Towards a better understanding of antimicrobial resistance dissemination:what can be learnt from studying model conjugative plasmids?

Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.

Construction and Identification of the Helper Plasmids for Reverse Genetic System of Rabies Virus Street Strain

To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.

Effect of excessive cadmium chloride on the plasmids of E.coli HB101 in vivo

After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa 5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl 2 constantly for 24h, these plasmids were isolated from E. coli, and the effect of excessive CdCl 2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl 2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl 2 did not inhibit the replication and amp + genes expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl 2 treatment for 24 hours might cause the sequences change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl 2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl 2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.

Microbial and enzyme technology: An efficient and convenient method for MiniPrep analysis of recombinant plasmids

Minipreparation (MiniPrep) analysis is an essential step for obtaining a recombinant plasmid that carries a DNA insert containing a gene of interest. The most commonly used method for this involves cultivation of transformed Escherichia coli (E. coli) in liquid medium, brief centrifugation for precipitation of bacterial pellets, and subsequent lysis of the pellets. This process is time-consuming and laborious, especially when the sample number is high. Here, we describe a more convenient method for MiniPrep analysis that utilizes solid medium-based cultivation of bacteria.

A simplified protocol for the semi-large scale recovery of plasmids from <i>Escherichia coli</i>grown on agar plates

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100-200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50-300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

Rapid Screening of Recombinant Plasmids by Direct Colony Quantitative Real-Time PCR

Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.

Minimizing endogenous cryptic plasmids to construct antibiotic-free expression systems for Escherichia coli Nissle 1917

The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.

Low Concentration of Wenyang Tonglin Decoction Promotes Conjugation and Transfer of Drug-Resistant Plasmids among Heterologous Strains

Objective To investigate the effect of low concentration of Wenyang Tonglin Decoction(WTD)on the binding conditions of R45 plasmid conjugative transfer under liquid phase conjugation and its mechanism.Methods Escherichia coli CP9(R45)and Staphylococcus aureus RN450RF were cultured in medium containing WTD,and their minimum inhibitory concentration(MIC)values were obtained.Using promoter fusion technology,E.coli CP9(R45)containing a promoter fusion was obtained.β-Galactosidase activity of TrfAp and TrbBp was tested,and the mRNA expression of regulatory factors(TrbA,KorA,and KorB)was detected by real-time fluorescent quantitative polymerase chain reaction.Results The MIC of E.coli CP9(R45)was 400 g/L and that of S.aureus RN450RF was 200 g/L.When the drug concentration in the culture medium was 200 g/L,the highest number of conjugants was(3.47±0.20)×10^(7) CFU/mL At 90 h of conjugation,the maximum number of conjugants was(1.15±0.06)×10^(8) CFU/mL When the initial bacterial concentration was 10^(8) CFU/mL,the maximum number of conjugants was(3.47±0.20)×10^(7) CFU/mL.When the drug concentration was 200 g/L,theβ-galactosidase activity of TrfAp and TrbBp significantly increased;the relative quantification of TrbA,KorA and KorB were significantly inhibited.Conclusion Low concentration of WTD promoted the development of bacterial resistance by affecting promoters and inhibiting the expression of regulatory factors.

Molecular characterization of bla_(NDM)-harboring plasmids reveal its rapid adaptation and evolution in the Enterobacteriaceae

Carbapenem is one of the few available drugs to treat multidrug-resistance Gram-negative bacteria infections.Recently,the plasmid-mediated spread of the carbapenem resistance gene bla_(NDM) poses a significant threat to public health,requiring global monitoring and surveillance.Here,we used both short-read(n=2461)and long-read(n=546)sequencing data to characterize the global distribution of bla_(NDM).We analyzed the replicon type of bla_(NDM)-positive plasmids and found that the dominant plasmid type was different in diverse geographical locations.Although bla_(NDM)gene has been transferred across diverse countries,its genetic backgrounds are highly conserved,and the mobile genetic element ISAba125,IS5,and IS26 may play an important role in the mobilization of bla_(NDM).A significant association was observed between host origin and gene presence/deletion variation on IncX3 plasmid,which may be a key factor in the bacterial adaption to diverse hosts.In this study,we analyzed the diversity,distribution and transmission of bla_(NDM)-positive plasmids from a global perspective,and emphasize the importance of plasmid analysis for understanding the evolution and adaptation of bla_(NDM)-positive plasmids and their co-evolution with bacterial genomes(resistome).

The function of three indigenous plasmids in Mesorhizobium huakuii 2020 and its symbiotic inter-action with Sym pJB5JI of Rhizobium leguminosarum

A Mesorhizobium huakuii strain 2020, isolated from a rice-growing field in southern China, contains three indigenous plasmids named p2020a, p2020b and p2020c, respectively. The plasmids were deleted via Tn5-sacB insertion, and two cured derivatives were obtained. Interestingly, the mutant 2020D29 curing of p2020c could significantly enhance the capacity of symbiotic nitrogen fixation. But the mutant 2020D8 curing of p2020b lost the ability to nodulate Astragalus sinicus. Furthermore, the third plasmid p2020a could be hardly eliminated, suggesting that some house-keeping genes necessary for strain growth located on this plasmid. Then the Sym plasmid pJB5JI of R. leguminosarum bv. viciae was transferred into 2020 and its cured derivatives. The pot plant test showed that the ability of competition and symbiotic nitrogen fixation of transconjugant 2020-137 (pJB5JI) was increased evidently in con-trast to 2020. pJB5JI could not restore the ability of 2020D8 to nodulate Astragalus sinicus. 2020D8-8 (pJB5JI) could form ineffective nodules on peas, which implied that the symbiotic plasmid pJB5JI could express its function at the chromosomal background of Mesorhizobium huakuii 2020. The plas-mid stability was checked in transconjugants under free-living and during symbiosis. The results indi-cated that pJB5JI failed to be detected in some nodule isolates. That Km resistance gene could be am-plified from all transconjugants and nodule isolates suggested that pJB5JI was fully or partially inte-grated into the chromosome of recipients.

Mobile genetic elements facilitate the transmission of antibiotic resistance genes in multidrug-resistant Enterobacteriaceae from duck farms

Multidrug-resistant(MDR)Enterobacteriaceae critically threaten duck farming and public health.The phenotypes,genotypes,and associated mobile genetic elements(MGEs)of MDR Enterobacteriaceae isolated from 6 duck farms in Zhejiang Province,China,were investigated.A total of 215 isolates were identified as Escherichia coli(64.65%),Klebsiella pneumoniae(12.09%),Proteus mirabilis(10.23%),Salmonella(8.84%),and Enterobacter cloacae(4.19%).Meanwhile,all isolates were resistant to at least two antibiotics.Most isolates carried tet(A)(85.12%),blaTEM(78.60%)and sul1(67.44%)resistance genes.Gene co-occurrence analysis showed that the resistance genes were associated with IS26 and integrons.A conjugative IncFII plasmid pSDM004 containing all the above MGEs was detected in Proteus mirabilis isolate SDM004.This isolate was resistant to 18 antibiotics and carried the blaNDM-5 gene.MGEs,especially plasmids,are the primary antibiotic resistance gene transmission route in duck farms.These findings provide a theoretical basis for the rational use of antibiotics in farms which are substantial for evaluating public health and food safety.

Remodeling tumor microenvironment using pH-sensitive biomimetic co-delivery of TRAIL/R848 liposomes against colorectal cancer

Background:Despite significant advancements in the development of anticancer therapies over the past few decades,the clinical management of colorectal cancer remains a challenging task.This study aims to investigate the inhibitory effects of cancer-targeting liposomes against colorectal cancer.Materials and Methods:Liposomes consisting of 3β-[N-(N′,N′-dimethylamino ethane)carbamoyl]-cholesterol(DC-CHOL),cholesterol(CHOL),and dioleoylphosphatidylethanolamine(DOPE)at a molar ratio of 1:1:0.5 were created and used as carriers to deliver an apoptosis-inducing plasmid encoding the tumor necrosis factor-related apoptosis-inducing ligand(pTRAIL)gene,along with the toll-like receptor(TLR7)agonist Rsiquimod(R848).The rationale behind this design is that pTRAIL can trigger cancer cell apoptosis by activating the DR4/5 receptor,while R848 can stimulate the immune microenvironment.Results:Experimental results demonstrated the synergistic effects of R848 and pTRAIL encapsulated by liposomes(RTL)in suppressing the proliferation of colorectal cancer cells.Moreover,further in vivo investigations revealed the strong anti-tumor efficacy of RTL in xenograft and orthotropic in situ models of colorectal cancer.Conclusions:These findings collectively highlight the therapeutic potential of R848/pTRAIL-loaded liposomes in the treatment of colorectal cancer.

In Vitro Antioxidant and Radio Protective Activities of Lycopene from Tomato Extract against Radiation—Induced DNA Aberration

Background: The accumulation of free radicals is linked to a number of diseases. Free radicals can be scavenged by antioxidants and reduce their harmful effects. It is therefore essential to look for naturally occurring antioxidants that come from plants, as synthetic antioxidants are toxic, carcinogenic and problematic for the environment. Lycopene is one of the carotenoids, a pigment that dissolves in fat and has antioxidant properties. Materials and Methods: The antioxidant and free radical scavenging activity were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The impact of lycopene on bacteria (E. coli) susceptibility to γ-radiation was examined by radio sensitivity assay. The study also examined the induction of strand breaks in plasmid pUC19 DNA and how lycopene extract protected the DNA from γ-radiation in vitro. Results: At varying concentrations, lycopene demonstrated its ability to scavenge free radicals such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH). IC50 for lycopene was determined at 112 μg/mL which was almost partial to IC50 of standard antioxidant L-ascorbic acid. The D10 value 180 Gy of E. coli was found to be >2-fold higher in the extract-containing lycopene sample than in the extract-free controls. The lycopene extracts inhibited the radiation-induced deterioration of the plasmid pUC19 DNA. At an IC50 concentration, lycopene provided the highest level of protection. Conclusion: Lycopene functions as an efficient free radical scavenger and possible natural antioxidant source. For cancer patients and others who frequently expose themselves to radiation, lycopene may be a useful plant-based pharmaceutical product for treating a variety of diseases caused by free radicals.

Characteristics of two transferable aminoglycoside resistance plasmids in Escherichia coli isolated from pig and chicken manure

Plasmid-mediated antibiotic resistance genes (ARGs) have recently become a more prominent concern in the global environment. However, the prevalence of aminoglycoside resistance plasmids in the livestock industry is under reported. In this study, two transferable aminoglycoside resistance plasmids, pRKZ3 and pKANJ7, isolated from pig and chicken manure, were characterized. Results showed that pRKZ3 (8236 bp) is a non-conjugative IncQ plasmid and contains genes encoding for plasmid replication and stabilization (repA, repB and repC), mobilization (mob), and antibiotic resistance (arr-3 and aacA). pKANJ7 (30142 bp) is a conjugative IncX plasmid which codes for a type IV secretion system (T4SS). Conjugative transfer experiments showed that the optimal mating time of pKANJ7 was 8 h under the starvation condition, but the number of tranconjugants increased with time under the nutrient condition. Statistical analysis indicated that the two plasmids had little impact on the growth of their hosts, but a relatively high level of fitness cost due to pKANJ7 was observed. We also found that the fitness cost of plasmids depended on their hosts. Compared with pKANJ7, the relative fitness cost index of pRKZ3 varied within a narrow range during the 10 days of competition. The low level of fitness cost of pRKZ3 might contribute to the persistence of the plasmid in the environment. Our study provides new information for understanding the characterizations of antibiotic resistance plasmids (ARPs) in manure sources and helps to clarify the transfer and persistence of ARPs in the environment following the application of manure.

Detection of Plasmids in Gloeocapsa sp. 589 and Gloeothece sp. G06

Since the first discovery of cyanobacterian plasmids in 1973, more than 60 strains have been found to have extra-chromosomal DNA, and their distribution covers almost all groups of the cyanobacteria. However, they were not discovered in the two unicellular genera Gloeocapsa and Gloeothece, and no further report was there on them ever since. Some species of these two genera having the ability to fix atmospheric nitrogen aerobically carry

Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

QuickChange mutagenesis is the method of choice for site-directed mutagenesis （SDM） of target sequences in a plasmid. It can be applied successfully to small plasmids （up to 10 kb）. However, this method cannot efficiently mutate bigger plasmids. Using KOD Hot Start polymerase in combination with high performance liquid chromatography （HPLC） purified primers, we were able to achieve SDM in big plasmids （up to 16 kb） involving not only a single base change but also multiple base changes. Moreover, only six polymerase chain reaction （PCR） cycles and 0.5μI of polymerase （instead of 18 PCR cycles and 1.0 μI of enzyme in the standard protocol） were sufficient for the reaction.

IDENTIFICATION OF THE fi FEATURE OF THE DRUG RESISTANT PLASMIDS pFD10 AND pFD11 IN E. COLI

This paper deals with a method of identifying, with transposon, the fertility inhibition feature of plasmids. Using the method of labelling the F′lac pro plasmid with transposon Tn5, we identified the fi feature of the drug resistant plasmids pFD10 and pFD11 with R144 as a control. The results obtained by this method are identical with that by the recombination method. However, the former is simple and more convenient. When the pFD10 plasmid coexisted with the F′lac pro plasmid, the fre-

Transport of antibiotic resistance plasmids in porous media and the influence of surfactants

Transport of engineered antibiotic resistance plasmids in porous media has been reported to potentially cause significant spreading of antibiotic resistance in the environment. In this work, transport of an indigenous resistance plasmid pK5 in porous media was investigated through packed column experiments. At identical ionic strengths in CaC12 solutions, the breakthroughs of pK5 from soil columns were very close to those from quartz sand columns, indicating that transport ofpK5 in quartz sand and soil was similar. A similarity in transport behavior was also tbund between pK5 and an engineered plasmid pBR322 that has approximately the same number of base pairs as pK5. The influence of surfactants, a major group of constituents in soil solutions, was examined using an engineered plasmid pcDNA3.1（＋）/myc-His A. The impact of an anionic surfactant, sodium dodecyl sulfate （SDS）, was negligible at concentrations up to 200 rag. L I. Cetyltrimethyl ammonium bromide （CTAB）, a cationic surfactant, was fbund to significantly enhance plasmid adsorption at high concentrations. However~ at environmentally relevant concentrations （ 〈 I mg. L I）, the effect of this surfactant was also minimal. The negligible impact of surfactants and the similarity between the transport of engineered and indigenous plasmids indicate that under environmentally relevant conditions, indigenous plasmids in soil also have the potential to transport over long distances and lead to the spreading of antibiotic resistance.