Developing Qualitative Plasmid DNA Reference Materials to Detect Mechanisms of Quinolone and Fluoroquinolone Resistance in Foodborne Pathogens

The aim of this study was to develop homogeneous and stable plasmid DNA reference materials for detecting the mechanisms of resistance to quinolones and fluoroquinolones in foodborne pathogens. The DNA fragments of 11 target genes associated with quinolone and fluoroquinolone resistance were artificially synthesized, inserted into plasmid vectors, and transferred into recipient cells. PCR and sequencing of DNA were performed to assess the genetic stability of the target DNA in recombinant Escherichia coli DH5α cells during subculturing for 15 generations. The limit of detection (LOD) of the target DNA was determined using PCR and real-time qualitative PCR (qPCR). The homogeneity and storage stability of plasmid DNA reference materials were evaluated in terms of plasmid DNA quantity, PCR-measured gene expression, and qPCR threshold cycle. All 11 target DNAs were successfully synthesized and inserted into vectors to obtain recombinant plasmids. No nucleotide mutations were identified in the target DNA being stably inherited and detectable in the corresponding plasmids during subculturing of recombinant strains. When the target DNA was assessed using PCR and qPCR, the LOD was ≤1.77 × 105 and 3.26 × 104 copies/μL, respectively. Further, when the reference materials were stored at 37 °C for 13 days, 4 °C for 90 days, and −20 °C for 300 days, each target DNA was detectable by PCR, and no mutations were found. Although the threshold cycle values of qPCR varied with storage time, they were above the LOD, and no significant differences were found in the quantity of each plasmid DNA at different timepoints. Further, the homogeneity and stability of the materials were highly consistent with the requirements of standard reference materials. To summarize, considering that our plasmid DNA reference materials conformed to standard requirements, they can be used to detect the mechanisms of quinolone and fluoroquinolone resistance in foodborne pathogens.

Characterization of a mcr-1 and CRISPR-Cas System Co-harboring Plasmid in a Carbapenemase-Producing High-Risk ST11 Klebsiella pneumoniae Strain

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring blaKPC–2 and blaOXA–48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

Recovery of Synthetic Zika Virus Based on Rio-U1 Isolate Using a Genetically Stable Two Plasmid System and cDNA Amplification

In 2016, the world experienced the unprecedented Zika epidemic. The ZIKV emerged as a major human pathogen due to its association with the impairment of perinatal development and Guillain–Barré syndrome. The occurrence of these severe cases of Zika points to the significance of studies for understanding the molecular determinants of flavivirus pathogenesis. Reverse genetics is a powerful method for studying the replication and determinants of pathogenesis, virulence, and viral attenuation of flaviviruses, facilitating the design of vaccines and therapeutics. However, the main hurdle in the development of infectious clones is the instability of full-length cDNA in Escherichia coli. Here, we described the development of a genetically stable and efficient infectious clone based on the ZIKV Rio-U1 isolated in the 2016 epidemic in Brazil. The employed strategy consisted of cloning the viral cDNA genome into two stable plasmid subclones and obtaining a high-quality cDNA template with increment in DNA mass for in vitro transcription by PCR amplification. The strategy for developing a ZIKV infectious cDNA clone designed in this study was successful, yielding a replicative and efficient clone-derived virus with high similarities with its parental virus, Rio-U1, by comparison of the proliferation capacity in mammal and insect cells. The infection of AG129 immunocompromised mice caused identical mortality rates, with similar disease progression and morbidity in the animals infected with the parental and the cDNA-derived virus. Histopathological analyses of mouse brains infected with the parental and the cDNA-derived viruses revealed a similar pathogenesis degree. We observed meningoencephalitis, cellular pyknosis, and neutrophilic invasion adjacent to the choroid plexus and perivascular cuffs with the presence of neutrophils. The developed infectious clone will be a tool for genetic and functional studies in vitro and in vivo to understand viral infection and pathogenesis better.

Evolving Populations in Biofilms Contain More Persistent Plasmids

Bacterial plasmids substantially contribute to the rapid spread of antibiotic resistance, which is a crisis in healthcare today. Coevolution of plasmids and their hosts promotes this spread of resistance by ameliorating the cost of plasmid carriage. However, our knowledge of plasmid–bacteria coevolution is solely based on studies done in well-mixed liquid cultures, even though biofilms represent the main way of bacterial life on Earth and are responsible for most infections. The spatial structure and the heterogeneity provided by biofilms are known to lead to increased genetic diversity as compared with well-mixed liquids. Therefore, we expect that growth in this complex environment could affect the evolutionary trajectories of plasmid–host dyads. We experimentally evolved Shewanella oneidensis MR-1 with plasmid pBP136Gm in biofilms and chemostats and sequenced the genomes of clones and populations. Biofilm populations not only maintained a higher diversity of mutations than chemostat populations but contained a few clones with markedly more persistent plasmids that evolved via multiple distinct trajectories. These included the acquisition of a putative toxin–antitoxin transposon by the plasmid and chromosomal mutations. Some of these genetic changes resulted in loss of plasmid transferability or decrease in plasmid cost. Growth in chemostats led to a higher proportion of variants with decreased plasmid persistence, a phenomenon not detected in biofilms. We suggest that the presence of more stable plasmid–host dyads in biofilms reflects higher genetic diversity and possibly unknown selection pressures. Overall, this study underscores the importance of the mode of growth in the evolution of antibiotic-resistant bacteria.

Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers. Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation. Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.

Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering

ABSTRACTBacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.

Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as an important human viral pathogen, causing congenital malformation including microcephaly among infants born to mothers infected with the virus during pregnancy. Phylogenetic analysis suggested that ZIKV can be classified into African and Asian lineages. In this study, we have developed a stable plasmid-based reverse genetic system for robust production of both ZIKV prototype African-lineage MR766 and clinical Asian-lineage FSS13025 strains using a tetracycline (Tet)-controlled gene expression vector. Transcription of the full-length ZIKV RNA is under the control of the Tet-responsive Ptight promoter at the 5′ end and an antigenomic ribozyme of hepatitis delta virus at the 3′ end. The transcription of infectious ZIKV RNA genome was efficiently induced by doxycycline. This novel ZIKV reverse genetics system will be valuable for the study of molecular viral pathogenesis of ZIKV and the development of new vaccines against ZIKV infection.

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

ABSTRACTThe KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequenceparSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacentmobCpromoter driving expression of themobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions inEscherichia coli, since its overexpression intransnegatively interferes with mini-RA3 replication and stable maintenance of RA3.

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

ABSTRACTLeptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including theparA,parB, andrepgenes into pMAT, a plasmid that cannot replicate inLeptospiraspp. and contains a backbone consisting of anaadAcassette,oriR6K, andoriTRK2/RP4. The inserted DNA segment was isolated from a 52-kb region withinLeptospiramayottensisstrain 200901116 that is not found in the closely related strainL. mayottensis200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use oftranscomplementation in the pathogenLeptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of theperRgene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel ofLeptospirastrains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation ofLeptospiraspp.

Isolation of the Bacillus thuringiensis plasmid carrying Bacthuricin F4 coding genes and evidence of its conjugative transfer

Introduction: Conjugation is an excellent natural mode of DNA transfer in vivo between bacteria, particularly when these conjugative elements carry technological traits such as bacteriocin encoding genes. In the present work, the bacteriocinogenic plasmid pIBF4 from Bacillus thuringiensis responsible of Bacthuricin F4 synthesis was isolated and characterized. Methodology: To isolate pIBF4, the total plasmid DNA from a non-bacteriocin transposant carrying the mini-Tn10 spectinomycin selective marker was extracted and used to transform Escherichia coli strain Top10. PIBF4 was extracted from the obtained transformant and then subjected to restriction enzyme analysis. Plasmid curing experiments were conducted to test the stability of pIBF4 at a stringent temperature of 42°C. Conjugative behavior of pIBF4 was assessed by mating experiments using the non-bacteriocin transposant mutant as a donor strain and several Bacillus thuringiensis strains as recipients. Results: The pIBF4 plasmid was isolated and had a molecular weight of 19.1 kb. Ninety-five percent of cells retained the pIBF4 plasmid after 200 generations, demonstrating its high stability. PIBF4 was successfully transferred to Bacillus thuringiensis HD1CryB strain with a transfer frequency of 1x10-8 transconjugants per donor cell. The study of the recipient host range revealed that pIBF4 is specifically transferable to Bacillus thuringiensis strains with variable transfer frequencies depending on the recipient host strain. Conclusion: Our results show that pIBF4 is a 19.1 kb highly stable plasmid transferable by conjugation to Bacillus thuringiensis strains with deferent transfer frequencies.