Molecular Characterization of the Convergent Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae Strain K1-ST23, Collected in Chile during the COVID-19 Pandemic.

The aim of this study was to investigate the genomic features of a carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) isolate (K-2157) collected in Chile. Antibiotic susceptibility was determined using the disk diffusion and broth microdilution methods. Whole-genome sequencing (WGS) and hybrid assembly were performed, using data generated on the Illumina and Nanopore platforms. The mucoid phenotype was analyzed using both the string test and sedimentation profile. The genomic features of K-2157 (e.g., sequence type, K locus, and mobile genetic elements) were retrieved using different bioinformatic tools. Strain K-2157 exhibited resistance to carbapenems and was identified as a high-risk virulent clone belonging to capsular serotype K1 and sequence type 23 (ST23). Strikingly, K-2157 displayed a resistome composed of ß-lactam resistance genes (blaSHV-190, blaTEM-1, blaOXA-9, and blaKPC-2), the fosfomycin resistance gene fosA, and the fluoroquinolones resistance genes oqxA and oqxB. Moreover, several genes involved in siderophore biosynthesis (ybt, iro, and iuc), bacteriocins (clb), and capsule hyperproduction (plasmid-borne rmpA [prmpA] and prmpA2) were found, which is congruent with the positive string test displayed by K-2157. In addition, K-2157 harbored two plasmids: one of 113,644 bp (KPC+) and another of 230,602 bp, containing virulence genes, in addition to an integrative and conjugative element (ICE) embedded on its chromosome, revealing that the presence of these mobile genetic elements mediates the convergence between virulence and antibiotic resistance. Our report is the first genomic characterization of a hypervirulent and highly resistant K. pneumoniae isolate in Chile, which was collected during the coronavirus disease 2019 (COVID-19) pandemic. Due to their global dissemination and public health impact, genomic surveillance of the spread of convergent high-risk K1-ST23 K. pneumoniae clones should be highly prioritized. IMPORTANCE Klebsiella pneumoniae is a resistant pathogen involved primarily in hospital-acquired infections. This pathogen is characterized by its notorious resistance to last-line antibiotics, such as carbapenems. Moreover, hypervirulent K. pneumoniae (hvKp) isolates, first identified in Southeast Asia, have emerged globally and are able to cause infections in healthy people. Alarmingly, isolates displaying a convergence phenotype of carbapenem resistance and hypervirulence have been detected in several countries, representing a serious threat to public health. In this work, we analyzed the genomic characteristics of a carbapenem-resistant hvKp isolate recovered in 2022 from a patient with COVID-19 in Chile, representing the first analysis of this type in the country. Our results will provide a baseline for the study of these isolates in Chile, which will support the adoption of local measures aimed at controlling their dissemination.

Low concentration quaternary ammonium compounds promoted antibiotic resistance gene transfer via plasmid conjugation.

During the pandemic of COVID-19, the amounts of quaternary ammonium compounds (QACs) used to inactivate the virus in public facilities, hospitals and households increased, which raised concerns about the evolution and transmission of antimicrobial resistance (AMR). Although QACs may play an important role in the propagation of antibiotic resistance gene (ARGs), the potential contribution and mechanism remains unclear. Here, the results showed that benzyl dodecyl dimethyl ammonium chloride (DDBAC) and didecyl dimethyl ammonium chloride (DDAC) significantly promoted plasmid RP4-mediated ARGs transfer within and across genera at environmental relevant concentrations (0.0004-0.4 mg/L). Low concentrations of QACs did not contribute to the permeability of the cell plasma membrane, but significantly increased the permeability of the cell outer membrane due to the decrease in content of lipopolysaccharides. QACs altered the composition and content of extracellular polymeric substances (EPS) and were positively correlated with the conjugation frequency. Furthermore, transcriptional expression levels of genes encode for mating pairing formation (trbB), DNA replication and translocation (trfA), and global regulators (korA, korB, trbA) are regulated by QACs. And we demonstrate for the first time that QACs decreased the concentration of extracellular AI-2 signals, which was verified to be involved in regulating conjugative transfer genes (trbB, trfA). Collectively, our findings underscore the risk of increased disinfectant concentrations of QACs on the ARGs transfer and provide new mechanisms of plasmid conjugation.

Genetic plurality of blaKPC-2-harboring plasmids in high-risk clones of Klebsiella pneumoniae of environmental origin.

International high-risk clones of Klebsiella pneumoniae are important human pathogens that are spreading to the environment. In the COVID-19 pandemic scenario, the frequency of carbapenemase-producing strains increased, which can contribute to the contamination of the environment, impacting the surrounding and associated ecosystems. In this regard, KPC-producing strains were recovered from aquatic ecosystems located in commercial, industrial, or agricultural areas and were submitted to whole-genome characterization. K. pneumoniae and Klebsiella quasipneumoniae subsp. quasipneumoniae strains were assigned to high-risk clones (ST11, ST340, ST307) and the new ST6325. Virulome analysis showed genes related to putative hypervirulence. Strains were resistant to almost all antimicrobials tested, being classified as extensively drug-resistant or multidrug-resistant. In this context, a broad resistome (clinically important antimicrobials and hazardous metal) was detected. Single replicon (IncX5, IncN-pST15, IncU) and multireplicon [IncFII(K1)/IncFIB(pQil), IncFIA(HI1)/IncR] plasmids were identified carrying the blaKPC-2 gene with Tn4401 and non-Tn4401 elements. An unusual association of blaKPC-2 and qnrVC1 and the coexistence of blaKPC-2 and mer operon (mercury tolerance) was found. Comparative analysis revealed that blaKPC-2-bearing plasmids were most similar to plasmids from Enterobacterales of Brazil, China, and the United States, evidencing the long persistence of plasmids at the human-animal-environmental interface. Furthermore, the presence of uncommon plasmids, displaying the interspecies, intraspecies, and clonal transmission, was highlighted. These findings alert for the spread of high-risk clones producing blaKPC-2 in the environmental sector and call attention to rapid dispersion in a post-pandemic world.

Comprehensive analysis of disinfectants on the horizontal transfer of antibiotic resistance genes.

The propagation of antimicrobial resistance (AMR) is constantly paralyzing our healthcare systems. In addition to the pressure of antibiotic selection, the roles of non-antibiotic compounds in disseminating antibiotic resistance genes (ARGs) are a matter of great concerns. This study aimed to explore the impact of different disinfectants on the horizontal transfer of ARGs and their underlying mechanisms. First, the effects of different kinds of disinfectants on the conjugative transfer of RP4-7 plasmid were evaluated. Results showed that quaternary ammonium salt, organic halogen, alcohol and guanidine disinfectants significantly facilitated the conjugative transfer. Conversely, heavy-metals, peroxides and phenols otherwise displayed an inhibitory effect. Furthermore, we deciphered the mechanism by which guanidine disinfectants promoted conjugation, which includes increased cell membrane permeability, over-production of ROS, enhanced SOS response, and altered expression of conjugative transfer-related genes. More critically, we also revealed that guanidine disinfectants promoted bacterial energy metabolism by enhancing the activity of electron transport chain (ETC) and proton force motive (PMF), thus promoting ATP synthesis and flagellum motility. Overall, our findings reveal the promotive effects of disinfectants on the transmission of ARGs and highlight the potential risks caused by the massive use of guanidine disinfectants, especially during the COVID-19 pandemic.

Genetic and enzymatic characterization of two novel blaNDM-36, -37 variants in Escherichia coli strains.

The widespread of different NDM variants in clinical Enterobacterales isolates poses a serious public health concern, which requires continuous monitoring. In this study, three E. coli strains carrying two novel blaNDM variants of blaNDM-36, -37 were identified from a patient with refractory urinary tract infection (UTI) in China. We conducted antimicrobial susceptibility testing (AST), enzyme kinetics analysis, conjugation experiment, whole-genome sequencing (WGS), and bioinformatics analysis to characterize the blaNDM-36, -37 enzymes and their carrying strains. The blaNDM-36, -37 harboring E. coli isolates belonged to ST227, O9:H10 serotype and exhibited intermediate or resistance to all ß-lactams tested except aztreonam and aztreonam/avibactam. The genes of blaNDM-36, -37 were located on a conjugative IncHI2-type plasmid. NDM-37 differed from NDM-5 by a single amino acid substitution (His261Tyr). NDM-36 differed from NDM-37 by an additional missense mutation (Ala233Val). NDM-36 had increased hydrolytic activity toward ampicillin and cefotaxime relative to NDM-37 and NDM-5, while NDM-37 and NDM-36 had lower catalytic activity toward imipenem but higher activity against meropenem in comparison to NDM-5. This is the first report of co-occurrence of two novel blaNDM variants in E. coli isolated from the same patient. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of NDM enzymes.

Disinfectants facilitate the transformation of exogenous antibiotic resistance genes via multiple pathways.

The prevalence and spread of multidrug-resistant (MDR) bacteria pose a global challenge to public health. Natural transformation is one of the essential ways for horizontal transfer of antibiotic resistance genes (ARGs). Although disinfectants are frequently used during COVID-19, little is known about whether these disinfectants are associated with the transformation of plasmid-borne ARGs. In our study, we assessed the effect of some disinfectants on bacterial transformation using resistance plasmids as extracellular DNA and E. coli DH5α as the recipient bacteria. The results showed that these disinfectants at environmentally relevant concentrations, including benzalkonium bromide (BB), benzalkonium chloride (BC) and polyhexamethylene guanidine hydrochloride (PHMG), significantly enhanced the transformation of plasmid-encoded ARGs. Furthermore, we investigated the mechanisms underlying the promotive effect of disinfectants on transformation. We revealed that the addition of disinfectants significantly increased the membrane permeability and promoted membrane-related genes expression. Moreover, disinfectants led to the boosted bacterial respiration, ATP production and flagellum motility, as well as increased expression of bacterial secretion system-related genes. Together, our findings shed insights into the spread of ARGs through bacterial transformation and indicate potential risks associated with the widespread use of disinfectants.

Comparison of truncated human angiotensin-converting enzyme 2 (hACE2) expression in pET28a(+) versus pET-SUMO vector and two Escherichia coli strains.

PURPOSE: Truncated human angiotensin-converting enzyme 2 (hACE2) expression rises a great scientific interest, considering its possible therapeutic and diagnostic applications. A promising research direction is the therapeutic use of smaller hACE2 versions with high binding affinity as decoy receptors for S1 glycoprotein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Another possible application is the use of these truncated versions for the functionalization of appropriate nanomaterials for constructing novel biosensors with a rapid and sensitive response for coronavirus disease 2019 (COVID-19) detection. The present study aimed to find a suitable system for high yield expression of different versions of truncated hACE2. MATERIALS AND METHODS: The encoding DNA for the hACE2 fragments (7-507 aa, 16-128 aa, and 30-357 aa) was obtained by PCR amplification using as template pcDNA3.1-hACE2 plasmid and further cloned into pET28a(+) and pET-SUMO vectors. The positive clones were selected and the correct DNA insertion was confirmed through gene sequencing. The truncated hACE2 proteins were further expressed in two E. coli strains, Rosetta(DE3) and BL21(DE3). RESULTS: For all three truncated hACE2 mini proteins, pET28a(+) does not lead to protein expression, regardless of the bacterial strain. The situation changes with the use of the pET-SUMO expression system when all hACE2 fragments are expressed, but with higher efficiency in E. coli BL21(DE3) than E. coli Rosetta. CONCLUSION: In the present study, we showed that different versions of recombinant hACE2 are successfully expressed in E. coli BL21(DE3) by using pET-SUMO expression system.

Immediate protein expression from exogenous mRNAs in embryonic brain.

mRNA vaccines for SARS-CoV-2 have been widely used and saving millions of people in the world. How efficiently proteins are produced from exogenous mRNAs in the embryonic brain, however, is less known. Here we show that protein expression occurs highly efficiently in neural stem cells, in a very narrow time window after mRNA electroporation in the embryonic mouse brain, where plasmids have been successfully transfected. Protein expression is detected 1 h and 12 h after the electroporation of mRNAs and plasmids, respectively. The delivery of exogenous mRNAs may be useful for not only vaccines but also functional analysis in the brain.

Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+  Escherichia coli strain. The fosA3+  E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+  E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.

Immune response induced in rodents by anti-CoVid19 plasmid DNA vaccine via pyro-drive jet injector inoculation.

There is an urgent need to stop the coronavirus disease 2019 (COVID-19) pandemic through the development of efficient and safe vaccination methods. Over the short term, plasmid DNA vaccines can be developed as they are molecularly stable, thus facilitating easy transport and storage. pVAX1-SARS-CoV2-co was designed for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) S protein. The antibodies produced led to immunoreactions against the S protein, an anti-receptor-binding-domain, and a neutralizing action of the pVAX1-SARS-CoV2-co, as previously confirmed. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine a pyro-drive jet injector (PJI) was used. An intradermally adjusted PJI demonstrated that the pVAX1-SARS-CoV2-co vaccine injection caused a high production of anti-S protein antibodies, triggered immunoreactions, and neutralized the actions against SARS-CoV-2. A high-dose pVAX1-SARS-CoV2-co intradermal injection using PJI did not cause any serious disorders in the rat model. A viral challenge confirmed that intradermally immunized mice were potently protected from COVID-19. A pVAX1-SARS-CoV2-co intradermal injection using PJI is a safe and promising vaccination method for overcoming the COVID-19 pandemic.

FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly.

Rapid and flexible plasmid construct generation at scale is one of the most limiting first steps in drug discovery projects. These hurdles can partly be overcome by adopting modular DNA design principles, automated sequence fragmentation, and plasmid assembly. To this end we have designed a robust, multimodule golden gate based cloning platform for construct generation with a wide range of applications. The assembly efficiency of the system was validated by splitting sfGFP and sfCherry3C cassettes and expressing them in E. coli followed by fluorometric assessment. To minimize timelines and cost for complex constructs, we developed a software tool named FRAGLER (FRAGment recycLER) that performs codon optimization, multiple sequence alignment, and automated generation of fragments for recycling. To highlight the flexibility and robustness of the platform, we (i) generated plasmids for SarsCoV2 protein reagents, (ii) automated and parallelized assemblies, and (iii) built modular libraries of chimeric antigen receptors (CARs) variants. Applying the new assembly framework, we have greatly streamlined plasmid construction and increased our capacity for rapid generation of complex plasmids.

In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression.

Ionizable cationic lipids play a critical role in developing new gene therapies for various biomedical applications, including COVID-19 vaccines. However, it remains unclear whether the formulation of lipid nanoparticles (LNPs) using DLin-MC3-DMA, an optimized ionizable lipid clinically used for small interfering RNA (siRNA) therapy, also facilitates high liver-selective transfection of other gene therapies such as plasmid DNA (pDNA). Here we report the first investigation into pDNA transfection efficiency in different mouse organs after intramuscular and intravenous administration of lipid nanoparticles (LNPs) where DLin-MC3-DMA, DLin-KC2-DMA or DODAP are used as the ionizable cationic lipid component of the LNP. We discovered that these three benchmark lipids previously developed for siRNA delivery followed an unexpected characteristic rank order in gene expression efficiency when utilized for pDNA. In particular, DLin-KC2-DMA facilitated higher in vivo pDNA transfection than DLin-MC3-DMA and DODAP, possibly due to its head group pKa and lipid tail structure. Interestingly, LNPs formulated with either DLin-KC2-DMA or DLin-MC3-DMA exhibited significantly higher in vivo protein production in the spleen than in the liver. This work sheds light on the importance of the choice of ionizable cationic lipid and nucleic acid cargo for organ-selective gene expression. The study also provides a new design principle towards the formulation of more effective LNPs for biomedical applications of pDNA, such as gene editing, vaccines and immunotherapies.

Degenerate sequence-based CRISPR diagnostic for Crimean-Congo hemorrhagic fever virus.

CRISPR (clustered regularly interspaced short palindromic repeats), an ancient defense mechanism used by prokaryotes to cleave nucleic acids from invading viruses and plasmids, is currently being harnessed by researchers worldwide to develop new point-of-need diagnostics. In CRISPR diagnostics, a CRISPR RNA (crRNA) containing a "spacer" sequence that specifically complements with the target nucleic acid sequence guides the activation of a CRISPR effector protein (Cas13a, Cas12a or Cas12b), leading to collateral cleavage of RNA or DNA reporters and enormous signal amplification. CRISPR function can be disrupted by some types of sequence mismatches between the spacer and target, according to previous studies. This poses a potential challenge in the detection of variable targets such as RNA viruses with a high degree of sequence diversity, since mismatches can result from target variations. To cover viral diversity, we propose in this study that during crRNA synthesis mixed nucleotide types (degenerate sequences) can be introduced into the spacer sequence positions corresponding to viral sequence variations. We test this crRNA design strategy in the context of the Cas13a-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) technology for detection of Crimean-Congo hemorrhagic fever virus (CCHFV), a biosafety level 4 pathogen with wide geographic distribution and broad sequence variability. The degenerate-sequence CRISPR diagnostic proves functional, sensitive, specific and rapid. It detects within 30-40 minutes 1 copy/µl of viral RNA from CCHFV strains representing all clades, and from more recently identified strains with new mutations in the CRISPR target region. Also importantly, it shows no cross-reactivity with a variety of CCHFV-related viruses. This proof-of-concept study demonstrates that the degenerate sequence-based CRISPR diagnostic is a promising tool of choice for effective detection of highly variable viral pathogens.

Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers.

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

A Single Dose of an MVA Vaccine Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Protein Neutralizes Variants of Concern and Protects Mice From a Lethal SARS-CoV-2 Infection.

We generated an optimized COVID-19 vaccine candidate based on the modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, termed MVA-CoV2-S(3P). The S(3P) protein was expressed at higher levels (2-fold) than the non-stabilized S in cells infected with the corresponding recombinant MVA viruses. One single dose of MVA-CoV2-S(3P) induced higher IgG and neutralizing antibody titers against parental SARS-CoV-2 and variants of concern than MVA-CoV2-S in wild-type C57BL/6 and in transgenic K18-hACE2 mice. In immunized C57BL/6 mice, two doses of MVA-CoV2-S or MVA-CoV2-S(3P) induced similar levels of SARS-CoV-2-specific B- and T-cell immune responses. Remarkably, a single administration of MVA-CoV2-S(3P) protected all K18-hACE2 mice from morbidity and mortality caused by SARS-CoV-2 infection, reducing SARS-CoV-2 viral loads, histopathological lesions, and levels of pro-inflammatory cytokines in the lungs. These results demonstrated that expression of a novel full-length prefusion-stabilized SARS-CoV-2 S protein by the MVA poxvirus vector enhanced immunogenicity and efficacy against SARS-CoV-2 in animal models, further supporting MVA-CoV2-S(3P) as an optimized vaccine candidate for clinical trials.

Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells.

Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

Optimization of Single-Dose VSV-Based COVID-19 Vaccination in Hamsters.

The ongoing COVID-19 pandemic has resulted in global effects on human health, economic stability, and social norms. The emergence of viral variants raises concerns about the efficacy of existing vaccines and highlights the continued need for the development of efficient, fast-acting, and cost-effective vaccines. Here, we demonstrate the immunogenicity and protective efficacy of two vesicular stomatitis virus (VSV)-based vaccines encoding the SARS-CoV-2 spike protein either alone (VSV-SARS2) or in combination with the Ebola virus glycoprotein (VSV-SARS2-EBOV). Intranasally vaccinated hamsters showed an early CD8+ T cell response in the lungs and a greater antigen-specific IgG response, while intramuscularly vaccinated hamsters had an early CD4+ T cell and NK cell response. Intranasal vaccination resulted in protection within 10 days with hamsters not showing clinical signs of pneumonia when challenged with three different SARS-CoV-2 variants. This data demonstrates that VSV-based vaccines are viable single-dose, fast-acting vaccine candidates that are protective from COVID-19.

Outbreak of NDM-1-Producing Escherichia coli in a Coronavirus Disease 2019 Intensive Care Unit in a Mexican Tertiary Care Center.

Emergency department areas were repurposed as intensive care units (ICUs) for patients with acute respiratory distress syndrome during the initial months of the coronavirus disease 2019 (COVID-19) pandemic. We describe an outbreak of New Delhi metallo-ß-lactamase 1 (NDM-1)-producing Escherichia coli infections in critically ill COVID-19 patients admitted to one of the repurposed units. Seven patients developed infections (6 ventilator-associated pneumonia [VAP] and 1 urinary tract infection [UTI]) due to carbapenem-resistant E. coli, and only two survived. Five of the affected patients and four additional patients had rectal carriage of carbapenem-resistant E. coli. The E. coli strain from the affected patients corresponded to a single sequence type. Rectal screening identified isolates of two other sequence types bearing blaNDM-1. Isolates of all three sequence types harbored an IncFII plasmid. The plasmid was confirmed to carry blaNDM-1 through conjugation. An outbreak of clonal NDM-1-producing E. coli isolates and subsequent dissemination of NDM-1 through mobile elements to other E. coli strains occurred after hospital conversion during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This emphasizes the need for infection control practices in surge scenarios. IMPORTANCE The SARS-CoV-2 pandemic has resulted in a surge of critically ill patients. Hospitals have had to adapt to the demand by repurposing areas as intensive care units. This has resulted in high workload and disruption of usual hospital workflows. Surge capacity guidelines and pandemic response plans do not contemplate how to limit collateral damage from issues like hospital-acquired infections. It is vital to ensure quality of care in surge scenarios.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Reverse Genetics with a Full-Length Infectious cDNA Clone of Bovine Torovirus.

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified in the new family Tobaniviridae. While reverse genetics systems have been established for various CoVs, none exist for ToVs. Here, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and hemagglutinin (HA)-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated a recombinant virus (rEGFP) wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. rEGFP expressed EGFP in infected cells but showed significantly lower levels of viral growth than wtBToV. Moreover, rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in nonstructural proteins (NSPs) that emerged during passage exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a phenotype similar to that of these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. IMPORTANCE ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae. Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, pathogenesis, and BToV vaccine development.