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.

Human Cell-Camouflaged Nanomagnetic Scavengers Restore Immune Homeostasis in a Rodent Model with Bacteremia.

Bloodstream infection caused by antimicrobial resistance pathogens is a global concern because it is difficult to treat with conventional therapy. Here, scavenger magnetic nanoparticles enveloped by nanovesicles derived from blood cells (MNVs) are reported, which magnetically eradicate an extreme range of pathogens in an extracorporeal circuit. It is quantitatively revealed that glycophorin A and complement receptor (CR) 1 on red blood cell (RBC)-MNVs predominantly capture human fecal bacteria, carbapenem-resistant (CR) Escherichia  coli, and extended-spectrum beta-lactamases-positive (ESBL-positive) E. coli, vancomycin-intermediate Staphylococcus aureus (VISA), endotoxins, and proinflammatory cytokines in human blood. Additionally, CR3 and CR1 on white blood cell-MNVs mainly contribute to depleting the virus envelope proteins of Zika, SARS-CoV-2, and their variants in human blood. Supplementing opsonins into the blood significantly augments the pathogen removal efficiency due to its combinatorial interactions between pathogens and CR1 and CR3 on MNVs. The extracorporeal blood cleansing enables full recovery of lethally infected rodent animals within 7 days by treating them twice in series. It is also validated that parameters reflecting immune homeostasis, such as blood cell counts, cytokine levels, and transcriptomics changes, are restored in blood of the fatally infected rats after treatment.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

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.

Engineered Ultra-High Affinity Synthetic Antibodies for SARS-CoV-2 Neutralization and Detection.

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus' impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC50-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (ß-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.

Cefepime/tazobactam compared with other tazobactam combinations against problem Gram-negative bacteria.

OBJECTIVES: Piperacillin/tazobactam has long been a broad-spectrum 'workhorse' antibiotic; however, it is compromised by resistance. One response is to re-partner tazobactam with cefepime, which is easier to protect, being less ß-lactamase labile, and to use a high-dose and prolonged infusion. On this basis, Wockhardt are developing cefepime/tazobactam (WCK 4282) as a 2+2 g q8h combination with a 90-min infusion. METHODS: The activity of cc cefepime/tazobactam was assessed, with other tazobactam combinations as comparators, against 1632 Enterobacterales, 745 Pseudomonas aeruginosa and 450 other non-fermenters, as submitted to the UK National Reference Laboratory. These were categorised by carbapenemase-gene detection and interpretive reading of phenotypes, with MICs determined by British Society for Antimicrobial Chemotherapy agar dilution. RESULTS: Although higher breakpoints may be justifiable, based on the pharmacodynamics, the results were reviewed against current cefepime criteria. On this basis, cefepime/tazobactam was broadly active against Enterobacterales with AmpC enzymes and extended-spectrum ß-lactamases (ESBLs), even when they had ertapenem resistance, suggesting porin loss. At 8+8 mg/L, activity extended to > 90% of Enterobacterales with OXA-48 and KPC carbapenemases, although the MICs for KPC producers belonging to the international Klebsiella pneumoniae ST258 lineage were higher; metallo-ß-lactamase producers remained resistant. Cefepime/tazobactam was less active than ceftolozane/tazobactam against Pseudomonas aeruginosa with AmpC de-repression or high-level efflux but achieved wider antipseudomonal coverage than piperacillin/tazobactam. Activity against other non-fermenters was species-specific. CONCLUSION: Overall, cefepime/tazobactam had a spectrum exceeding those of piperacillin/tazobactam and ceftolozane/tazobactam and resembling or exceeding that of carbapenems. Used as a 'new-combination of old-agents' it has genuine potential to be 'carbapenem-sparing'.

Spread of OXA-48-producing Klebsiella pneumoniae among COVID-19-infected patients: The storm after the storm.

The impact of secondary infections by multidrug-resistant bacteria in COVID-19- infected patients has yet to be evaluated. Here, we report the clinical and molecular features of an outbreak of seven patients carrying CTX-M-15- and OXA-48-producing Klebsiella pneumoniae belonging to ST326 during COVID-19 pandemic in an ICU in northern Spain. Those patients were admitted to beds close to each other, two of them developed ventilator-associated pneumonia (VAP), one exhibited primary bacteremia and the remaining four were considered to be colonized. None of them was colonized prior to admission to the ICU an all, except one of those who developed VAP, were discharged. Hydroxychloroquine and lopinavir/ritonavir were administered to all of them as COVID-19 therapy and additionally, three of them received tocilizumab and corticosteroids, respectively. Reusing of personal protective equipment due to its initial shortage, relaxation in infection control measures and negative-pressure air in ICU rooms recommended for the protection of health care workers (HCWs), could have contributed to this outbreak. Maximization of infection control measures is essential to avoid secondary infections by MDR bacteria in COVID-infected patients.