The ongoing COVID-19 pandemic reminds human beings of the challenging nature of the virus SARS-CoV-2. Likewise, bacterial resistance constitutes another potential threat to human health globally. It is the time to prepare for the worst scenario that antibiotic-resistant pathogens could cause 10 million deaths by 2050. Six major types of bacterial pathogens are abbreviated as ESKAPE (i.e., gram-positive Enterococcus faecium, Staphylococcus aureus, gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) due to their capability of escaping the action of conventional antibiotics. These pathogens utilize different mechanisms to mitigate the killing effects of antibiotics. To identify potent antimicrobials, numerous approaches are under exploration. This chapter summarizes antimicrobial peptides (AMPs), their engineered analogs, synthetic mimics, conjugates, combinations, formulated nanoparticles, and surface immobilized forms that have demonstrated activity against the ESKAPE pathogens. Also discussed are mechanisms of bacterial killing, and the potential of AMPs as future antibiotics.

The use of implanted medical devices improves the quality of patients' lives. Both polymers (e.g. polyethylene terephthalate) and metals (e.g. titanium and steel) are used to fabricate these devices. An unwanted accompanying problem with such implants is microbial infection (similar to 3%). Bacteria are able to colonize the surface of implants and form difficult-to-remove polymicrobial bio-films. This chapter reviews an alternative approach, which prevents biofilm formation by immobilization of antimicrobial peptides on biomaterial surfaces. These peptides are usually short, cationic and can rapidly eliminate bacteria, viruses, fungi and parasites. We highlight antimicrobial immobilization methods, physical characterization approaches and biological activity assays for surface-coated peptides, and discuss current achievements as well as existing issues with the development of antibiofilm surfaces.

This review deals with the design and application strategies of new antibiotics based on naturally occurring antimicrobial peptides (AMPs). The initial candidate can be designed based on three-dimensional structure or selected from a library of peptides from natural or laboratory sources followed by optimization via structure-activity relationship studies. There are also advanced application strategies such as induction of AMP expression from host cells by various factors (e.g., metals, amino acids, vitamin D and sunlight), the use of engineered probiotic bacteria to deliver peptides, the design of prodrug and peptide conjugates to improve specific targeting. In addition, combined uses of newly developed AMPs with existing antimicrobial agents may provide a practical avenue for effective management of antibiotic resistant bacteria (superbugs), including biofilms. Finally, we highlight AMPs already in use or under clinical trials.

Clostridium difficile , a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. Ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for P. aeruginosa but are absent in mammals. One such enzyme, type II dehydroquinase (DHQase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. This step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aromatic metabolites. The recombinant expression, purification and crystal structure of catalytically active DHQase from P. aeruginosa (PaDHQase) are presented. Cubic crystals belonging to space group F23, with unit-cell parameters a=b=c=125.39 Å, were obtained by vapor diffusion in sitting drops and the structure was refined to an R factor of 16% at 1.74 Å resolution. PaDHQase is a prototypical type II DHQase with the classical flavodoxin-like α/β topology.

Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.

Pseudomonas aeruginosa is a major cause of opportunistic infection and is resistant to most antibiotics. As part of efforts to generate much-needed new antibiotics, structural studies of enzymes that are critical for the virulence of P. aeruginosa but are absent in mammals have been initiated. 2-Keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8Ps), also known as 2-dehydro-3-deoxyphosphooctonate aldolase, is vital for the survival and virulence of P. aeruginosa. This enzyme catalyzes a key step in the synthesis of the lipopolysaccharide (LPS) of most Gram-negative bacteria: the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate to produce 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P). This step is vital for the proper synthesis and assembly of LPS and the survival of P. aeruginosa. Here, the recombinant expression, purification and crystal structure of KDO8Ps from P. aeruginosa are presented. Orthorhombic crystals were obtained by vapor diffusion in sitting drops in the presence of 1 mM phosphoenlpyruvate. The structure reveals the prototypical α/β TIM-barrel structure expected from this family of enzymes and contains a tetramer in the asymmetric unit.