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We characterized all ceftazidime-avibactam-resistant KPC-producing K. pneumoniae (KPC-Kp) isolates recovered from both patients and environmental samples at the ICU of our hospital in 2020, during the COVID-19 pandemic initiation. Antimicrobial susceptibility testing (Sensititre-EUMDROXF; disk-diffusion) and WGS analysis (Illumina-Novaseq/Miseq; Oxford Nanopore ® -MinION) were performed. Ten patients (16% of ICU patients) were colonized/infected by a ceftazidime-avibactam-resistant KPC-Kp isolate (March-December), six of them during/after treatment with ceftazidime-avibactam. Two ceftazidime-avibactam-resistant KPC-Kp were also recovered from two ICU sinks (July-September). All isolates belonged to ST307 clone and had identical resistance gene content. Six KPC-variants were detected in patient isolates (KPC-62, KPC-92, KPC-150, KPC-66, KPC-53, KPC-46). KPC-92 and KPC-66 variants were also detected in sink isolates. Regardless of the origin (patients or sinks), KPC-92-, KPC-150 and KPC-62-Kp isolates combining altered porin proteins also exhibited increased/resistant MIC values to cefiderocol, cefepime-taniborbactam, aztreonam-avibactam, meropenem-vaborbactam and/or imipenem-relebactam. A cgMLST analysis demonstrated the clonal spread of KPC-ST307-Kp within the ICU, between patients and the hospital environment. Clustering was observed mainly by KPC variants (KPC-92, KPC-62). A variant calling analysis and plasmid characterization showed possible transmission between patients and sinks. Our results suggest that the patient care environment likely contributed to persistence and spread of last-line antibiotics-resistant KPC-Kp within the ICU during the COVID-19 pandemic.

PurposeTo characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates.MethodsCPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing.ResultsA reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers.ConclusionsThe reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice. IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines. Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans , indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans , whereas it would only survive a single virus infection cycle in vivo . This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection. Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans , whereas it would only survive a single virus infection cycle in vivo . This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

A SARS-CoV lacking the full-length E gene (SARS-CoV-[increment]E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-[increment]E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-[increment]E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.