Antimicrobial resistance is a critical global challenge in the 21st century, validating Sir Alexander Fleming’s warning about the misuse of antibiotics leading to resistant microbes. With a dwindling arsenal of effective antibiotics, it is imperative to concentrate on alternative antimicrobial strategies. Previous studies have not comprehensively discussed the advantages and limitations of various strategies, including bacteriophage therapy, probiotics, immunotherapies, photodynamic therapy, essential oils, nanoparticles and antimicrobial peptides (AMPs) within a single review. This review addresses that gap by providing an overview of these various non-antibiotic antimicrobial strategies, highlighting their pros and cons, with a particular emphasis on antimicrobial peptides (AMPs). We explore the mechanism of action of AMPs against bacteria, viruses, fungi and parasites. While these peptides hold significant promise, their application in mainstream drug development is hindered by challenges such as low bioavailability and potential toxicity. However, advancements in peptide engineering and chemical modifications offer solutions to enhance their clinical utility. Additionally, this review presents updates on strategies aimed at improving the cost, stability and selective toxicity of AMPs through the development of peptidomimetics. These molecules have demonstrated effective activity against a broad range of pathogens, making them valuable candidates for integration into surface coatings to prevent device-associated infections. Furthermore, we discuss various approaches for attaching and functionalising these peptides on surfaces. Finally, we recommend comprehensive in vivo studies to evaluate the efficacy of AMPs and their mimetics, investigate their synergistic combinations with other molecules and assess their potential as coatings for medical devices.

Purposes: This study compared antimicrobial resistance between exoU and exoS Pseudomonas aeruginosa strains isolated from microbial keratitis (MK) and examined their resistance genotypes. Methods: The presence of exoU and exoS was determined in 187 MK isolates using PCR. Minimum inhibitory concentrations of ciprofloxacin, levofloxacin, gentamicin, and tobramycin were measured. Whole genome sequencing of 39 isolates was used to identify resistance genes via Resfinder. Mutations in key genes, including DNA gyrase, topoisomerase IV, efflux pumps, and DNA repair systems, were analyzed using Geneious Prime. Functional effects of novel SNPs were predicted using SIFT. Results: Antibiotic resistance was significantly higher in exoU than exoS: 38.2% vs. 20.5% for ciprofloxacin, 29.1% vs. 12.1% for levofloxacin, 40% vs. 23.5% for gentamicin, and 29.1% vs. 14.4% for tobramycin (all p < 0.05). ExoU isolates exclusively had mutations in GyrA (Thr83Ile) and ParC (Ser87Ile), as well as in efflux pump regulators MexZ (Gly89Ser), NalC (Asp79Glu) and MexS (Val73Ala) (p < 0.01). They also more frequently harbored the acquired resistance genes aph(6)-Id (55% vs. 0%) and aph(3’’)-Ib (60% vs. 5.3%) and had higher mutation rates in DNA repair genes mutL (70% vs. 15.8%) and mutS (45% vs. 5.3%) (p < 0.01). Mutations in gyrA, parC, efflux pump (mexB, mexD, mexY) and regulator (mexZ, nalC, mexS) genes correlated with fluoroquinolone resistance (R ≥ 0.33; p ≤ 0.04). Possession of aph(3’’)-Ib, aph(6)- Id and SNPs in efflux pump regulators mexZ and parR were associated with aminoglycoside resistance. Conclusion: ExoU strains exhibited more resistance genes and mutations, contributing to higher resistance to fluoroquinolones and aminoglycosides.

Purpose: This study aimed to investigate differences in virulence factors between exoU and exoS lineages of Pseudomonas aeruginosa from microbial keratitis (MK). Method: Initially, the whole genome sequence (WGS) data of 20 exoU and 19 exoS P. aeruginosa keratitis isolates from India and Australia retrieved from NCBI genome database was assessed for the presence of different virulence genes using the Virulence Factors Database (VFDB). To confirm the VFDB results, a separate set of keratitis isolates consisting of 148 P. aeruginosa (36 from India and 112 from Australia) were screened by PCR for the presence of exoU , exoS , and the virulence genes found to be significantly different in the VFDB analysis. Flagellar length was measured by transmission electron microscopy (TEM) and phospholipase D (PLD) activity was determined by Amplex Red Phospholipase D Assay Kit. Results: From 327 virulence-associated genes, the VFDB analysis identified significant differences in four virulence factor genes pilA , pldA , algP , and flaG between the exoU and exoS groups (all p < 0.05).When combining PCR and VFDB data from 187 keratitis isolates, pldA (83.6% vs. 31.8%, p < 0.01) and flaG (80% vs. 55.3%, p < 0.01) showed significantly higher prevalence in the exoU than the exoS . Similar trends were observed among Australian isolates while within the Indian isolates, only pldA (77.3% vs. 35.3%, p < 0.01) differed significantly. No association was found between flagellar length and flaG , but PLD activity correlated with the presence of the pldA . Conclusion: The pldA  and flaG genes might be relevant virulence factors for the exoU group, potentially associated with the severity of MK.

Background: Microbial keratitis (MK) is a rapid and devastating infection that can result reduced vision, with lack of treatment potentially resulting in stromal necrosis and even permanent vision loss. Pseudomonas aeruginosa is a common cause of MK and its rise in antibiotic resistance has made it increasingly difficult to treat. Purpose: This review aims to provide a better understanding of the resistance mechanisms of P. aeruginosa and highlights major adaptations to combat fluoroquinolones, aminoglycosides, β-lactams and polymyxin antibiotics commonly used in MK, and addresses the global resistance profiles of P. aeruginosa keratitis. Method: A narrative review was conducted using PubMed, Scopus, Web of Science, MEDLINE, and Google Scholar. Search terms included “ Pseudomonas aeruginosa ”, “microbial keratitis”, “antibiotic resistance”, antibiotic class-specific resistance terms, “surveillance studies”, and “regional resistance patterns” to consolidate current information of the various intrinsic, acquired and adaptive resistance mechanisms of P. aeruginosa conferred across fluoroquinolones, aminoglycosides, β-lactams and polymyxin along with resistance profile of keratitis isolates across continents. Results: P. aeruginosa displays complex resistance mechanisms, including intrinsic efflux systems, reduced porin permeability, enzymatic drug inactivation, horizontal gene transfer, and target-site mutations, contributing to MDR in MK. Resistance patterns vary markedly by region, with higher resistance to fluoroquinolones, cephalosporins, and aminoglycosides reported in Asia, while Europe and North America showed lower rates. Australian isolates demonstrate heterogeneous resistance, retaining susceptibility to aminoglycosides. Conclusion: Future studies comparing resistance mechanisms and data of P. aeruginosa across regions will be essential to identify geographical variations, inform region-specific surveillance, guide targeted therapies to improve interventions of MK.

The aim of this study was to determine differences in susceptibility of exoU and exoS Pseudomonas aeruginosa to multi-purpose disinfecting solutions (MPDS) and antibiotics and their association with disinfectant resistance genes. A total of 16 exoU and 24 exoS P. aeruginosa keratitis isolates were tested for susceptibility against four multipurpose disinfecting solutions (OPTI-FREE PureMoist, Acuvue RevitaLens, Biotrue, and Renu Advanced Formula) using minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. Antibiotic susceptibility to ciprofloxacin, levofloxacin, gentamicin, and tobramycin was determined by MIC. Whole genome sequences from 27 isolates were utilized to identify disinfectant resistance genes and single nucleotide polymorphisms (SNPs) in the genes using the Comprehensive Antibiotic Resistance Database (CARD) and Geneious Prime respectively. Polymerase chain reaction (PCR) was used to detect the disinfectant resistance genes qacE, qacEΔ1, and sugE1. Correlation between disinfectant resistance genes and their SNPs with disinfectant and antibiotic MICs was determined by Spearman's Rho. All MPDS were bactericidal at full strength, but upon dilution OPTI-FREE Puremoist and Renu Advanced Formula were the most active against the exoU and exoS groups correspondingly. ExoU showed significantly higher resistance than exoS (p ≤ 0.04) to levofloxacin (37.5 % vs. 8.3 %), gentamicin (43.8 % vs. 0 %), and tobramycin (37.5 % vs. 0 %). The qacEΔ1 and sugE1 genes along with several SNPs in the resistance genes were more common in the exoU group. SNPs in disinfectant resistance genes correlated with MPDS MICs/MBCs, notably mexQ Arg1036His (R ≥ -0.38, p ≤ 0.04), Gly505Asp (R ≥ 0.40, p ≤ 0.04) and opmD Gly269Ser (R ≥ 0.43, p ≤ 0.03). QacEΔ1 and sugE1 were linked to increased MIC (R ≥ 0.54, p < 0.01) and MBC (R ≥ 0.50, p < 0.01) for Renu Advanced Formula. Also, multiple SNPs and qacEΔ1 and sugE1 showed moderate to large positive associations with antibiotic resistance. ExoU strains had distinct MPDS susceptibility patterns, with higher disinfectant resistance genes prevalence and mutations, contributing to MPDS and antibiotics resistance.

Background: Pseudomonas aeruginosa is a versatile Gram-negative pathogen that thrives in diverse environments. This pathogen causes a range of infections, including microbial keratitis (MK), a sight-threatening corneal infection. Central to its virulence are six specialized protein secretion systems (Types 1-6) that span the complex cell envelope. One-step systems (Types 1, 3, 4, 6) directly translocate effectors across both of its cell membranes, while two-step systems (Types 2, 5) first export substrates into the periplasm via the Sec or Tat pathways before outer-membrane release. These molecular machines deliver toxins, enzymes and competitive effectors that facilitate tissue damage, immune evasion, nutrient acquisition, biofilm formation and interbacterial killing. Their expression and activity are often coordinated by quorum-sensing networks (Las, Rhl, Pqs, Iqs). Although various secretion systems and their effectors have been characterized, the specific contributions of each system to corneal infection have yet to be comprehensively reviewed. Purposes and Methods: PubMed and Google Scholar were searched to synthesize current knowledge of the structure, regulation, and substrates of these secretion systems, to highlight their contributions to keratitis pathogenesis, and to evaluate emerging anti-virulence strategies targeting these pathways as novel therapeutics. Results: The T3SS system and its effectors ExoU and ExoS play dominant roles in keratitis severity while other secretion systems further enhance virulence by facilitating toxin release, biofilm development, and interbacterial competition. Regulatory interactions with quorum-sensing pathways amplify their impact during infection. Conclusion: Understanding the functional roles of all six secretion pathways and their regulatory mechanisms will be critical for identifying and developing novel anti-virulence therapeutics for MK.