly identifying the offending pathogens. This review will focus on the biology, disease pathology, and detection methodologies used for the Borreliaceae family which includes the Lyme disease agent Borreliella burgdorferi. Previous work revealed that Borreliaceae genomes differ from most bacteria in that they are composed of large numbers of replicons, both linear and circular, with the main chromosome being the linear with telomeric-like termini. While these findings are novel, additional gene-specific analyses of each class of these multiple replicons are needed to better understand their respective roles in metabolism and pathogenesis of these enigmatic spirochetes. Historically, such studies were challenging due to a dearth of both analytic tools and a sufficient number of high-fidelity genomes among the various taxa within this family as a whole to provide for discriminative and functional genomic studies. Recent advances in long-read whole-genome sequencing, comparative genomics, and machine-learning have provided the tools to better understand the fundamental biology and phylogeny of these genomically-complex pathogens while also providing the data for the development of improved diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

This study provides a thorough investigation of a diverse set of antimicrobial resistant (AMR) Staphylococcus aureus isolates collected from a broad range of ready-to-eat (RTE) food in various geographic regions of Russia ranging from Pskov to Kamchatka. Thirty-five isolates were characterized using the whole genome sequencing (WGS) analysis in terms of clonal structure, the presence of resistance and virulence determinants, as well as plasmid replicon sequences and CRISPR/Cas systems. To the best of our knowledge, this is the first WGS-based surveillance of Russian RTE food-associated S. aureus isolates. The isolates belonged to fifteen different multilocus sequence typing (MLST)-based types with a predominant being the ones of clonal complex (CC) 22. The isolates studied can pose a threat to public health since about 40% of the isolates carried at least one enterotoxin gene, and 70% of methicillin-resistant (MRSA) isolates carried a tsst1 gene encoding a toxin that may cause severe acute disease. In addition, plasmid analysis revealed some important characteristics, e.g., Rep5 and Rep20 plasmid replicons were a "signature" of MRSA CC22. By analyzing the isolates belonging to the same/single strain based on cgMLST analysis, we were able to identify the differences in their accessory genomes marking their dynamics and plasticity. This data is very important since S. aureus isolates studied and RTE food, in general, represent an important route of transmission and dissemination of multiple pathogenic determinants. We believe that the results obtained will facilitate performing epidemiological surveillance and developing protection measures against this important pathogen in community settings. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC = 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China. [ABSTRACT FROM AUTHOR]

This study aimed to characterize the changes in fecal carriage of Extended-Spectrum β-Lactamase (ESBL) producing Enterobacterales (ESBL-PE) in a single Dutch veal calves. During the rearing period at the Dutch veal farm, a decrease in fecal carriage of cefotaxime-resistant Escherichia coli isolates was observed after 2 weeks at the veal farm, while an increase of cefotaxime-resistant Klebsiella pneumoniae isolates was demonstrated. E. coli and K. pneumoniae were isolated from rectal swabs collected from 110 veal calves in week 2, 6, 10, 18, and 24 after their arrival at the farm. ESBL-PE isolates were selectively cultured and identified by MALDI-TOF. ESBL genes were characterized by RT-PCR, PCRs, and amplicon sequencing. A total of 80 E. coli and 174 K. pneumoniae strains were isolated from 104 out of 110 veal calves. The prevalence of ESBL- E. coli decreased from week 2 (61%) to week 6 (7%), while an unexpected increase in ESBL- K. pneumoniae colonization was detected in week 6 (80%). The predominant ESBL genes detected in E. coli isolates were bla CTX-M-15 and the non-ESBL gene bla TEM-1a, while in K. pneumoniae bla CTX-M-14 gene was detected in all isolates. Four cefotaxime-resistant K. pneumoniae isolates were randomly selected and characterized in deep by transformation, PCR-based replicon typing, and whole-genome sequencing (WGS). The clonal relatedness of a subgroup of nine animals carrying K. pneumoniae ESBL genes was investigated by Multi Locus sequence typing (MLST). In four ESBL- K. pneumoniae isolates, bla CTX-M-14 was located on IncFIIK and IncFIINK plasmid replicons and the isolates were multi-drug resistant (MDR). MLST demonstrated a clonal spread of ESBL- K. pneumoniae ST107. To the best of our knowledge, this is the first study to report a change in fecal carriage of ESBL-PE over time in the same veal calf during the rearing period. [ABSTRACT FROM AUTHOR]

Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana. These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration. [ABSTRACT FROM AUTHOR]

Partitioning systems ensure the stable inheritance of bacterial low-copynumber replicons, such as chromosomes, chromids, and megaplasmids. These loci consist of two genes encoding partition proteins A and B, and at least one parS centromere-like sequence. In chromids and megaplasmids, partitioning systems are often located in the vicinity of replication systems. An extreme example of this co-localization are alphaproteobacterial repABC replicons, where the partition (repAB) and replication (repC) genes form a single operon, with parS sequences usually positioned in close proximity to these genes. In this study, we characterized a more complex repABC system found in Paracoccus aminophilus (Rhodobacterales) megaplasmid pAMI4 (438 kb). Besides the repABC operon with a single parS site, this replicon has a 2-kb non-coding locus positioned 11.5 kb downstream of repC, which contains three additional parS repeats (3parS). We demonstrated that 3parS is bound by partition protein B in vitro and is essential for proper pAMI4 partitioning in vivo. In search of similar loci, we conducted a comparative analysis of parS distribution in other repABC replicons. This revealed different patterns of parS localization in Rhodobacterales and Rhizobiales. However, in both these taxonomic orders, parS sites are almost always located inside or close to the repABC operon. No other 3parS-like loci were found in the closest relatives of pAMI4. Another evolutionarilyindependent example of such a locus was identified as a conserved feature in chromosome 2 of Allorhizobium vitis and related replicons. [ABSTRACT FROM AUTHOR]

Self-amplifying (sa) RNA molecules—“replicons”—derived from the genomes of positive-sense RNA viruses are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a nonmonotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-saRNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of Lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as 1/10 of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements. IMPORTANCE Positive-sense RNA viruses often have a cytotoxic effect on their host cell because of the strength of their RNA replicase proteins, even though only one copy of their genome begins the viral life cycle in each cell. Noninfectious forms of them—replicons—which include just their RNA replication-related genes, are also strongly self-amplifying and cytotoxic. Accordingly, when replicons fused with nonviral genes of interest are transfected into cells to amplify expression of proteins of interest, one needs to keep the replicon “dose” sufficiently low. We demonstrate how to control the number of RNA replicons getting into transfected cells and that there is a sweet spot for the replicon dose that optimizes protein expression and cell viability. Examples are given for the case of Nodamura viral replicons with fluorescent protein reporter genes in a variety of mammalian cell lines, quantified by flow cytometry and live/dead cell assays. [ABSTRACT FROM AUTHOR]

Translation of plant plus-strand RNA viral genomes that lack a 59 cap frequently requires the use of cap-independent translation enhancers (CITEs) located in or near the 39 untranslated region (UTR). 39CITEs are grouped based on secondary structure and ability to interact with different translation initiation factors or ribosomal subunits, which assemble a complex at the 39 end that is nearly always transferred to the 59 end via a long-distance kissing-loop interaction between sequences in the 39CITE and 59 hairpins. We report here the identification of a novel 39CITE in coat protein- deficient RNA replicons that are related to umbraviruses. Umbra-like associated RNAs (ulaRNAs), such as citrus yellow vein-associated virus (CYVaV), are a new type of subviral RNA that do not encode movement proteins, coat proteins, or silencing suppressors but can independently replicate using their encoded RNA-dependent RNA polymerase. An extended hairpin structure containing multiple internal loops in the 39 UTR of CYVaV is strongly conserved in the most closely related ulaRNAs and structurally resembles an I-shaped structure (ISS) 39CITE. However, unlike ISS, the CYVaV structure binds to eIF4G and no long-distance interaction is discernible between the CYVaV ISS-like structure and sequences at or near the 59 end. We also report that the ;30- nucleotide (nt) 59 terminal hairpin of CYVaV and related ulaRNAs can enhance translation of reporter constructs when associated with either the CYVaV 39CITE or the 39CITEs of umbravirus pea enation mosaic virus (PEMV2) and even independent of a 39CITE. These findings introduce a new type of 39CITE and provide the first information on translation of ulaRNAs. IMPORTANCE Umbra-like associated RNAs (ulaRNAs) are a recently discovered type of subviral RNA that use their encoded RNA-dependent RNA polymerase for replication but do not encode any coat proteins, movement proteins, or silencing suppressors yet can be found in plants in the absence of any discernible helper virus. We report the first analysis of their translation using class 2 ulaRNA citrus yellow vein-associated virus (CYVaV). CYVaV uses a novel eIF4G-binding I-shaped structure as its 39 cap-independent translation enhancer (39CITE), which does not connect with the 59 end by a long-distance RNA:RNA interaction that is typical of 39CITEs. ulaRNA 59 terminal hairpins can also enhance translation in association with cognate 39CITEs or those of related ulaRNAs and, to a lesser extent, with 39CITEs of umbraviruses, or even independent of a 39CITE. These findings introduce a new type of 39CITE and provide the first information on translation of ulaRNAs. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) represents an increasing issue worldwide, spreading not only in humans and farmed animals but also in wildlife. One of the most relevant problems is represented by Extended-Spectrum Beta-Lactamases (ESβLs) producing Escherichia coli because they are the cause of important infections in human. Wild boars (Sus scrofa) as a source of ESβLs attracted attention due to their increasing density and their habits that lead them to be at the human-livestock-wildlife interface. The aim of this study was to increase the knowledge about the ESβLs E. coli strains carried by wild boars living in a particularly high-density area of Northern Italy. The analysis of 60 animals allowed to isolate 16 ESβL-producing E. coli strains (prevalence 23.3%), which were characterised from a phenotypical and molecular point of view. The overall analysis revealed that the 16 isolates were all not only ESβL producers but also multidrug resistant and carried different types of plasmid replicons. The genome analysis performed on a subset of isolates confirmed the heterogeneity observed with pulsed-field gel electrophoresis (PFGE) and highlighted the presence of two pandemic sequence types, ST131 and ST10, with different collections of virulence factors. The genomic context of ESβL genes further evidenced that all of them were surrounded by transposons and insertion sequences, suggesting the possibility to exchange AMR genes. Overall, this study shows the worrying dissemination of ESβL-producing E. coli in wild boars in Northern Italy, suggesting the role of these animals as a spreader of AMR and their inclusion in surveillance programmes, to shed light on the "One Health" complex interactions. [ABSTRACT FROM AUTHOR]

Copiotrophic marine bacteria of the Roseobacter group (Rhodobacterales, Alphaproteobacteria) are characterized by a multipartite genome organization. We sequenced the genomes of Sulfitobacter indolifex DSM 14862T and four related plasmid-rich isolates in order to investigate the composition, distribution, and evolution of their extrachromosomal replicons (ECRs). A combination of long-read PacBio and short-read Illumina sequencing was required to establish complete closed genomes that comprised up to twelve ECRs. The ECRs were differentiated in stably evolving chromids and genuine plasmids. Among the chromids, a diagnostic RepABC-8 replicon was detected in four Sulfitobacter species that likely reflects an evolutionary innovation that originated in their common ancestor. Classification of the ECRs showed that the most abundant plasmid system is RepABC, followed by RepA, DnaA-like, and RepB. However, the strains also contained three novel plasmid types that were designated RepQ, RepY, and RepW. We confirmed the functionality of their replicases, investigated the genetic inventory of the mostly cryptic plasmids, and retraced their evolutionary origin. Remarkably, the RepY plasmid of S. pontiacus DSM 110277 is the first high copy-number plasmid discovered in Rhodobacterales. [ABSTRACT FROM AUTHOR]