Introduction: Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16–20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described.Methods: Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller–Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well.Results: The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively.Discussion: In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method’s uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

Antimicrobial peptides (AMPs) are a diverse group of small, naturally occurring molecules that orchestrate the innate immune response of various organisms, from microorganisms to humans. Characterized by their broad-spectrum activity against bacteria, fungi and viruses, AMPs are increasingly recognized for their potential as novel therapeutic agents in the face of rising antibiotic resistance. Here, we present several newly designed AMPs, one of which, DTN6, exerts significant activity against several organisms with MIC values as low as 0.5 µg/mL. The D-TN6 peptide influences both bacteria and yeasts. Scanning electron microscopy and transmission electron microscopy results showed that the bacterial membrane is affected by D-TN6, which is resistant to proteases and is effective against antibiotic-resistant pathogens with hemolytic activity and low toxicity. The D-TN6 peptide is effective in vivo against standard S. aureus strains in wounds. Thus, D-TN6 is a potent antibiotic candidate with a broad spectrum of activity. Overall, AMPs are a promising tool for the development of next-generation antimicrobial agents that could mitigate global health threats posed by multidrug-resistant pathogens.

M13 bacteriophages (phages) are used as very important tools in molecular biology, biotechnology and, nanotechnology. Many methods have been developed so far for the purification of these phages. However, it is important that phages retain their infecting properties, especially in biotechnological applications such as phage display technology. The most widely used is the PEG precipitation method, but it has some limitations such as impurities and reduced infectivity. To overcome them, we developed a new method for purification of M13 bacteriophages using syringe filters made of cellulose acetate membranes with a pore diameter of 0.22 µm. Phages were aggregated so that they could remain on the filters and for this purpose, the pH of the phage cultures was reduced to 3. The phage cultures were filtered and then the phages were recovered in tris-buffered saline (TBS) buffer (pH 10.5) by reversing the filter. The recovery rate was 250% higher than the standard PEG method. This new Faj-elek method offers an alternative to existing methods, allowing cheaper, easier and faster purification of M13 phages using syringe filters available in every research laboratory.

Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in Escherichia coli and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3′ ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3′ end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication.

Objectives: The escalation of multidrug-resistant (MDR) clinically relevant bacterial isolates, including Escherichia coli and key ESKAPE pathogens, represents a critical global healthcare threat. While antimicrobial peptides (AMPs) offer promising alternatives, metabolic instability often limits their clinical use. This study investigated the therapeutic potential, antimicrobial efficacy, and resistance dynamics of peptide D-TN6 against highly resistant bacterial strains. Methods: D-TN6 efficacy was evaluated against 164 clinical isolates, including pan-drug-resistant, carbapenem-resistant, and polymyxin-resistant phenotypes. A 20-passage serial induction assay compared resistance development kinetics of D-TN6 against gentamicin over an extended period. Results: D-TN6 demonstrated potent efficacy against MRSA (minimum inhibitory concentration (MIC)₉₀: 1 µg/mL) and remained effective against polymyxin B-resistant Klebsiella pneumoniae (MIC₉₀: 8 µg/mL) and pan-drug-resistant Acinetobacter baumannii (MIC₉₀: 8 µg/mL). In resistance assays, while the gentamicin MIC increased 256-fold by the 17th passage, the D-TN6 MIC remained constant at 1 µg/mL throughout the 20-passage study. Conclusions: These findings underscore D-TN6 as a robust therapeutic candidate. Its efficacy against MDR strains and its feature of low propensity for resistance development—likely due to membrane disruption—position D-TN6 as a promising solution for life-threatening infections where conventional last-resort agents fail.