Plasmid Group Research Articles

Understanding the genetic basis of the incompatibility of IncK1 and IncK2 plasmids

Antimicrobial resistance is a persistent challenge in human and veterinary medicine, which is often encoded on plasmids which are transmissible between bacterial cells. Incompatibility is the inability of two plasmids to be stably maintained in one cell which is caused by the presence of identical or closely related shared determinants between two plasmids originating from partition or replication mechanisms. For I-complex plasmids in Enterobacteriacae, replication- based incompatibility is caused by the small antisense RNA stem-loop structure called RNAI. The I-complex plasmid group IncK consists of two compatible subgroups, IncK1 and IncK2, for which the RNAI differs only by five nucleotides. In this study we focussed on the interaction of the IncK1 and IncK2 RNAI structures by constructing minireplicons containing the replication region of IncK1 or IncK2 plasmids coupled with a kanamycin resistance marker. Using minireplicons excludes involvement of incompatibility mechanisms other than RNAI. Additionally, we performed single nucleotide mutagenesis targeting the five nucleotides that differ between the IncK1 and IncK2 RNAI sequences of these minireplicons. The obtained results show that a single nucleotide change in the RNAI structure is responsible for the compatible phenotype of IncK1 with IncK2 plasmids. Only nucleotides in the RNAI top loop and interior loop have an effect on minireplicon incompatibility with wild type plasmids, while mutations in the stem of the RNAI structure had no significant effect on incompatibility. Understanding the molecular basis of incompatibility is relevant for future in silico predictions of plasmid incompatibility.

Genome-Based Characterization of Multidrug-Resistant Escherichia coli Isolated from Clinical Bovine Mastitis.

Mastitis occurrence in dairy cows is a broad topic that involves several sectors, from antimicrobial resistance and virulence of strains to economic implications and cattle management practices. Here, we assessed the molecular characterization (antimicrobial resistance determinants, virulence genes, sequences type, serotypes, and plasmid types) of 178 Escherichia coli strains isolated from milk samples from cows with clinical mastitis using a genome-based k-mers approach. Of these, 53 (29.8%) showed multidrug resistance by disc diffusion. We selected eight multidrug-resistant mastitis-associated E. coli for whole-genome sequencing and molecular characterization based on raw data using k-mers. We assessed antimicrobial resistance genes, virulence factors, serotypes, Multilocus Sequence Typing (MLST), and plasmid types. The most antimicrobial resistance gene found were blaTEM-1B (7/8), tetA (6/8), strA (6/8), strB (6/8), and qnrB19 (5/8). A total of 25 virulence factors were detected encoding adhesins, capsule, enzymes/proteins, increased serum survival, hemolysin, colicins, and iron uptake. These virulence factors were associated with Extraintestinal Pathogenic E. coli. Three pandemic clones were found: ST10, ST101, and ST69. Two E. coli were assigned in the O117 serogroup and one in the O8:H25 serotype. The most common plasmid groups were IncFII (7/8) and IncFIB (6/8). Our findings contribute to the knowledge of virulence mechanisms, epidemiological aspects, and antimicrobial resistance determinants of E. coli strains obtained from clinical mammary infections of cows.

Protection of H2S against Hypoxia/Reoxygenation Injury in Rat Hippocampal Neurons through Inhibiting Phosphorylation of ROCK2 at Thr436 and Ser575.

H2S (hydrogen sulfide) protects cerebral vasodilatation and endothelial cells against oxygen-glucose deprivation/reoxygenation injury via the inhibition of the RhoA-ROCK pathway and ROCK2 expression. However, the inhibitory mechanism of H2S on ROCK2 expression is still unclear. The study aimed to investigate the target and mechanism of H2S in inhibition of ROCK2. His-ROCK2wild protein was constructed, expressed, and was used for phosphorylation assay in vitro. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the potential phosphorylation sites of ROCK2. Recombinant ROCK2wild-pEGFP-N1, ROCK2T436A-pEGFP-N1, and ROCK2S575F-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK2 expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca2+ were detected to evaluate the neuroprotective mechanism of H2S. Phosphorylation at Thr436 and Ser575 of ROCK2 was observed by mass spectrometry when Polo-like kinase 1 (PLK1) and protein kinase A (PKA) were added in vitro, and NaHS significantly inhibited phosphorylation at Thr436 and Ser575. Additionally, NaHS significantly inhibited the expression of ROCK2 and recombinant proteins GFP-ROCK2, GFP-ROCK2T436A, and GFP-ROCK2S575F in transfected RHNs. Compared with empty plasmid, GFP-ROCK2T436A, and GFP-ROCK2S575F groups, NaHS significantly inhibited the release of LDH, NSE, and Ca2+ and promoted ROCK2 activity in the GFP-ROCK2wild group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK2 protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK2T436A, and the GFP-ROCK2S575F group, NaHS had more significant inhibitory effects on hypoxia/reoxygenation (H/R) injury-induced cell viability reduction and increased LDH and NSE release in the GFP-ROCK2wild group. Exogenous H2S protected the RHNs against H/R injury via Thr436 and Ser575 of ROCK2. These findings suggested that Thr436 and Ser575 may be the dominant sites that mediated the effect of NaHS on protecting RHNs against H/R injury.

Ozonated oil alleviates dinitrochlorobenzene-induced allergic contact dermatitis via inhibiting the FcεRI/Syk signaling pathway.

Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms. Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions. Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1β, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1β, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05). Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.

High prevalence of GR2 and GR4 plasmids in Acinetobacter baumannii strains from Brazil.

Acinetobacter baumannii is Gram-negative pathogen with extensive role in healthcare-associated infections (HAIs). Plasmids in this species are important carriers of antimicrobial resistance genes. In this work we investigated the plasmids of 227 Brazilian A. baumannii genomes. A total of 389 plasmid sequences with 424 Rep proteins typed to 22 different homology groups (GRs) were identified. The GR2 plasmid group was the most predominant (40.6%), followed by the GR4 group (16.7%), representing ∼57% of all plasmids. There is a wide distribution of plasmids among the isolates and most strains carry more than one plasmid. Our analyses revealed a significant prevalence of GR4 plasmids in Brazilian A. baumannii genomes carrying several antimicrobial resistance genes, notably to carbapenem (39,43%). These plasmids harbor a MOBQ relaxase that might confer increased spreading potential in the environment. Most plasmids of the predominant groups belong to the same plasmid taxonomic unit (PTU-Pse7) and have a AbkA/AbkB toxin-antitoxin system that has a role in plasmid stability and dissemination of carbapenem resistance genes. The results of this work should contribute to our understanding of the molecular content of plasmids in a large and populous country, highlighting the importance of genomics for enhanced epidemiological surveillance.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Characterization of a Pathogenic &lt;em&gt;Escherichia coli&lt;/em&gt; Strain Derived from &lt;em&gt;Oreochromis&lt;/em&gt; spp. Farms Using Whole-Genome Sequencing

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish variety cultured. Because aquaculture practices are susceptible to microbial contamination derived from anthropogenic sources, extensive antibiotic usage is needed, leading to aquaculture systems becoming an important source of antibiotic-resistant and pathogenic bacteria of clinical relevance such as Escherichia coli (E. coli). Here, the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain, recovered from inland farmed Oreochromis spp., were elucidated through whole-genome sequencing (WGS) and in silico analysis. Antimicrobial susceptibility testing (AST) and WGS were performed. Furthermore, phylogenetic group, serotype, multilocus sequence typing (MLST), acquired antimicrobial resistance, virulence, plasmid, and prophage content were determined using diverse available web tools. The E. coli isolate only exhibited intermediate susceptibility to ampicillin and was characterized as ONT:H21-B1-ST40 strain by WGS-based typing. Although only a single antimicrobial resistance-related gene was detected [mdf(A)], several virulence-associated genes (VAGs) from the atypical enteropathogenic E. coli (aEPEC) pathotype were identified. Additionally, the cargo of plasmid replicons from large plasmid groups and 18 prophage-associated regions were detected. In conclusion, the WGS characterization of an aEPEC isolate, recovered from a fish farm in Sinaloa, Mexico, allows insights into its pathogenic potential and the possible human health risk of consuming raw aquacultural products. It is necessary to exploit next-generation sequencing (NGS) techniques for studying environmental microorganisms and to adopt a one health framework to learn how health issues originate.

Comparative genomic analysis of plasmids harboring bla OXA-48-like genes in Klebsiella pneumoniae.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious medical problem worldwide. Acquired OXA-48-like carbapenemases encoded by plasmids are important causes of carbapenem resistance in K. pneumoniae. To explore the links between plasmids and bla OXA-48-like genes in K. pneumoniae, we systematically analyzed the variants of bla OXA-48-like plasmid replicon types, phylogenetic patterns, geographic distribution, conjugative transfer regions, and the genetic environments surrounding bla OXA-48-like of 191 bla OXA-48-like-harboring plasmids, which were identified from 4451 plasmids of K. pneumoniae downloaded from GenBank. Our results showed that seven different variants of bla OXA-48-like genes were identified from the 191 bla OXA-48-like-harboring plasmids in K. pneumoniae, with bla OXA-48, bla OXA-232, and bla OXA-181 being highly prevalent. In K. pneumoniae, bla OXA-48 was mainly carried by the composite transposon Tn1999.2 located on IncL/M-type conjugative plasmids, which were mainly geographically distributed in Switzerland, Germany, and China. In K. pneumoniae, the blaOXA-232 gene was mainly carried by 6.1-kb ColKP3-type mobilizable plasmids, which were mainly isolated in India. In K. pneumoniae, bla OXA-181 was mainly carried by a group of 50-kb ColKP3-IncX3 hybrid conjugative plasmids and a group of small ColKP3-type mobilizable plasmids with lengths of 5.9-9.3 kb, the former was sporadically discovered in China, South Korea, India, and Czech Republic, while the latter was almost all isolated in India. In addition, five bla OXA-245-harboring 65.9-kb IncL plasmids of K. pneumoniae isolated in Spain were found to have the genetic context of bla OXA-245 more complicated than that of bla OXA-48-harboring IncL/M-type plasmids, with two copies of IS1R inserted both upstream and downstream of bla OXA-245-lysR. These findings enhance our understanding of the genetic diversity of bla OXA-48-like-harboring plasmids in K. pneumoniae.

Overexpression of Numb gene can inhibit the progression of biliary liver fibrosis in adult liver

Objective: To investigate whether the overexpression of Numb gene can effectively intervene the progression of cholestatic liver fibrosis (CLF) in adult liver. Methods: Twenty-four SD rats were randomly divided into sham operation (Sham, n=6), common bile duct ligation (BDL, n=6), empty vector plasmid (Numb-EV, n=6) and numb gene overexpression group (Numb-OE, n=6). The CLF model was prepared by common bile duct ligation. Simultaneously, the model was established, and the adeno-associated virus (AAV) carrying the cloned numb gene was injected into the rats' spleens. Samples were collected at the end of four weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), serum total bilirubin (TBil), serum total bile acid (TBA), liver histopathology, liver tissue hydroxyproline (Hyp) content, and alpha smooth muscle actin (α-SMA), cytokeratin (CK) 7, and CK19 expression conditions were determined in liver tissue. An analysis of variance was used to compare the means of multiple groups. Results: Compared with the Sham group, the Numb mRNA level in the rat liver tissue was significantly decreased in the BDL group (0.872±0.237 vs. 0.452±0.147, P=0.003). Compared with the Numb-EV group, the Numb mRNA level in the liver tissue was significantly increased in the Numb-OE group (0.487±0.122 vs. 1.094±0.345, P＜0.01). Compared with the Sham group, the Hyp content (μg/L) (288.46±49.49 vs. 901.98±271.85, P＜0.01) and the α-SMA mRNA level (0.858±0.234 vs. 8.976±1.398, P＜0.01) were significantly higher in the BDL group. Compared with the Numb-EV group, the Hyp content (864.32±113.54 vs. 580.44±171.77, P=0.039), the α-SMA mRNA level (6.138±1.443 vs. 1.322±0.859, P＜0.01) and the protein levels were significantly reduced in the Numb-OE group. Compared with the Sham group, the serum ALT, AST, TBil, and TBA levels were significantly increased in the BDL group (P＜0.01), and the ALB content was significantly decreased (P＜0.01). Compared with the Numb-EV group, AST and TBil levels were significantly reduced in the Numb-OE group (P＜0.01), as were the ALT and TBA levels (P＜0.05); however, the ALB content was significantly increased (P＜0.01), and the differences were statistically significant. Compared with the Sham group, the mRNA expression levels of CK7 and CK19 were significantly increased in the BDL group (1.40±0.42 vs. 43.78±7.56; 1.11±0.51 vs. 363.81±134.84, P＜0.01). The mRNA expression levels of CK7 and CK19 were significantly reduced in the OE group (343.19±81.22 vs. 3.22±2.34; 40.53±14.02 vs. 15.68±9.36,P＜0.01). Conclusion: Overexpression of the Numb gene can inhibit CLF progression in the adult liver, which may become a new target for CLF therapy.

A widespread group of large plasmids in methanotrophic Methanoperedens archaea

Anaerobic methanotrophic (ANME) archaea obtain energy from the breakdown of methane, yet their extrachromosomal genetic elements are little understood. Here we describe large plasmids associated with ANME archaea of the Methanoperedens genus in enrichment cultures and other natural anoxic environments. By manual curation we show that two of the plasmids are large (155,605 bp and 191,912 bp), circular, and may replicate bidirectionally. The plasmids occur in the same copy number as the main chromosome, and plasmid genes are actively transcribed. One of the plasmids encodes three tRNAs, ribosomal protein uL16 and elongation factor eEF2; these genes appear to be missing in the host Methanoperedens genome, suggesting an obligate interdependence between plasmid and host. Our work opens the way for the development of genetic vectors to shed light on the physiology and biochemistry of Methanoperedens, and potentially genetically edit them to enhance growth and accelerate methane oxidation rates.

A Trojan horse biomimetic delivery system using mesenchymal stem cells for HIF-1α siRNA-loaded nanoparticles on retinal pigment epithelial cells under hypoxia environment.

To demonstrate the feasibility of mesenchymal stem cell (MSC)-mediated nano drug delivery, which was characterized by the "Trojan horse"-like transport of hypoxia-inducible factor-1α small interfering RNA (HIF-1α siRNA) between MSCs and retinal pigment epithelial cells (RPE) under hypoxia environment. Plasmid and lentivirus targeting the human HIF-1α gene were designed and constructed. HIF-1α siRNA was encapsulated into poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) through the water-in-oil-in-water (w/o/w) multiple emulsion technique. The effect of PLGA-NPs uptake on the expression of HIF-1α mRNA was tested in RPE cells by real-time quantitative polymerase chain reaction (qPCR) and additional transfected conditions were used as control, including lentivirus group, nude plasmid group and blank PLGA group. MSCs were transfected with the NPs and the transfection efficacy was evaluated by flow cytometry. Transwell co-culture system of transfected MSCs and RPE cells was constructed under hypoxia environment. The effects of MSC-loaded HIF-1α siRNA PLGA-NPs on proliferation, apoptosis, and migration of RPE cells were then evaluated. The effect of transfected MSCs on HIF-1α expression of RPE cells was analyzed by using qPCR at the time points 24h, 3d, and 7d. The average diameter of PLGA-NPs loaded with HIF siRNA was 314.1 nm and the zeta potential was -0.36 mV. The transfection efficiency of PLGA-NPs was 67.3%±5.2% into MSCs by using flow cytometry. Compared with the lentivirus group, the PLGA-NPs loaded with HIF-1α siRNA can effectively reduce the expression of HIF-1α mRNA up to 7d in RPE (0.63±0.05 at 7d, P<0.001). In the Transwell co-culture system of transfected MSCs and RPE, the abilities of proliferation (2.34±0.17, 2.40±0.28, 2.47±0.24 at 48h, F=0.23, P=0.80), apoptosis (14.83%±2.43%, 12.94%±2.19%, 12.39%±3.21%; F=0.70, P=0.53) and migration (124.5±7.78, 119.5±5.32, 130±9.89, F=1.33, P=0.33) of the RPE cells had no differences between MSC-loaded HIF-1α siRNA PLGA-NPs and other groups. The inhibition of PLGA on the HIF-1α mRNA expression in RPE cells could continue until the 7th day, the level of HIF-1α mRNA was lower than that of other groups (F=171.98, P<0.001). The delivery of PLGA-NPs loaded with HIF-1α siRNA carried by MSCs is found to be beneficial temporally for HIF-1α mRNA inhibition in RPE cells under hypoxia environment. The MSC-based bio-mimetic delivery of HIF-1α siRNA nanoparticles is a potential method for therapy against choroidal neovascularization.

Effects of transcription factor SIX2 gene on the proliferation of bovine skeletal muscle satellite cells

Objective: To investigate the effect of SIX2 gene on the proliferation of bovine skeletal muscle satellite cells. Methods: Bovine skeletal muscle satellite cells were used as experimental materials, and the expression of SIX2 gene in bovine skeletal muscle satellite cells was detected by real-time quantitative PCR at 24 h, 48 h, and 72 h of proliferation. The SIX2 gene overexpression vector was constructed by homologous recombination. The SIX2 gene overexpression plasmid and the control empty plasmid were transfected into bovine skeletal muscle satellite cells, and each group had three complex Wells. The cell viability was detected by MTT assay at 24 h, 48 h and 72 h after transfection. At 48 h after transfection, the cell cycle was detected by flow cytometry, and the expressions of cell proliferation marker genes were detected by real-time quantitative PCR (qRT-PCR) and Western blot. Results: With the proliferation of bovine skeletal muscle satellite cells, the expression of SIX2 mRNA was increased. Compared with the control group, the expressions of SIX2 mRNA and protein in the SIX2 gene overexpression plasmid group were increased by 18 and 2.6 times, respectively (P＜0.01). The cell viability of the SIX2 gene overexpression plasmid group was increased (P＜0.01), the proportion of G1 cells was decreased by 24.6%, and the proportion of S phase and G2 phase cells was increased by 20.3% and 4.31%, respectively (P＜0.01). The mRNA and protein expressions of Pax7 gene were increased by 15.84 and 1.22 times, respectively, and the mRNA and protein expressions of proliferation marker genes PCNA and CCNB1 were increased by 4.82, 2.23,1.55 and 1.46 times, respectively (P＜0.01). Conclusion: Overexpression of SIX2 gene promotes the proliferation of bovine skeletal muscle satellite cells.

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Global Distribution and Diversity of Prevalent Sewage Water Plasmidomes.

ABSTRACTSewage water from around the world contains an abundance of short plasmids, several of which harbor antimicrobial resistance genes (ARGs). The global dynamics of plasmid-derived antimicrobial resistance and functions are only starting to be unveiled. Here, we utilized a previously created data set of 159,332 assumed small plasmids from 24 different global sewage samples. The detailed phylogeny, as well as the interplay between their protein domains, ARGs, and predicted bacterial host genera, were investigated to understand sewage plasmidome dynamics globally. A total of 58,429 circular elements carried genes encoding plasmid-related features, and MASH distance analyses showed a high degree of diversity. A single (yet diverse) cluster of 520 predicted Acinetobacter plasmids was predominant among the European sewage water. Our results suggested a prevalence of plasmid-backbone gene combinations over others. This could be related to selected bacterial genera that act as bacterial hosts. These combinations also mirrored the geographical locations of the sewage samples. Our functional domain network analysis identified three groups of plasmids. However, these backbone domains were not exclusive to any given group, and Acinetobacter was the dominant host genus among the theta-replicating plasmids, which contained a reservoir of the macrolide resistance gene pair msr(E) and mph(E). Macrolide resistance genes were the most common in the sewage plasmidomes and were found in the largest number of unique plasmids. While msr(E) and mph(E) were limited to Acinetobacter, erm(B) was disseminated among a range of Firmicutes plasmids, including Staphylococcus and Streptococcus, highlighting a potential reservoir of antibiotic resistance for these pathogens from around the globe.IMPORTANCE Antimicrobial resistance is a global threat to human health, as it inhibits our ability to treat infectious diseases. This study utilizes sewage water plasmidomes to identify plasmid-derived features and highlights antimicrobial resistance genes, particularly macrolide resistance genes, as abundant in sewage water plasmidomes in Firmicutes and Acinetobacter hosts. The emergence of macrolide resistance in these bacteria suggests that macrolide selective pressure exists in sewage water and that the resident bacteria can readily acquire macrolide resistance via small plasmids.

GINS2 Is Downregulated in Peripheral Blood of Patients with Intervertebral Disk Degeneration and Promotes Proliferation and Migration of Nucleus Pulposus Cells.

GINS complex subunit 2 (GINS2) regulates the migration, invasion, and growth of cells in many malignant and chronic diseases. In the present study, we aimed to investigate the expression of GINS2 in the peripheral blood and nucleus pulposus (NP) cells of patients with intervertebral disk degeneration (IDD). GINS2 expression was detected using bioinformatics tools from the GEO public repository and validated using peripheral blood samples from IDD patients and healthy participants. GINS2 clinical significance was explored by the receiver operating curve (ROC) utilizing area under the curve (AUC). Moreover, the influences of GINS2 on cell viability, migration, and invasion were explored by MTT, wound healing, and transwell assays, whereas cell apoptosis was determined by flow cytometry. Expression levels of GINS2 in the peripheral blood were significantly lower in IDD patients than in healthy participants. Moreover, ROC obtained a significantly higher AUC of GINS2 in IDD patients. Further, overexpressed GINS2 increased the proliferation, migration, and invasion of NP cells while overexpressed GINS2 decreased the apoptotic property of cells compared to the NC plasmid and control groups. In conclusion, GINS2 might be a potential therapeutic target of IDD.

Comparative genomic analysis of clinical Acinetobacter nosocomialis isolates from Terengganu, Malaysia led to the discovery of a novel tetracycline-resistant plasmid

To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance. Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform. The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes. Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

A bla SIM-1 and mcr-9.2 harboring Klebsiella michiganensis strain reported and genomic characteristics of Klebsiella michiganensis.

As a newly emerging Klebsiella pathogen, more and more Klebsiella michiganensis drug resistant strains have been reported in recent years, which posed serious threats to public health. Here we first reported a multidrug-resistant K. michiganensis strain 12084 with two bla SIM-1 and one mcr-9.2 genes isolated from the sputum specimen of a patient in the Second Affiliated Hospital of Zhejiang University School of Medicine and analyzed its genetic basis and drug-resistance phenotypes. Genetic analysis showed that this strain harbored three different incompatibility groups (IncHI2, IncHI5, and IncFIIpKPHS2:IncFIB-4.1) of plasmids (p12084-HI2, p12084-HI5, and p12084-FII). A total of 26 drug-resistance genes belonging to 12 classes of antibiotics were identified, most of which (24) were located on two plasmids (p12084-HI2 and p12084-HI5). Interestingly, two bla SIM-1 genes were identified to locate on p12084-HI2 and p12084-HI5, respectively, both of which were embedded in In630, indicating their genetic homogeny. It was noting that one bla SIM-1 gene was situated in a novel unit transposon (referred to as Tn6733) on the p12084-HI5 plasmid. We also discovered an mcr-9.2 gene on the p12084-HI2 plasmid. To the best of our knowledge, this is the first report of a bla SIM-1 and mcr-9.2 harboring K. michiganensis strain. We then investigated the population structure/classification, and antibiotic resistance for all 275 availably global K. michiganensis genomes. Population structure revealed that K. michiganensis could be divided into two main clades (Clade 1 and Clade 2); the most popular ST29 was located in Clade 1, while other common STs (such as ST50, ST27, and ST43) were located in Clade 2. Drug-resistance analysis showed 25.5% of the K. michiganensis strains (70/275) harboring at least one carbapenemase gene, indicating severe drug resistance of K. michiganensis beyond our imagination; this is a dangerous trend and should be closely monitored, especially for ST27 K. michiganensis with the most drug-resistant genes among all the STs. Overall, we reported a bla SIM-1 and mcr-9.2 harboring K. michiganensis strain, and further revealed the population structure/classification, and drug-resistance of K. michiganensis, which provided an important framework, reference, and improved understanding of K. michiganensis.

Role and mechanism of miR-548-3p/DAG1 in the occurrence and malignant transformation of laryngeal carcinoma.

The AMC-HN-8 cell line and the primary human laryngeal epi-thelial cell lines were utilized in this work to explore the molecular mecha-nism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548-3p overexpression plasmid, interference plasmid and blank plasmid were con-structed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Nor-mal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation charac-teristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteris-tics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

Molecular Epidemiology of Plasmid-Mediated Types 1 and 3 Fimbriae Associated with Biofilm Formation in Multidrug Resistant Escherichia coli from Diseased Food Animals in Guangdong, China.

ABSTRACTTypes 1 and 3 fimbriae in Enterobacteriaceae play versatile roles in bacterial physiology including attachment, invasion, cell motility as well as with biofilm formation and urinary tract infections. Herein, we investigated the prevalence and transmission of plasmid-mediated types 1 and 3 fimbriae from 1753 non-duplicate Enterobacteriaceae from diseased food Animals. We identified 123 (7.01%) strong biofilm producers and all was identified as E. coli. WGS analysis of 43 selected strong biofilm producers revealed that they harbored multiple ARGs, including ESBLs, PMQR and mcr-1. The gene clusters mrkABCDF and fimACDH encoding types 1 and 3 fimbriae, respectively, were identified among 43 (34.96%) and 7 (5.7%) of 123 strong biofilm isolates, respectively. These two operons were able to confer strong biofilm-forming ability to an E. coli weak-biofilm forming laboratory strain. Plasmid analysis revealed that mrk and fim operons were found to co-exist with ARGs and were primarily located on IncX1 and IncFII plasmids with similar backbones, respectively. mrkABCDF operons was present in all of 9457 Klebsiella pneumoniae using archived WGS data, and shared high homology to those on plasmids of 8 replicon types and chromosomes from 6 Enterobacteriaceae species from various origins and countries. In contrast, fimACDH operons was present in most of Enterobacter cloacae (62.15%), and shared high homology to those with only a small group of plasmids and Enterobacteriaceae species. This is the first comprehensive report of the prevalence, transmission and homology of plasmid-encoded type 1 and 3 fimbriae among the Enterobacteriaceae. Our findings indicated that plasmid-encoded mrkABCDF and fimACDH were major contributors to enhanced biofilm formation among E. coli and these two operons, in particular mrk could be as a potential anti-biofilm target.IMPORTANCE Biofilms allow bacteria to tolerate disinfectants and antimicrobials, as well as mammalian host defenses, and are therefore difficult to treat clinically. Most research concerning biofilm-related infections is typically focused on chromosomal biofilm-associated factors, including types 1 and 3 fimbriae of biofilm-forming Enterobacterium. However, the transmission and homology of the mobile types 1 and 3 fimbriae among Enterobacteriaceae is largely unknown. The findings revealed that the plasmid-encoded type 3 fimbriae encoded by mrkABCDF and type 1 fimbriae encoded by fimACDH were major contributors to enhancing biofilm formation among strong biofilm E. coli from diseased food producing animals. Additionally, mrk operon with high homology at an amino acid sequence was present both on plasmids of various replicon types and on chromosomes from diverse Enterobacteriaceae species from numerous origins and countries. These findings provide important information on the transmission of the mobile types 1 and 3 fimbriae among Enterobacteriaceae, indicating a potential antibiofilm target.

Protective immunity against spring viremia of carp virus by mannose modified chitosan loaded DNA vaccine

Spring viremia of carp virus (SVCV) usually be considered as one of the serious in viral diseases of aquaculture, and DNA vaccine with novel delivery mechanism or adjuvant has proven to be a promising and effective strategy to control aquatic animal diseases. In this study, the mannose-modified chitosan, a carrier system for vaccine delivery, were used to developed a chitosan-encapsulated DNA vaccine (CS-M-G) against SVCV, then investigated immune response induced by the vaccine. Our results showed that CS-M-G was confirmed the spherical or elliptical with even distribution and ranging from approximately 50 to 150 nm in size, the expression of the antigen gene could still be detected after 21 d post vaccination. The CS-M-G induces the highest antibody levels in the 20 μg dose group which is about 3 times than naked plasmid group at 21 d post vaccination, and still hold a higher level than control group at 28 d post vaccination. On the side, strongest protection with relative percent survival of 62.1% in the 20 μg CS-M-G group, which could produce significantly higher enzyme activities and up-regulated expression of immune-associated genes than control group. Thus, our results indicate that DNA vaccine loaded with mannose-modified chitosan induces strong immune response and provided an effective protection against SVCV infection, may be helpful and extended for developing more aquatic animal vaccines in the future.