Plasmid Stability Research Articles

Genomic insights into the emergence and spread of NDM-1-producing Vibrio spp. isolates in China.

Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health. To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio. A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains. In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts. The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide.

Design of a novel switchable antibody display system in Pichiapastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomycescerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichiapastoris (Komagataellaphaffii). Establishment of surface display in P.pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments(DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P.pastoris formaldehyde dehydrogenase (FLD1) promoter, S.cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyceslactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S.cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P.pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Substrate-inducible and antibiotic-free high-level 4-hydroxyvaleric acid production in engineered Escherichia coli

In this study, we developed a levulinic acid (LA)-inducible and antibiotic-free plasmid system mediated by HpdR/P hpdH and infA-complementation to produce 4-hydroxyvaleric acid (4-HV) from LA in an engineered Escherichia coli strain. The system was efficiently induced by the addition of the LA substrate and resulted in tight dose-dependent control and fine-tuning of gene expression. By engineering the 5′ untranslated region (UTR) of hpdR mRNA, the gene expression of green fluorescent protein (GFP) increased by at least two-fold under the hpdH promoter. Furthermore, by evaluating the robustness and plasmid stability of the proposed system, the engineered strain, IRV750f, expressing the engineered 3-hydroxybutyrate dehydrogenase (3HBDH∗) and formate dehydrogenase (CbFDH), produced 82 g/L of 4-HV from LA, with a productivity of 3.4 g/L/h and molar conversion of 92% in the fed-batch cultivation (5 L fermenter) without the addition of antibiotics or external inducers. Overall, the reported system was highly beneficial for the large-scale and cost-effective microbial production of value-added products and bulk chemicals from the renewable substrate, LA.

Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6

BackgroundBacterial cells often harbor multiple plasmids, but the interaction between these plasmids is poorly understood. Pseudomonas putida ND6, one of the typical naphthalene-degrading bacteria, contains two annotated plasmids, pND6-1 and pND6-2. The conjugative plasmid pND6-2 can be used as a helper to mobilize the naphthalene-degrading plasmid pND6-1. The role of pND6-1 in functions other than naphthalene degradation has never been explored. Therefore, it is of great significance to study the possible effect of pND6-1 on the transfer rate or maintenance of pND6-2. ResultsTo investigate the interaction between pND6-1 and pND6-2, we successfully cured plasmid pND6-1 using the incompatibility method, generated the pND6-1-null strain ND6dp1, and first classified it into the IncP-7 group by curing experimental evidence. Subsequently, the pND6-1-cured mutant showed an insignificant increase in cell growth compared with the wild ND6, indicating that a lower cost was imposed on the host by pND6-1. The influences of pND6-1 on the stability and conjugation of plasmid pND6-2 were subsequently explored. The results showed that the presence of pND6-1 enhanced the stability of pND6-2 in natural host ND6 and heterologous host P. putida KT2440. In addition, the presence of pND6-1 in wild-type hosts promoted intra-specific and inter-specific conjugative transfer efficiency of pND6-2 by 3.9–6.3-fold with P. putida, P. resinovans, and P. fluorescens as the recipients, respectively. ConclusionsCollectively, these results suggest that the plasmid pND6-1 improves the pND6-2 plasmid's stability and transfer rate in the ND6 strain with the selected recipient strains.How to cite: Wang S, Liu M, Wang D, et al. Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.08.002.

A One-Health Sampling Strategy to Explore the Dissemination and Relationship Between Colistin Resistance in Human, Animal, and Environmental Sectors in Laos

This study was designed to investigate the molecular epidemiology of mobile colistin resistance ( mcr ) using a “One-Health” approach in Laos and to predict whether any dominant plasmid backbone and/or strain type influences the dissemination of mcr . We collected 673 samples from humans (rectal normal flora), poultry, and the environment (water, flies, birds, etc.) in Vientiane, Lao People’s Democratic Republic (Laos), from May to September 2018. A total of 238 Escherichia coli ( E. coli ) isolated from non-duplicative samples, consisting of 98 MCR-positive E. coli (MCRPEC) (“ mcr ” denotes the gene encoding mobile colistin resistance, and “MCR” denotes the subsequent protein encoded by mcr ) and 140 MCR-negative E. coli (MCRNEC), were characterized by phenotype and Illumina sequencing. A subset of MCRPEC was selected for MinION sequencing, conjugation assay, plasmid stability, and growth kinetics in vitro . The prevalence of MCRPEC was found to be 14.6% (98/673), with the highest prevalence in human rectal swabs (45.9% (45/98), p < 0.0001, odds ratio (OR): 0.125, 95% confidence interval (CI): 0.077–0.202). The percentages of MCRPEC from other samples were 14.3% (2/14) in dog feces, 12.0% (24/200) in flies, 11.0% (11/100) in chicken meat, 8.9% (8/90) in chicken cloacal, 8.0% (4/50) in chicken caeca, and 7.5% (4/53) in wastewater. MCRPEC was significantly more resistant to co-amoxiclav, sulfamethoxazole-trimethoprim, levofloxacin, ciprofloxacin, and gentamicin than MCRNEC ( p < 0.05). Genomic analysis revealed the distribution of MCRPEC among diverse clonal types. The putative plasmid Inc types associated with mcr-1 were IncX4, IncHI2, IncP1, IncI2, and IncFIA, and those associated with mcr-3 were IncFII, IncFIA, IncFIB, IncP1, and IncR. Recovery of highly similar plasmids from both flies and other sampling sectors implied the role of flies in the dissemination of mcr-1 . mcr -positive plasmids were shown to be conjugative, and a significantly high transfer rate into a hypervirulent clone ST1193 was observed. Plasmids containing mcr irrespective of Inc type were highly stable and invariably did not exert a fitness effect upon introduction into a new host. These findings signify the urgent need for a standard infection control program to radically decontaminate the source of resistance.

Frequent convergence of mcr-9 and carbapenemase genes in Enterobacter cloacae complex driven by epidemic plasmids and host incompatibility

ABSTRACT Convergence of mcr and carbapenemase genes has been sporadically detected in Enterobacter cloacae complex (ECC) with an upward trend. However, the state of the epidemic and underlying mechanism of such convergence has been poorly understood. In this study, the co-occurrence of MCR and carbapenemases was systematically analyzed in 230 clinical ECC isolates collected between 2000 and 2018 together with a global dataset consisting of 3,559 ECC genomes compiled from GenBank. We identified 48 mcr-9/mcr-10-positive isolates (MCR-ECC) (20.9%) in our collection, and a comparable ratio of MCR-ECC (720/3559, 20.2%) was detected in the global dataset. A high prevalence of carbapenemase-producing MCR-ECC (MCR-CREC) was further identified in the MCR-ECC of both datasets (16/48, 33.3%; 388/720, 53.9%), demonstrating a frequent convergence of mcr-9/10 and carbapenemase genes in ECC worldwide. An epidemic IncHI2/2A plasmid with a highly conserved backbone was identified and largely contributed to the dissemination of mcr-9 in ECC worldwide. A highly conserved IncX3-type NDM-1-carrying plasmid and IncN-type IMP-4-carrying plasmid were additionally detected in MCR-CREC isolated in China. Our surveillance data showed that MCR-CREC emerged (in 2013) much later than MCR-ECC (in 2000), indicating that MCR-CREC could be derived from MCR-ECC by additional captures of carbapenemase-encoding plasmids. Tests of plasmid stability and incompatibility showed that the mcr-9/mcr-10-encoding plasmids with the NDM-1-encoding plasmids stably remained in ECC but incompatible in Escherichia coli, suggesting that the convergence was host-dependent. The findings extend our concern on the convergence of resistance to the last resort antibiotics and highlight the necessity of continued surveillance in the future.

Plasmid and chromosomal copies of blaCMY-2 mediate resistance to third-generation cephalosporins in Escherichia coli from food animals in China

The use of antimicrobials in food animals is the major determinant for the propagation of resistant bacteria in the animal reservoir. The objective of this study was to investigate the presence and distribution of third-generation cephalosporin (3GC) -resistant and plasmid-mediated AmpC (pAmpC)-producing Escherichia coli isolated from food animals in Southern China. In total, 744 3GC-resistant and 40 blaCMY-2-positive E. coli strains were recovered from 1656 food animal fecal samples across five rearing regions. The blaCMY-2 genes were located on IncC, IncFIB or IncI1 type plasmids in 12 E. coli isolates. In the other 22 isolates, S1-PFGE and hybridization analyses revealed that the blaCMY-2 gene was chromosomally located and demonstrated a high prevalence of the chromosomally encoded blaCMY-2 gene in E. coli. Plasmid stability and growth curve experiments demonstrated that IncI1, IncC and IncFIB plasmids can exist stably in the host bacteria and with a low growth burden and may be the reason these plasmids can be widely disseminated in breeding environments. Whole genome sequencing indicated that ISEcp1 and IS1294 played important roles in blaCMY-2 transfer to both plasmids and the chromosome. Our study confirmed that blaCMY-2 mediated resistance of food animal-derived E. coli to 3GC and highlights the urgent need for appropriate monitoring programmes.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Large-Scale Analysis of Fitness Cost of tet(X4)-Positive Plasmids in Escherichia coli.

Tigecycline is one of important antimicrobial agents for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence and prevalence of plasmid-mediated tigecycline resistance gene tet(X4) are threatening human and animal health. Fitness cost elicited by resistance plasmids is a key factor affecting the maintenance and transmission of antibiotic resistance genes (ARGs) in the host. A comparative analysis of the fitness cost of different types of tet(X4)-positive plasmids is helpful to understand and predict the prevalence of dominant plasmids. In this study, we performed a large-scale analysis of fitness cost of tet(X4)-positive plasmids origin from clinical isolates. These plasmids were successfully electroporated into a reference strain Escherichia coli TOP10, and a series of transformants carrying the tet(X) gene were obtained. The effects of tet(X4)-positive plasmids on the growth rate, plasmid stability, relative fitness, biofilm formation, and virulence in a Galleria mellonella model were evaluated. Consequently, we found that these plasmids resulted in varying degrees of fitness cost on TOP10, including delayed bacterial growth and attenuated virulence. Out of these plasmids, tet(X4)-harboring IncFII plasmids showed the lowest fitness cost on the host. Furthermore, by means of experimental evolution in the presence of commonly used drugs in clinic, the fitness cost of tet(X4)-positive plasmids was substantially alleviated, accompanied by increased plasmid stability. Collectively, our data reveal the differential fitness cost caused by different types of tet(X4)-positive plasmids and suggest that the wide use of tetracycline antibiotics may promote the evolution of plasmids.

Construction of an Antibiotic-Free Vector and its Application in the Metabolic Engineering of Escherichia Coli for Polyhydroxybutyrate Production.

An antibiotic- and inducer-free culture condition was proposed for polyhydroxybutyrate (PHB) production in recombinant Escherichia coli. First, antibiotic-free vectors were constructed by installing the plasmid maintenance system, alp7, hok/sok, and the hok/sok and alp7 combination into the pUC19 vector. The plasmid stability test showed that pVEC02, the pUC19 vector containing the hok/sok system, was the most effective in achieving antibiotic-free cultivation in the E. coli B strain but not in the K strain. Second, the putative phaCAB operon derived from Caldimonas manganoxidans was inserted into pVEC02 to yield pPHB01 for PHB production in E. coli BL21 (DE3). The putative phaCAB operon was first shown function properly for PHB production and thus, inducer-free conditions were achieved. However, the maintenance of pPHB01 in E. coli requires antibiotics supplementation. Finally, an efficient E. coli ρ factor-independent terminator, thrLABC (ECK120033737), was inserted between the phaCAB operon and the hok/sok system to avoid possible transcriptional carry-over. The newly constructed plasmid pPHB01-1 facilitates an antibiotic- and inducer-free culture condition and induces the production of PHB with a concentration of 3.0 on0.2 g/L, yield of 0.26 /L0.07 g/g-glucose, and content of 44 /g3%. The PHB production using E. coli BL21 (DE3)/pPHB01-1 has been shown to last 84 and 96 h in the liquid and solid cultures.

Optimization and characterization of immobilized E. coli for engineered thermostable xylanase excretion and cell viability

There are many parameters that may have influenced the properties of cell during immobilization process. Particularly, the immobilization methods, carrier materials, and enzyme loading amount that have been proved to be important for immobilization process. The physiological responses of microorganisms are depending on the immobilization technique used. Typical alterations to the micro-environment of the immobilized cell involved the altered water activity, presence of ionic charges, cell confinement and modified surface tension. In this study, the graphene oxide was selected as a suitable carrier for immobilization process of recombinant E.coli and adsorption was chosen as an appropriate method to improve the production of engineered thermostable xylanase . High level production of thermostable xylanase by immobilized recombinant cell in the 5 L bioreactor was studied by using optimum research surface methodology (RSM) conditions was studied. The immobilization of E. coli onto nanoparticle matrix manages to improve the cell performance by improving the protein expression, reduced the occurrences of cell lysis as well as improved the plasmid stability of the host cell. Thus, immobilization contributes a physical support for both whole cells as well as enzymes to develop a better operative achievement system for industrialized fields and give rise to the biological advancement existing enzyme for instance xylanase.

Coexistence of bla NDM-1 and bla IMP-4 in One Novel Hybrid Plasmid Confers Transferable Carbapenem Resistance in an ST20-K28 Klebsiella pneumoniae.

ObjectivesWe identified a novel hybrid plasmid simultaneously carrying blaNDM-1 and blaIMP-4 in an ST20-K28 carbapenem-resistant Klebsiella pneumoniae (CRKP) strain AZS099 and reported its detailed genetic and phenotypic characterization.MethodsAntimicrobial susceptibility was characterized using broth microdilution method. Complete genome characteristics and plasmid detailed analysis were carried out by PacBio Sequel and Illumina sequencing and further bioinformatics analysis. Conjugation assay, S1-PFGE, Southern blot, plasmid stability, and fitness cost were conducted to the phenotypic characterization of this novel hybrid plasmid.ResultsAZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. Susceptibility testing showed that AZS099 was resistant to almost all β-lactams examined, including cephalosporins, combinations of β-lactams and β-lactamase inhibitors, carbapenems, and aztreonam. PacBio and Illumina sequencing together with S1-PFGE and Southern blot showed that blaNDM-1 and blaIMP-4 were simultaneously located on a 296 kb IncFIB(K)/IncHI1B/IncX3 plasmid (pAZS099-NDM-IMP), which consists of four main parts that came from four different types of plasmids. The region harboring blaIMP-4 is located in a class 1 integron designated as In0, which is located in an IS6100-IS26 transposon-like structure with a total length of ~5 kb. The region harboring blaNDM-1 is located in the Tn125 transposon remnant. Conjugation and transformation assay confirmed that the plasmid pAZS099-NDM-IMP has the potential for horizontal transfer and displayed high stability (retention rate > 95%). Furthermore, growth curve assessment confirmed that the presence of pAZS099-NDM-IMP exhibits no growth pressure on bacteria.ConclusionOur research reported a hybrid plasmid coharboring blaNDM-1 and blaIMP-4 in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid could threaten the control of antimicrobial resistance and should be closely supervised.

Emergence of a Hypervirulent Tigecycline-Resistant Klebsiella pneumoniae Strain Co-producing bla NDM-1 and bla KPC-2 With an Uncommon Sequence Type ST464 in Southwestern China.

Emergence of blaNDM–1 and blaKPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both blaNDM–1 and blaKPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained blaNDM–1, blaKPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying blaNDM–1 and blaKPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying blaNDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR, rcsA, lon, and csrD genes of AHSWKP25. Our results highlighted the potential of blaNDM–1 and blaKPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

A platform method for plasmid isoforms analysis by capillary gel electrophoresis.

In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4kb). In addition, 6.5kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.

Phenotypic and Genotypic Characterization of Multidrug-Resistant Enterobacter hormaechei Carrying qnrS Gene Isolated from Chicken Feed in China.

ABSTRACTMultidrug resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. The plasmid-mediated quinolone resistance (PMQR) gene qnrS is prevalent in Enterobacteriaceae. However, the qnrS gene is rarely found in Enterobacter hormaechei (E. hormaechei). Here, we reported one multidrug resistant E. hormaechei strain M1 carrying the qnrS1 and blaTEM-1 genes. This study was to analyze the characteristics of MDR E. hormaechei strain M1. The E. hormaechei strain M1 was identified as Enterobacter cloacae complex by biochemical assay and 16S rRNA sequencing. The whole genome was sequenced by the Oxford Nanopore method. Taxonomy of the E. hormaechei was based on multilocus sequence typing (MLST). The qnrS with the other antibiotic resistance genes were coexisted on IncF plasmid (pM1). Besides, the virulence factors associated with pathogenicity were also located on pM1. The qnrS1 gene was located between insertion element IS2A (upstream) and transposition element ISKra4 (downstream). The comparison result of IncF plasmids revealed that they had a common plasmid backbone. Susceptibility experiment revealed that the E. hormaechei M1 showed extensive resistance to the clinical antimicrobials. The conjugation transfer was performed by filter membrane incubation method. The competition and plasmid stability assays suggested the host bacteria carrying qnrS had an energy burden. As far as we know, this is the first report that E. hormaechei carrying qnrS was isolated from chicken feed. The chicken feed and poultry products could serve as a vehicle for these MDR bacteria, which could transfer between animals and humans through the food chain. We need to pay close attention to the epidemiology of E. hormaechei and prevent their further dissemination.IMPORTANCE Enterobacter hormaechei is an opportunistic pathogen. It can cause infections in humans and animals. Plasmid-mediated quinolone resistance (PMQR) gene qnrS can be transferred intergenus, which is leading to increase the quinolone resistance levels in Enterobacteriaceae. Chicken feed could serve as a vehicle for the MDR E. hormaechei. Therefore, antibiotic-resistance genes (ARGs) might be transferred to the intestinal flora after entering the gastrointestinal tract with the feed. Furthermore, antibiotic-resistant bacteria (ARB) were also excreted into environment with feces, posing a huge threat to public health. This requires us to monitor the ARB and antibiotic-resistant plasmids in the feed. Here, we demonstrated the characteristics of one MDR E. hormaechei isolate from chicken feed. The plasmid carrying the qnrS gene is a conjugative plasmid with transferability. The presence of plasmid carrying antibiotic-resistance genes requires the maintenance of antibiotic pressure. In addition, the E. hormaechei M1 belonged to new sequence type (ST). These data show the MDR E. hormaechei M1 is a novel strain that requires our further research.

Deletion of FTL_1199 to determine the role of this gene in erythrocyte invasion by Francisella tularensis.

Francisella tularensis is a bacterium that induces the zoonotic disease tularemia. In the course of infection, F. tularensis bacteria invade erythrocytes, a phenomenon that heightens the colonization of ticks after a blood meal. To better understand the mechanism of erythrocyte invasion, we hypothesized that transcription of bacterial genes significant in erythrocyte invasion would be upregulated upon exposure to these host cells. An RNA-seq unveiled that transcription of 7% of F. tularensis genes augment when in erythrocyte presence. Of these, we pinpointed three putative transcriptional regulators, namely FTL_0671, FTL_1199, and FTL_1665. The goal was to delete FTL_1199 in F. tularensis LVS. Splicing by overlap extension PCR amplified and duplicated the up and downstream (~500 bp each) regions of the target gene in tandem into a shuttle vector that is insecure within F. tularensis. This newly generated plasmid, pDEL1199, was mobilized inside of F. tularensis by conjugation. Merodiploid strains generated by homologous recombination were isolated and transformed with pGUTS – a stable plasmid that encodes a homing endonuclease (I-SceI) and a kanamycin resistance cassette. Expression of I-SceI within the merodiploid produces a double-stranded break in pDEL1199 that had previously integrated in the chromosome. This breakage resulted in a second recombination that either ensued to wild-type or deletion of FTL_1199 deduced through a PCR. Finally, in DFTL_1199 strains, pGUTS was cured by successive cultivation in the absence of selection followed by replica-plating on chocolate II agar ± kanamycin. Gentamicin protection assays involving F. tularensis DFTL_1199 suggest that FTL_1199 is important in erythrocyte invasion.&#x0D; &#x0D; (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, R15HL14735 from NHLBI, and funds from the NASA West Virginia Space Grant Consortium).

Co-transfer of last-line antibiotic resistance and virulence operons by an IncFIBk-FII-X3-ColKP3 hybrid plasmid in Klebsiella pneumoniae.

To characterize a clinical Klebsiella pneumoniae isolate from China co-harbouring tet(X4), blaOXA-181 and the aerobactin operon on an IncFIBk-FII-X3-ColKP3 hybrid plasmid. A tigecycline-resistant strain was recovered from the intestinal sample of a patient. It was subjected to antimicrobial susceptibility testing, conjugation assay, virulence testing, WGS, bioinformatics analysis, plasmid stability testing and fitness cost testing. The strain K. pneumoniae T877 was resistant to tigecycline, intermediate to piperacillin/tazobactam and ertapenem, and positive for tet(X), blaOXA-181 and the virulence-associated operon iutAiucABCD, which were located on the same plasmid, named pKPT877-hybrid. It was 99.96% identical to the IncFIBk-FII plasmid pSCH6109-Vir (accession number CP050860) from K. pneumoniae strain SCH6109 at 96% coverage with the absence of a 50 kb region on pKPT877-hybrid; this region was highly homologous to the 51 kb IncX3-ColKP3-type, blaOXA-181-carrying plasmid pOXA181-191773 (accession number CP080367). Plasmid pKPT877-hybrid was conjugatively transferable to the ST11 K. pneumoniae strains FJ8 and KP04. pKPT877-hybrid did not have a significant impact on the fitness cost and could be maintained stably in T877. We report for the first time (to the best of our knowledge) the co-transfer of last-line antibiotic resistance determinants [tet(X4) and blaOXA-181] and the aerobactin operon (iutAiucABCD) by a mobile IncFIBk-FII-X3-ColKP3 hybrid plasmid, which can be stably maintained in K. pneumoniae strains, even in the absence of antibiotic selective pressure. Once the plasmid transfers to a K. pneumoniae with porin deficiency, the strain might have high levels of resistance to carbapenems and tigecycline, which are the last line of defence against infections. Heightened and continuous efforts are needed to control its dissemination.

Linear plasmids in Klebsiella and other Enterobacteriaceae.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella , first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K . pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin–antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Construction and Application of a Plasmid-Based Signal Peptide Library for Improved Secretion of Recombinant Proteins with Priestia megaterium.

The secretion of recombinant proteins plays an important role in their economic production and purification. The secretion efficiency depends on the responsible signal peptide (SP) in combination with the target protein and the given host and cannot be predicted so far. Due to its high plasmid stability, the lack of alkaline extracellular proteases and only few contaminating extracellular host proteins, Priestia megaterium provides a promising alternative to common Bacillus species. For the development of an easy and fast cloning and screening system to identify the SP best suited to a distinct protein, a plasmid-based SP library containing all predicted 182 Sec-dependent SPs from P. megaterium was established. The splitting of the SPs into 10 groups of individual multi-SP plasmids (pMSPs) allows their grouped amplification and application in screening approaches. The functionality of the whole library was demonstrated by enhancing the amount of the already well-secreted α-amylase AmyE by 1.6-fold. The secretion of a novel penicillin G acylase, which remained as insoluble protein inside the cells, as its native SP is unsuitable for secretion in P. megaterium, could be enhanced even up to 29-fold. Overall, only around 170 recombinant P. megaterium clones based on 50 inserted SPs had to be screened to achieve sufficient amounts for further enzyme characterizations. Thus, this newly developed plasmid-based genetic tool applicable for P. megaterium and also other Bacillus species facilitates the identification of suitable SPs for secretion of recombinant proteins.

Plasmid-mediated ciprofloxacin, carbapenem and colistin resistance of a foodborne Escherichia coli isolate

Multidrug resistant (MDR) Escherichia coli poses great threat to human and animal health. To investigate the genetic characteristics of conjugative plasmids that encode ciprofloxacin, carbapenem and colistin resistance in a MDR E. coli strain (974) isolated from pork samples in China, the E. coli strain 974 was subjected to conjugation study, S1-PFGE, Southern hybridization and was sequenced by Illumina Nextseq 500 and MinION Platforms. The ciprofloxacin-resistance-encoding plasmid, p974-IncF, was found to harbor multiple mobile elements that in turn contain genes resistant to β-lactams, quinolone, florephenicol, tetracycline, aminoglycosides, sulfonamides, streptomycin, trimethoprim and chloramphenicol. The backbone of blaNDM-1-bearing plasmid, p974-NDM, was found consist of a 17.3kb resistance region and a 56.7kb of transfer region. However, the classical ISCR1 mobile element and class I integron were missing in plasmid p974-NDM. Resembling the backbone of other IncI2 plasmids, the mcr-1-bearing plasmid, p974-MCR, contained genes that encode replication function, plasmid stability, proteins related to type IV secretion complex, as well as two pilus-encoding genes, which are conserved among all IncI2 plasmids. Enhanced surveillance efforts and limitation of usage of antibiotics are urgently warranted to control the dissemination of these resistance determinants.