Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics for treating infections caused by drug-resistant bacteria; yet, many peptides are limited by toxicity to eukaryotic cells and instability in biological environments. Conjugation to linear polymers that reduce cytotoxicity and improve stability, however, often decreases antimicrobial activity. In this work, we combine the biocompatibility advantages of poly(ethylene glycol) (PEG) with the efficacy merits of nonlinear polymer architectures that accommodate multiple AMPs per molecule. By conjugating a chemokine-derived AMP, stapled Ac-P9, to linear and star-shaped PEG with various arm numbers and lengths, we investigated the role of molecular architecture in solution properties (i.e., ζ-potential, size, and morphology) and performance (i.e., antimicrobial activity, hemolysis, and protease resistance). Linear, 4-arm, and 8-arm conjugates with 2-2.5 kDa PEG arms were found to form nanoscale structures in solution with lower ζ-potentials relative to the unconjugated AMP, suggesting that the polymer partially shields the cationic AMP. Reducing the length of the PEG arms of the 8-arm conjugate to 1.25 kDa appeared to better reveal the peptide, seen by the increased ζ-potential, and promote assembly into particles with a larger size and defined spherical morphology. The antimicrobial effects exerted by the short 8-arm conjugate rivaled that of the unconjugated peptide, and the AMP constituents of the short 8-arm conjugate were protected from proteolytic degradation. All other conjugates examined also imparted a degree of protease resistance, but exhibited some reduced level of antimicrobial activity as compared to the AMP alone. None of the conjugates caused significant cytotoxic effects, which bodes well for their future potential to treat infections. While enhancing proteolytic stability often comes with the cost of lower antimicrobial activity, we have found that presenting AMPs at high density on a neutral nonlinear polymer strikes a favorable balance, exhibiting both enhanced stability and high antimicrobial activity.

Conjugating antimicrobial peptides (AMPs) to nonlinear polymers is a promising strategy to overcome the translational challenges of AMPs toward treating infections caused by antibiotic-resistant bacteria. Nonlinear polymers, and therefore conjugates, can be prepared with various architectures (e.g., star-shaped, comb-like, hyperbranched, etc.), however, the effects of polymer architecture on antimicrobial performance and related properties, like size and morphology in solution and secondary structure, are not yet well-understood. Here, we compare conjugates of the human chemokine-derived AMP stapled P9 with poly(ethylene glycol) (PEG) prepared in two of the major nonlinear architectures: star-shaped and comb-like. At comparable molecular weights and compositions (peptide wt %), comb-like conjugates afford increased helicity, solubility, antimicrobial activity, and proteolytic stability compared to star-shaped analogs. We then leveraged the expansive design space of comb-like architectures to prepare conjugates with different backbone lengths and PEG side chain lengths, with shorter PEG side chains leading to increased helicity, yet potentially less shielding from proteolytic degradation and the longest backbone lengths furnishing the most potent antimicrobial activity. Both comb-like and star-shaped conjugates display high zeta potential, indicating that the cationic AMPs were accessible for electrostatic interactions with bacterial membranes. Yet, the comb-like conjugates showed a higher fraction of unimolecular structures indicative of a lower propensity for supramolecular assembly that could be encumbering the desired AMP-bacteria interactions in the star-shaped conjugates. Together, our work shows comb-like AMP-polymer conjugates to outperform analogous star-shaped conjugates, while adding design flexibility to access an expansive range of monomer chemistries, monomer distributions, and backbone lengths to modulate performance-determining properties and ultimately furnish an effective suite of AMP-polymer materials as alternatives to conventional antibiotics for combatting bacterial infections.

Klebsiella pneumoniae carbapenemases (KPCs) are a family of serine β-lactamases that confer broad antibiotic resistance by hydrolyzing virtually all β-lactam (BL) agents. Contemporary β-lactamase inhibitors (BLIs) such as avibactam were developed to neutralize the activity of KPCs and other clinically important carbapenemases. Ceftazidime-avibactam (CZA), a BL/BLI combination in which the cephalosporin ceftazidime is protected from KPC-mediated hydrolysis, demonstrated improved outcomes in early clinical use. However, CZA-resistant isolates soon emerged. Herein, we describe a challenging clinical case in which high-level resistance to CZA evolved during therapy for a complicated infection caused by carbapenem-resistant Serratia marcescens harboring bla _KPC-2 . Whole-genome sequencing, analysis of antibiotic resistance genes, and phenotypic susceptibility assays of serial S. marcescens isolates revealed that resistance arose via a 45-nucleotide in-frame duplication within bla _KPC-2 , yielding KPC variant 44 ( bla _KPC-44 ). Notably, the evolution of bla _KPC-44 not only conferred resistance to CZA but also marked cross-resistance to meropenem-vaborbactam and imipenem-relebactam while remaining susceptible to cefiderocol. To our knowledge, this report represents the first description of bla _KPC-44 emerging outside of K. pneumoniae and among the few documenting a CZA-resistance-conferring KPC variant emerging in a non -K . pneumoniae Enterobacterales species. Ultimately, the evolved strain persisted despite therapy throughout a fatal clinical course, underscoring the potential for CZA selective pressure to drive treatment-emergent resistance to multiple contemporary BL/BLI agents.

The continued emergence and spread of antimicrobial resistance among pathogenic bacteria are ever-growing threats to health and economy. Here, we report the draft genomes for 45 Enterobacterales clinical isolates, including historical and contemporary drug-resistant organisms, obtained in Pakistan between 1998 and 2016: 5 Serratia , 3 Salmonella , 3 Enterobacter , and 34 Klebsiella .