In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

Twenty-eight years of GM Food and feed without harm: why not accept them?

Since the first genetically engineered or modified crops or organisms (GMO) were approved for commercial production in 1995, no new GMO has been proven to be a hazard or cause harm to human consumers. These modifications have improved crop efficiency, reduced losses to insect pests, reduced losses to viral and microbial plant pathogens and improved drought tolerance. A few have focused on nutritional improvements producing beta carotene in Golden Rice. Regulators in the United States and countries signing the CODEX Alimentarius and Cartagena Biosafety agreements have evaluated human and animal food safety considering potential risks of allergenicity, toxicity, nutritional and anti-nutritional risks. They consider risks for non-target organisms and the environment. There are no cases where post-market surveillance has uncovered harm to consumers or the environment including potential transfer of DNA from the GMO to non-target organisms. In fact, many GMOs have helped improve production, yield and reduced risks from chemical insecticides or fungicides. Yet there are generic calls to label foods containing any genetic modification as a GMO and refusing to allow GM events to be labeled as organic. Many African countries have accepted the Cartagena Protocol as a tool to keep GM events out of their countries while facing food insecurity. The rationale for those restrictions are not rational. Other issues related to genetic diversity, seed production and environmental safety must be addressed. What can be done to increase acceptance of safe and nutritious foods as the population increases, land for cultivation is reduced and energy costs soar?

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo.

Development of pBACpAK entrapment vector derivatives to detect intracellular transfer of mobile genetic elements within chloramphenicol resistant bacterial isolates.

Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3')-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.

Toxicology and digestibility of Chlamydomonas debaryana green algal biomass.

There is an economic interest, both for food security and for the non-meat-eating population, in the development of novel, sustainable sources of high-quality protein. The green algae Chlamydomonas reinhardtii has already been developed for this purpose, and the closely related species, Chlamydomonas debaryana, is a complementary source that also presents some additional advantages, such as reduced production cost. To determine whether C. debaryana may have a similar safety profile to that of C. reinhardtii, a wild type strain was obtained, designated TS04 after confirmation of its identity, and subjected to a battery of preclinical studies. Genetic toxicity was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in a mouse model. No genotoxic potential (e.g., mutagenicity and clastogenicity) was observed in these tests under the employed conditions up to maximum recommended concentrations or doses. To assess general toxicity, a 90-day repeated-dose oral toxicity study was conducted in rats. No mortality or adverse effects were observed, and no target organs were identified up to the maximum feasible dose, due to solubility, of 4,000 mg/kg bw/day. The no-observed-adverse-effect level was determined as the highest dose tested. A digestibility study in simulated gastric fluid was conducted and determined that TS04 has low allergenic potential, exhibiting rapid digestion of proteins. Due to the negative results of our evaluation, it is reasonable to proceed with further development and additional investigations to contribute towards a safety assessment of the proposed use in food for human consumption.

Intracellular Transposition of Mobile Genetic Elements Associated with the Colistin Resistance Gene mcr-1.

Mobile colistin resistance (mcr) genes are often located on conjugative plasmids, where their association with insertion sequences enables intercellular and intracellular dissemination throughout bacterial replicons and populations. Multiple mcr genes have been discovered in every habitable continent, in many bacterial species, on both plasmids and integrated into the chromosome. Previously, we showed the intercellular transfer of mcr-1 on an IncI1 plasmid, pMCR-E2899, between strains of Escherichia coli. Characterizing the intracellular dynamics of mcr-1 transposition and recombination would further our understanding of how these important genes move through bacterial populations and whether interventions can be put in place to stop their spread. In this study, we aimed to characterize transfer events from the mcr-1-containing transposon Tn7511 (ISApl1-mcr-1-pap2-ISApl1), located on plasmid pMCR-E2899, using the pBACpAK entrapment vector. Following the transformation of pBACpAK into our DH5α-Azir/pMCR-E2899 transconjugant, we captured ISApl1 in pBACpAK multiple times and, for the first time, observed the ISApl1-mediated transfer of the mcr-1 transposon (Tn7511) into the chromosome of E. coli DH5α. Whole-genome sequencing allowed us to determine consensus insertion sites of ISApl1 and Tn7511 in this strain, and comparison of these sites allowed us to explain the transposition events observed. These observations reveal the consequences of ISApl1 transposition within and between multiple replicons of the same cell and show mcr-1 transposition within the cell as part of the novel transposon Tn7511. IMPORTANCE By analyzing the intracellular transfer of clinically relevant transposons, we can understand the dissemination and evolution of drug resistance conferring mobile genetic elements (MGEs) once a plasmid enters a cell following conjugation. This knowledge will help further our understanding of how these important genes move through bacterial populations. Utilizing the pBACpAK entrapment vector has allowed us to determine the mobility of the novel mcr-1-containing transposon Tn7511.

Safety evaluation of Neurospora crassa mycoprotein for use as a novel meat alternative and enhancer.

Cultivation of filamentous fungi to produce sustainable, nutrient rich meat replacements has recently attracted significant commercial and research interest. Here, we report evidence for the safety and nutritional value of Neurospora crassa mycoprotein, a whole mycelium food ingredient produced by fermentation and minimal downstream processing. N. crassa has a long history of human use in fermented foods and in molecular biology research. A survey of studies that used N. crassa in animal feed revealed no adverse effects to the health of the animals. Furthermore, a review of the literature found no reports of confirmed allergenicity or toxicity in humans involving N. crassa. Genomic toxigenicity analysis and in vitro testing did not identify any toxins in N. crassa mycoprotein. Two independent genomic allergenicity studies did not identify proteins that would be considered a particular risk for allergenic potential. Furthermore, nutritional analysis demonstrated that N. crassa mycoprotein is a good source of complete protein and is rich in fiber, potassium, and iron. Taken together, the presented data and the history of human use without evidence of human or animal harm indicate that foods containing N. crassa can generally be regarded as safe.

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate.

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.

Development of a Sequence Searchable Database of Celiac Disease-Associated Peptides and Proteins for Risk Assessment of Novel Food Proteins.

Celiac disease (CeD) is an autoimmune enteropathy induced by prolamin and glutelin proteins in wheat, barley, rye, and triticale recognized by genetically restricted major histocompatibility (MHC) receptors. Patients with CeD must avoid consuming these proteins. Regulators in Europe and the United States expect an evaluation of CeD risks from proteins in genetically modified (GM) crops or novel foods for wheat-related proteins. Our database includes evidence-based causative peptides and proteins and two amino acid sequence comparison tools for CeD risk assessment. Sequence entries are based on the review of published studies of specific gluten-reactive T cell activation or intestinal epithelial toxicity. The initial database in 2012 was updated in 2018 and 2022. The current database holds 1,041 causative peptides and 76 representative proteins. The FASTA sequence comparison of 76 representative CeD proteins provides an insurance for possible unreported epitopes. Validation was conducted using protein homologs from Pooideae and non-Pooideae monocots, dicots, and non-plant proteins. Criteria for minimum percent identity and maximum E-scores are guidelines. Exact matches to any of the 1,041 peptides suggest risks, while FASTA alignment to the 76 CeD proteins suggests possible risks. Matched proteins should be tested further by CeD-specific CD4/8+ T cell assays or in vivo challenges before their use in foods.

Safety evaluation of Fy Protein™ (Nutritional Fungi Protein), a macroingredient for human consumption.

Fy Protein™ (Nutritional Fungi Protein) is a macro-ingredient produced from the fermentation of the fungal microorganism Fusarium strain flavolapis, isolated from springs in Yellowstone National Park. Fy Protein contains all of the essential amino acids plus fiber, fat, carbohydrates, vitamins, and minerals and is developed as an alternative to animal-based protein foods such as meat and dairy. Fy Protein's nutritional, digestibility, genotoxicity, allergenicity, toxicity, secondary metabolites, and pathogenicity were evaluated. Fy Protein did not show mutagenic or genotoxic potential in in vitro tests. In an allergenicity review, Fy Protein was found to be of low allergenic potential. In a 90-day sub chronic dietary study in rats, administration of Fy Protein did not produce any significant toxicologic manifestations, and the no observed effect level (NOAEL) was the highest-level fed of 150,000 ppm (15% in the diet). Regulated secondary metabolites from fungi (termed mycotoxins) were non-detectable and below regulated levels using quantitative analytical techniques. A literature review was completed to identify the potential human pathogenicity of Fusarium sp., showing that Fusarium rarely infects humans, with infections seldom developing even in immunocompromised individuals. The results of these studies confirm that Fy Protein from fermented F. str. flavolapis has low toxicological, genotoxic, pathogenic, and allergenic potential under the conditions tested and anticipated use.

Challenges in Gluten Analysis: A Comparison of Four Commercial Sandwich ELISA Kits.

Gluten is composed of prolamin and glutelin proteins from several related grains. Because these proteins are not present in identical ratios in the various grains and because they have some differences in sequence, the ability to accurately quantify the overall amount of gluten in various food matrices to support gluten-free labeling is difficult. Four sandwich ELISAs (the R-Biopharm AG R5 RIDASCREEN®, the Neogen Veratox® R5, the Romer Labs AgraQuant® G12, and the Morinaga Wheat kits) were evaluated for their performance to quantify gluten concentrations in various foods and ingredients. The Morinaga and AgraQuant® G12 tests yielded results comparable to the two R5 kits for most, but not for certain, foods. The results obtained with the Morinaga kit were lower when compared to the other kits for analyzing powders of buckwheat and several grass-based products. All four kits were capable of detecting multiple gluten-containing grain sources including wheat, rye, barley, semolina, triticale, spelt, emmer, einkorn, Kamut™, and club wheat. Users of the ELISA kits should verify the performance in their hands, with matrices that are typical for their specific uses. The variation in results for some food matrices between test methods could result in trade disputes or regulatory disagreements.

Health System Approach to Improve Chronic Obstructive Pulmonary Disease Care after Hospital Discharge: Stepped-Wedge Clinical Trial.

Rationale: Patients discharged from the hospital for chronic obstructive pulmonary disease (COPD) exacerbation have impaired quality of life and frequent readmission and death. Clinical trials to reduce readmission demonstrate inconsistent results, including some demonstrating potential harms. Objectives: We tested whether a pragmatic proactive interdisciplinary and virtual review of patients discharged after hospitalization for COPD exacerbation would improve quality of life, using the Clinical COPD Questionnaire, and reduce all-cause 180-day readmission and/or mortality. Methods: We performed a stepped-wedge clinical trial. We enrolled primary care providers and their patients after hospital discharge for COPD at two Department of Veterans Affairs medical centers and 10 outpatient clinics. A multidisciplinary team reviewed health records and developed treatment recommendations delivered to primary care providers via E-consult. We facilitated uptake by entering recommendations as unsigned orders that could be accepted, modified, or canceled. Providers and patients made all final treatment decisions. Measurements and Main Results: We enrolled 365 primary care providers. Over a 30-month period, 352 patients met eligibility criteria, with 191 (54.3%) patients participating in the control and 161 (45.7%) in the intervention. The intervention led to clinically significant better Clinical COPD Questionnaire scores (-0.47; 95% confidence interval [CI], -0.85 to -0.09; 52.6% missing) but did not reduce 180-day readmission and/or mortality (adjusted odds ratio, 0.83; 95% CI, 0.49 to 1.38), in part because of wide CIs. Among the 161 patients in the intervention group, we entered 519 recommendations as unsigned orders, of which 401 (77.3%) were endorsed. Conclusions: A pragmatic health system-level intervention that delivered proactive specialty supported care improved quality of life but did not reduce 180-day readmission or death. Clinical trial registered with www.clinicaltrials.gov (NCT02021955).

Subchronic feeding, allergenicity, and genotoxicity safety evaluations of single strain bacterial protein.

Microbial proteins are potentially important alternatives to animal protein. A safety assessment was conducted on a Clostridium protein which can serve as a high-quality protein source in human food. A battery of toxicity studies was conducted comprising a 14-day dose-range finding dietary study in rats, 90-day dietary study in rats and in vitro genotoxicity studies. The allergenic potential was investigated by bioinformatics analysis. In the 90-day feeding study, rats were fed diets containing 0, 5.0, 7.5, and 10% Clostridium protein. The Clostridium protein-containing diets were well-tolerated and no adverse effects on the health or growth were observed. Significant reductions in neutrophil counts were observed in all female rats compared to controls, which were slightly outside of reference ranges. These effects were not deemed to be adverse due to the absence of comparable findings in male rats and high physiological variability of measured values within groups. A No-Observed-Adverse-Effect-Level (NOAEL) of at least 10% Clostridium protein, the highest dose tested and corresponding to 5,558 and 6,671 mg/kg body weight/day for male and female rats, respectively, was established. No evidence of genotoxicity was observed and the allergenic potential was low. These results support the use of Clostridium protein as a food ingredient.

Cannabis-related allergies: An international overview and consensus recommendations.

Cannabis is the most widely used recreational drug in the world. Cannabis sativa and Cannabis indica have been selectively bred to develop their psychoactive properties. The increasing use in many countries has been accelerated by the COVID-19 pandemic. Cannabis can provoke both type 1 and type 4 allergic reactions. Officially recognized allergens include a pathogenesis-related class 10 allergen, profilin, and a nonspecific lipid transfer protein. Other allergens may also be relevant, and recognition of allergens may vary between countries and continents. Cannabis also has the potential to provoke allergic cross-reactions to plant foods. Since cannabis is an illegal substance in many countries, research has been hampered, leading to challenges in diagnosis since no commercial extracts are available for testing. Even in countries such as Canada, where cannabis is legalized, diagnosis may rely solely on the purchase of cannabis for prick-to-prick skin tests. Management consists of avoidance, with legal issues hindering the development of other treatments such as immunotherapy. Education of healthcare professionals is similarly lacking. This review aimed to summarize the current status of cannabis allergy and proposes recommendations for the future management of this global issue.

Evaluation of the genotoxic potential of protoporphyrin IX and the safety of a protoporphyrin IX-rich algal biomass.

Chlamydomonas reinhardtii is a nonpathogenic, nontoxigenic green algae used as a sustainable source of protein in foods. In order to mimic meat-like qualities, a strain rich in protoporphyrin IX (PPIX), an endogenous heme/chlorophyll precursor, was developed using an evolution and selection strategy, and investigations were carried out to evaluate the safety of the novel strain, C. reinhardtii (red), strain TAI114 (TAI114). Digestibility and proteomic evaluations were conducted to determine whether any potentially allergenic or toxic proteins occurred as the result of the mutation process. The genotoxic potential of pure PPIX was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test. Finally, the novel TAI114 biomass was evaluated for general toxicity and identification of target organs in a 90-day repeated-dose oral toxicity study in rats. All proteins were rapidly degraded in pepsin at pH 2.0 suggesting low allergenic potential. The proteomic evaluation indicated that TAI114 biomass contains typical C. reinhardtii proteins. PPIX was unequivocally negative for genotoxic potential and no target organs or adverse effects were observed in rats up to the maximum feasible dose of 4000 mg/kg bw/day TAI114 biomass, which was determined to be the no-observed-adverse-effect-level (NOAEL). These results support the further development and risk characterization of TAI114 biomass as a novel ingredient for use in the meat analogue category of food.

First identification of bla NDM-5 producing Escherichia coli from neonates and a HIV infected adult in Tanzania.

Introduction. Carbapenem-resistant members of the family Enterobacteriaceae are emerging as a global public-health threat and cause substantial challenges in clinical practice.Gap Statement. There is a need for increased and continued genomic surveillance of antimicrobial resistance genes globally in order to detect outbreaks and dissemination of clinically important resistance genes and their associated mobile genetic elements in human pathogens.Aim. To describe the resistance mechanisms of carbapenem-resistant Escherichia coli.Methods. Rectal swabs from neonates and newly diagnosed human immunodeficiency virus (HIV) infected adults were collected between April 2017 and May 2018 and screened for faecal carriage of carbapenamases and OXA-48 producing members of the family Enterobacteriaceae. Bacterial isolates were identified using matrix assisted laser desorption ionization time of flight mass spectrometry. Antimicrobial susceptibility testing was performed by E-test. Whole genomes of carbapenem-resistant E. coli were investigated using a hybrid assembly of Illumina and Oxford Nanopore Technologies sequencing reads.Results. Three carbapenem-resistant E. coli were detected, two from neonates and one from an HIV infected adult. All three isolates carried bla NDM-5. Two E. coli from neonates belonged to ST167 and bla NDM-5 co-existed with bla CTX-M-15 and bla OXA-01, and all were carried on IncFIA type plasmids. The E. coli from the HIV infected adult belonged to ST2083, and carried bla NDM-5 on an IncX3 type plasmid and bla CMY-42 on an IncI type plasmid. All bla NDM-5 carrying plasmids contained conjugation related genes. In addition, E. coli from the HIV infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). One E. coli from a neonate also carried one extra plasmid Col(BS512). All three E. coli harboured resistance genes to fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline, carried on the IncFIA type plasmid. Furthermore, E. coli from the neonates carried a chloramphenicol resistance gene (catB3), also on the IncFIA plasmid. All three isolates were susceptible to colistin.Conclusion. This is the first report, to our knowledge, from Tanzania detecting bla NDM-5 producing E. coli. The carbapenemase gene was carried on an IncFIA and IncX3 type plasmids. Our findings highlight the urgent need for a robust antimicrobial resistance (AMR) surveillance system to monitor and rapidly report on the incidence and spread of emerging resistant bacteria in Tanzania.

Intracellular Transposition and Capture of Mobile Genetic Elements following Intercellular Conjugation of Multidrug Resistance Conjugative Plasmids from Clinical Enterobacteriaceae Isolates.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation and are therefore important agents of ARG dissemination. Detection and characterization of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination and allow us to determine interventional strategies to interrupt this process. Entrapment vectors can be used to capture active MGEs, as they contain a positive selection genetic system conferring a selectable phenotype upon the insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK. In this study, we aimed to extend the utilization of pBACpAK to other bacterial taxa. We utilized an MGE-free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG-containing plasmids following conjugation from clinical Enterobacteriaceae donors. Following the conjugative transfer of multiple conjugative plasmids and screening for tetracycline resistance in these transconjugants, we captured several insertion sequence (IS) elements and novel transposons (Tn7350 and Tn7351) and detected the de novo formation of novel putative composite transposons where the pBACpAK-located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid, as well as the ISKpn14-mediated integration of an entire 119-kb, blaNDM-1-containing conjugative plasmid from Klebsiella pneumoniae. IMPORTANCE By analyzing transposition activity within our MGE-free recipient, we can gain insights into the interaction and evolution of multidrug resistance-conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons, and cointegration and/or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow fine-scale experimental studies of factors affecting intracellular transposition and MGE formation in and from ARG-encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.