Plasmid Group Research Articles

Analysis of Acinetobacter P-type type IV secretion system-encoding plasmid diversity uncovers extensive secretion system conservation and diverse antibiotic resistance determinants.

Acinetobacter baumannii is globally recognized as a multi-drug-resistant pathogen of critical concern due to its capacity for horizontal gene transfer and resistance to antibiotics. Phylogenetically diverse Acinetobacter species mediate human infection, including many considered as important emerging pathogens. While globally recognized as a pathogen of concern, pathogenesis mechanisms are poorly understood. P-type type IV secretion systems (T4SSs) represent important drivers of pathogen evolution, responsible for horizontal gene transfer and secretion of proteins that mediate host-pathogen interactions, contributing to pathogen survival, antibiotic resistance, virulence, and biofilm formation. Genes encoding a P-type T4SS were previously identified on plasmids harboring the carbapenemase gene blaNDM-1 in several clinically problematic Acinetobacter; however, their prevalence among the genus, geographical distribution, the conservation of T4SS proteins, and full capacity for resistance genes remain unclear. Using systematic analyses, we show that these plasmids belong to a group of 53 P-type T4SS-encoding plasmids in 20 established Acinetobacter species, the majority of clinical relevance, including diverse A. baumannii sequence types and one strain of Providencia rettgeri. The strains were globally distributed in 14 countries spanning five continents, and the conjugative operon's T4SS proteins were highly conserved in most plasmids. A high proportion of plasmids harbored resistance genes, with 17 different genes spanning seven drug classes. Collectively, this demonstrates that P-type T4SS-encoding plasmids are more widespread among the Acinetobacter genus than previously anticipated, including strains of both clinical and environmental importance. This research provides insight into the spread of resistance genes among Acinetobacter and highlights a group of plasmids of importance for future surveillance.

Effects of PTPN6 Gene Knockdown in SKM-1 Cells on Apoptosis, Erythroid Differentiation and Inflammations

Objective: Protein tyrosine phosphatase non-receptor type 6 (PTPN6) is a cytoplasmic phosphatase that acts as a key regulatory protein in cell signaling to control inflammation and cell death. In order to investigate the role of PTPN6 in hematologic tumor myelodysplastic syndrome (MDS), this study infected SKM-1 cell line (MDS cell line) with packaged H_PTPN6-shRNA lentivirus to obtain H_PTPN6-shRNA SKM-1 stable strain. The effect of PTPN6 knockdown on apoptosis, erythroid differentiation, and inflammations in SKM-1 cell line was examined. Methods: The stable knockdown SKM-1 cell line was validated using qPCR and Western blot assays. The proliferation activity, apoptosi, erythroid differentiation, and inflammatory cytokines in SKM-1 cells were assessed before and after transfection. Results: qPCR confirmed that the expression level of H_PTPN6-shRNA in SKM-1 cells was significantly reduced, and Western blot showed that the protein expression level of H_PTPN6-shRNA in SKM-1 cells was also significantly reduced. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell viability. Flow cytometry revealed that the apoptosis rate of cells in the PTPN6 knockdown group was 0.8%, lower than the 2.7% observed in the empty plasmid group; the expression rate of the erythroid differentiation marker CD235a was 13.2%, lower than the 25.0% observed in the empty plasmid group. The expression levels of the proinflammatory factors IL-6 and IL-8 increased, and the expression levels of the inhibitor factor IL-4 decreased. Conclusions: The PTPN6 gene was successfully knocked down using lentivirus-mediated transduction, and the constructed cell line was validated using PCR and Western blot. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell proliferation viability. Flow cytometry analysis of apoptosis and erythroid differentiation indicated that PTPN6 knockdown inhibits apoptosis and erythroid differentiation in SKM-1 cells and also alters the level of inflammations in the bone marrow microenvironment. It suggests that the PTPN6 gene acts as a tumor suppressor in myelodysplastic syndrome cells, influencing hematopoietic cell apoptosis, erythroid differentiation, and inflammations. This provides a reliable experimental basis for further in-depth studies on the mechanism of PTPN6 in MDS and related pharmacological research.

ADAM8 promotes alcoholic liver fibrosis through the MAPK signaling pathway

The effect and molecular regulatory mechanism of A Disintegrin and Metalloproteinase 8 (ADAM8) were explored in alcoholic liver fibrosis (ALF). C57BL/6N male mice were randomly divided into control, alcohol, and ADAM8-sgRNA3 plasmid groups. The control group received control liquid diet, while the alcohol and ADAM8-sgRNA3 plasmid groups were given alcohol liquid feed diet combined with ethanol gavage treatment for 8 weeks to induce ALF modeling. In addition, the ADAM8-sgRNA3 plasmid group was injected with the effective ADAM8-sgRNA3 plasmid, while the alcohol and control group mice were injected with an equivalent amount of physiological saline. LX-2 human hepatic stellate cells were divided into control, alcohol, si-ADAM8-2, and si-ADAM8-NC groups and induced for 48 h for model establishment in vitro. Serological detection, pathological staining, Western blotting, qRT-PCR and CCK8 assay were performed for experiments. Compared with the alcohol group, ADAM8 mRNA, protein and, positive area rate, serological indicators, pathological changes, and the expression of liver fibrosis marker and MAPK signaling pathway-related factors in the ADAM8-sgRNA3 plasmid group significantly decreased in vivo. Compared with the alcohol group, ADAM8 mRNA and protein expression, cell viability, and the expression of liver fibrosis markers and MAPK signaling pathway-related factors (p-ERK1/2, PCNA, Bcl-2, p-c-Jun, TGFβ1, p–p38 MAPK and HSP27) reduced significantly in the si-ADAM8-2 group. Therefore, ADAM8 promotes ALF through the MAPK signaling pathway, a promising target for treating ALF.

High-throughput single-cell sequencing of activated sludge microbiome

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

Towards quantifying plasmid similarity.

Plasmids are extrachromosomal replicons which can quickly spread resistance and virulence genes between clinical pathogens. From the tens of thousands of currently available plasmid sequences we know that overall plasmid diversity is structured, with related plasmids sharing a largely conserved 'backbone' of genes while being able to carry very different genetic cargo. Moreover, plasmid genomes can be structurally plastic and undergo frequent rearrangements. So, how can we quantify plasmid similarity? Answering this question requires practical efforts to sample natural variation as well as theoretical considerations of what defines a group of related plasmids. Here we consider the challenges of analysing and rationalising the current plasmid data deluge to define appropriate similarity thresholds.

Research note: characteristics of blaNDM and mcr-1 co-producing Escherichia coli from retail chicken meat

Carbapenems and colistin are vital antimicrobials used to treat Enterobacteriaceae-caused infections. The present study aimed to characterize the coexistence mechanism of carbapenem and colistin resistance in an Escherichia coli isolated from retail chicken meat. A total of 4 E. coli isolates co-harboring carbapenem resistance gene blaNDM (2 E. coli isolates with blaNDM-5 and 2 with blaNDM-9) and colistin resistance gene mcr-1. Antimicrobial susceptibility testing exhibited that all the 4 E. coli strains had multidrug resistance profile and consistent with the resistance genes they carried. MLST showed that 3 E. coli isolates belonged to a pathogenic E. coli lineage ST354, which is closely associated with human infections and pose a serious threat to public health. Whole genome sequencing (WGS) showed that 4 mcr-1-positive plasmids with sizes of 60.4 kb-67.4 kb all belonged to the IncI2 type. A total of 5 blaNDM-harboring plasmids ranged from 99.0 kb∼138.3 kb, among which 4 plasmids belonged to unknow type and only pCS5L-NDM belonged to IncFIA/IncFIB group of hybrid plasmids, a novel carrier for blaNDM. Comparative analysis exhibited that the mcr-1 or blaNDM-carrying plasmids of E. coli strains from chicken meat showed high identity with that from Enterobacteriaceae of human origin, which indicated the risk of mcr-1 or blaNDM dissemination from retail meat to human. The simultaneous occurrence of mcr-1 and blaNDM in E. coli emphasizes the significant of antimicrobial resistance surveillance in retail meat.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Chromosomal and plasmid localization of ileS2 in high-level mupirocin-resistant Staphylococcus pseudintermedius and Staphylococcus aureus isolated from canine and feline origins.

To characterize the mobile genetic elements and genetic localization of ileS2 in high-level mupirocin-resistant (Hi-MupR) methicillin-resistant Staphylococcus pseudintermedius (MRSP) and MRSA isolates recovered from canine and feline clinical samples. The identification of bacterial species and presence of mecA and ileS2 genes in MRSP and MRSA isolates were performed using MALDI-TOF MS and PCR, respectively. Antimicrobial resistance (AMR) phenotypes were determined by broth microdilution assays. The genome characteristics, ileS2-containing elements and staphylococcal cassette chromosome mec (SCCmec) were illustrated using complete circular genomes obtained from hybrid assembly of Illumina short-reads and Oxford Nanopore Technologies long-reads. These were analysed through phylogenetic and bioinformatics approaches. A total of 18 MRSP clinical isolates and four MRSA clinical isolates exhibited the Hi-MupR phenotype and carried multiple AMR genes, including mecA and ileS2 genes. MRSP ST182-SCCmec V (n = 6) and ST282-ΨSCCmec57395-t10 (n = 4) contained the ileS2 transposable unit associated with IS257 on the chromosome. Three MRSA ST398-SCCmec V-t034/t4652 isolates carried ∼42 kb pSK41-like ileS2 plasmids, whereas similar ileS2 plasmids lacking tra genes were found in MRSP ST282-ΨSCCmec57395-t72/t21 isolates. Furthermore, a new group of ileS2 plasmids, carried by MRSP ST45-ΨSCCmec57395, ST433-ΨSCCmecKW21-t05 and ST2165-SCCmec IV-t06, and by one MRSA ST398-SCCmec V-t034 strain, shared the plasmid backbone with the cfr/fexA-carrying plasmid pM084526_1 in MRSA ST398. This study provides the first evidence of ileS2 integration into the S. pseudintermedius chromosome, which is a rare occurrence in staphylococcal species, and plasmids played a pivotal role in dissemination of ileS2 in both staphylococcal species.

Preparation of interleukin-1β-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice

Objective: To prepare interleukin-1β-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1β-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1β-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1β-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1β (IL-1β) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1β antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 μg/ml and 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis.

Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 μmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 μmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Characterization of the diversity of type IV secretion system-encoding plasmids in Acinetobacter

ABSTRACT The multi-drug resistant pathogen Acinetobacter baumannii has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, A. baumannii has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant A. baumannii ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the E. coli F-plasmid, and plasmids with homologous gene clusters have been identified in several A. baumannii sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of Acinetobacter and one strain of Klebsiella pneumoniae from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent A. baumanii, and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among Acinetobacter than previously thought, and that they represent an important potential reservoir for future clinical concern.

The plasmidome associated with Gram-negative bloodstream infections: A large-scale observational study using complete plasmid assemblies

Plasmids carry genes conferring antimicrobial resistance and other clinically important traits, and contribute to the rapid dissemination of such genes. Previous studies using complete plasmid assemblies, which are essential for reliable inference, have been small and/or limited to plasmids carrying antimicrobial resistance genes (ARGs). In this study, we sequenced 1,880 complete plasmids from 738 isolates from bloodstream infections in Oxfordshire, UK. The bacteria had been originally isolated in 2009 (194 isolates) and 2018 (368 isolates), plus a stratified selection from intervening years (176 isolates). We demonstrate that plasmids are largely, but not entirely, constrained to a single host species, although there is substantial overlap between species of plasmid gene-repertoire. Most ARGs are carried by a relatively small number of plasmid groups with biological features that are predictable. Plasmids carrying ARGs (including those encoding carbapenemases) share a putative ‘backbone’ of core genes with those carrying no such genes. These findings suggest that future surveillance should, in addition to tracking plasmids currently associated with clinically important genes, focus on identifying and monitoring the dissemination of high-risk plasmid groups with the potential to rapidly acquire and disseminate these genes.

Genomic investigation of multispecies and multivariant blaNDM outbreak reveals key role of horizontal plasmid transmission.

New Delhi metallo-β-lactamases (NDMs) are major contributors to the spread of carbapenem resistance globally. In Australia, NDMs were previously associated with international travel, but from 2019 we noted increasing incidence of NDM-positive clinical isolates. We investigated the clinical and genomic epidemiology of NDM carriage at a tertiary-care Australian hospital from 2016 to 2021. We identified 49 patients with 84 NDM-carrying isolates in an institutional database, and we collected clinical data from electronic medical record. Short- and long-read whole genome sequencing was performed on all isolates. Completed genome assemblies were used to assess the genetic setting of blaNDM genes and to compare NDM plasmids. Of 49 patients, 38 (78%) were identified in 2019-2021 and only 11 (29%) of 38 reported prior travel, compared with 9 (82%) of 11 in 2016-2018 (P = .037). In patients with NDM infection, the crude 7-day mortality rate was 0% and the 30-day mortality rate was 14% (2 of 14 patients). NDMs were noted in 41 bacterial strains (ie, species and sequence type combinations). Across 13 plasmid groups, 4 NDM variants were detected: blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-7. We noted a change from a diverse NDM plasmid repertoire in 2016-2018 to the emergence of conserved blaNDM-1 IncN and blaNDM-7 IncX3 epidemic plasmids, with interstrain spread in 2019-2021. These plasmids were noted in 19 (50%) of 38 patients and 35 (51%) of 68 genomes in 2019-2021. Increased NDM case numbers were due to local circulation of 2 epidemic plasmids with extensive interstrain transfer. Our findings underscore the challenges of outbreak detection when horizontal transmission of plasmids is the primary mode of spread.

A comprehensive list of genes required for the efficient conjugation of plasmid Rts1 was determined by systematic deletion analysis.

While conjugation-related genes have been identified in many plasmids by genome sequencing, functional analyses have not yet been performed in most cases, and a full set of conjugation genes has been identified for only a few plasmids. Rts1, a prototype IncT plasmid, is a conjugative plasmid that was originally isolated from Proteus vulgaris. Here, we conducted a systematic deletion analysis of Rts1 to fully understand its conjugation system. Through this analysis along with complementation assays, we identified 32 genes that are required for the efficient conjugation of Rts1 from Escherichia coli to E. coli. In addition, the functions of the 28 genes were determined or predicted; 21 were involved in mating-pair formation, three were involved in DNA transfer and replication, including a relaxase gene belonging to the MOBH12 family, one was involved in coupling, and three were involved in transcriptional regulation. Among the functionally well-analysed conjugation systems, most of the 28 genes showed the highest similarity to those of the SXT element, which is an integrative conjugative element of Vibrio cholerae. The Rts1 conjugation gene set included all 23 genes required for the SXT system. Two groups of plasmids with conjugation systems nearly identical or very similar to that of Rts1 were also identified.

Identification of the reporter gene combination that shows high contrast for cellular level MRI.

Currently, we can label the certain cells by transducing specific genes, called reporter genes, and distinguish them from other cells. For example, fluorescent protein such as green fluorescence protein (GFP) is commonly used for cell labeling. However, fluorescent protein is difficult to observe in living animals. We can observe the reporter signals of the luciferin-luciferase system from the outside of living animals using in vivo imaging systems, although the resolution of this system is low. Therefore, in this study, we examined the reporter genes, which allowed the MRI-mediated observation of labeled cells in living animals. As a preliminary stage of animal study, we transduced some groups of plasmids that coded the protein that could take and store metal ions to the cell culture, added metal ions solutions, and measured their T1 or T2 relaxation values. Finally, we specified the best reporter gene combination for MRI, which was the combination of transferrin receptor, DMT1, and Ferritin-M6A for T1WI, and Ferritin-M6A for T2WI.

Contribution of Native Plasmids of Pantoea vagans C9-1 to Epiphytic Fitness and Fire Blight Management on Apple and Pear Flowers and Fruits.

Pantoea vagans C9-1 (C9-1) is a biological control bacterium that is applied to apple and pear trees during bloom for suppression of fire blight, caused by Erwinia amylovora. Strain C9-1 has three megaplasmids: pPag1, pPag2, and pPag3. Prior bioinformatic studies predicted these megaplasmids have a role in environmental fitness and/or biocontrol efficacy. Plasmid pPag3 is part of the large Pantoea plasmid (LPP-1) group that is present in all Pantoea spp. and has been hypothesized to contribute to environmental colonization and persistence, while pPag2 is less common. We assessed fitness of C9-1 derivatives cured of pPag2 and/or pPag3 on pear and apple flowers and fruit in experimental orchards. We also assessed the ability of a C9-1 derivative lacking pPag3 to reduce populations of E. amylovora on flowers and disease incidence. Previously, we determined that tolerance to stresses imposed in vitro was compromised in derivatives of C9-1 lacking pPag2 and/or pPag3; however, in this study, the loss of pPag2 and/or pPag3 did not consistently reduce the fitness of C9-1 on flowers in orchards. Over the summer, pPag3 contributed to survival of C9-1 on developing apple and pear fruit in two of five trials, whereas loss of pPag2 did not significantly affect survival of C9-1. We also found that loss of pPag3 did not affect C9-1's ability to reduce E. amylovora populations or fire blight incidence on apple flowers. Our findings partially support prior hypotheses that LPP-1 in Pantoea species contributes to persistence on plant surfaces but questions whether LPP-1 facilitates host colonization.

Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy