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Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs' minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the presence of ESKAPE organisms on the hands of students working in the intensive care unit (ICU) at a veterinary academic hospital. Methods: A cross-sectional study was conducted among students working in an ICU at a veterinary academic hospital in South Africa. Students were sampled before the start of the ICU shift using a modified glove-juice method. Standard microbiological techniques and a series of polymerase chain reaction (PCR) assays were used to identify and characterize the bacteria. All the isolates were tested for resistance against a specific panel of antibiotics using the disk diffusion method. Proportions of bacterial species and their antimicrobial-susceptibility profiles were calculated. Results: At screening, all the veterinary students (n = 62) carried at least one of the ESKAPE organisms on their hands. Escherichia coli was the most isolated organism (76%, 47/62), followed by P. aeruginosa (48%, 30/62), A. baumannii (47%, 29/62), E. faecium (35%, 22/62), K. pneumoniae (27%, 17/62), and S. aureus (24%, 15/62). A reduced proportion of isolates were recovered from the samples, E. coli (26%, 12/47), E. faecium (23%, 5/22), P. aeruginosa (43%, 13/30), A. baumannii (24%,7/29), K. pneumoniae (41%, 7/17), and S. aureus (20%, 3/15). Most of the organisms showed a high proportion of resistance to at least one antibiotic. Multidrug resistance was reported among just over half (56%, 5/9) of E. coli, 40% (2/5) of E. faecium, 100% (13/13) of P. aeruginosa, and 33% (1/3) of S. aureus isolates. Conclusion: Students working in the ICU carry several organisms belonging to the ESKAPE group of organisms before contact with patients. Moreover, MDR resistance was common among this group of organisms. The findings of the present study underscore the importance of infection prevention and control (IPC) strategies to help reduce the likelihood of the spread of these organisms to personnel, owners, family members, and patients. [ABSTRACT FROM AUTHOR]

Introduction: This study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge. Methods: The selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their a-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK. Results: Firstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their a-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 µM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations. Discussion: Assessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs. [ABSTRACT FROM AUTHOR]

Background: The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms. Methods: In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties. Results: The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3–4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25–30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with <50% PI incorporation for the L-RWV peptides. By contrast, most H-RWV peptides displayed almost up to 100% PI incorporation. The results suggest that membrane perturbation is not the primary killing mechanism of the L-amphipathic RWV peptides, in contrast to the H-RWV peptides. Conclusions: Taken together, the data indicate that both types of amphipathicity may provide different ideal pharmacological properties that deserve further investigation. [ABSTRACT FROM AUTHOR]

Abstract Objective This study aimed to investigate the presence of ESKAPE organisms on the hands of students working in the intensive care unit (ICU) at a veterinary academic hospital. Methods A cross-sectional study was conducted among students working in an ICU at a veterinary academic hospital in South Africa. Students were sampled before the start of the ICU shift using a modified glove-juice method. Standard microbiological techniques and a series of polymerase chain reaction (PCR) assays were used to identify and characterize the bacteria. All the isolates were tested for resistance against a specific panel of antibiotics using the disk diffusion method. Proportions of bacterial species and their antimicrobial-susceptibility profiles were calculated. Results At screening, all the veterinary students (n = 62) carried at least one of the ESKAPE organisms on their hands. Escherichia coli was the most isolated organism (76%, 47/62), followed by P. aeruginosa (48%, 30/62), A. baumannii (47%, 29/62), E. faecium (35%, 22/62), K. pneumoniae (27%, 17/62), and S. aureus (24%, 15/62). A reduced proportion of isolates were recovered from the samples, E. coli (26%, 12/47), E. faecium (23%, 5/22), P. aeruginosa (43%, 13/30), A. baumannii (24%,7/29), K. pneumoniae (41%, 7/17), and S. aureus (20%, 3/15). Most of the organisms showed a high proportion of resistance to at least one antibiotic. Multidrug resistance was reported among just over half (56%, 5/9) of E. coli, 40% (2/5) of E. faecium, 100% (13/13) of P. aeruginosa, and 33% (1/3) of S. aureus isolates. Conclusion Students working in the ICU carry several organisms belonging to the ESKAPE group of organisms before contact with patients. Moreover, MDR resistance was common among this group of organisms. The findings of the present study underscore the importance of infection prevention and control (IPC) strategies to help reduce the likelihood of the spread of these organisms to personnel, owners, family members, and patients.

Abstract Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels.

Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels. [ABSTRACT FROM AUTHOR]

Introduction: Bacterial infection and biofilm formation contribute to impaired healing in chronic diabetic wounds. Staphylococcus aureus and Pseudomonas aeruginosa are found in human diabetic wound biofilms. They may develop antibiotic resistance, increasing the urgency for alternative or complementary therapies. Diabetic wound healing may be improved with the use of biomedically engineered scaffolds, which can also serve as delivery systems for antibacterial compounds. Manuka honey is a potent antibacterial and wound care agent due to its high osmolarity, low pH, and constituents (such as methylglyoxal). Honey exhibits bacteriostatic and bactericidal effects, modulates the expression of biofilm forming genes, and restores antibiotic susceptibility in previously drug resistant pathogens. Methods: In this study, we created a dermal regeneration template (DRT) composed of polycaprolactone-gelatin (PCL-gelatin) and Manuka honey to retain honey in the wound and also provide a scaffold for tissue regeneration. Results and discussion: Soluble Manuka honey inhibited the planktonic and biofilm growth of both S. aureus (UWH3) and P. aeruginosa (PA14) co-cultures. Manuka honey embedded PCL-gelatin scaffolds did not exhibit bacteriostatic or bactericidal effects on cocultures of UHW3 and PA14; however, they promoted the expression of AgrA, a gene associated with dispersal of S. aureus biofilms. [ABSTRACT FROM AUTHOR]

Background: The emergence of multidrug-resistant pathogens through excessive and indiscriminate use of antibiotics, together with the lack of highly efficient treatment options for bacterial infections, has raised the development of novel antimicrobial agents to top priority. In this context, cryptic host defense peptides (cHDPs) are being explored as a novel class of antimicrobial agents. In this study, short peptides were designed from the long nonantibacterial protein ToAP2 and analysed for their positive net charge, hydrophobicity, hydrophobic moment, hydrophobic and hydrophilic planes. Methods: From the designed fragments, five 15 amino acid fragments were synthesised by solid-phase peptide synthesis (SPPS) and analysed for antimicrobial activity against ESKAPE pathogens. All the peptides were subject to cytotoxicity, mode of action, structure and function studies to find the best template for further optimisation. Results: Among them, two peptides, FKL15 and SKL15, showed better efficiency in killing P. aeruginosa under physiological salt and plasma conditions with no cytotoxicity issues. Further, the peptides destroyed the bacterial membranes and adopted a random coil structure in the presence of the bacteria. Conclusions: The data indicates that FKL15 and SKL15 are promising antimicrobial peptides against antibiotic-resistant bacteria with great potential to develop as drugs with high economic value. [ABSTRACT FROM AUTHOR]

Background Lysins (cell wall hydrolases) targeting gram-negative organisms require engineering to permeabilize the outer membrane and access subjacent peptidoglycan to facilitate killing. In the current study, the potential clinical utility for the engineered lysin CF-370 was examined in vitro and in vivo against gram-negative pathogens important in human infections. Methods Minimum inhibitory concentration (MICs) and bactericidal activity were determined using standard methods. An in vivo proof-of-concept efficacy study was conducted using a rabbit acute pneumonia model caused by Pseudomonas aeruginosa. Results CF-370 exhibited potent antimicrobial activity, with MIC50/90 values (in µg/mL) for: P aeruginosa , 1/2; Acinetobacter baumannii , 1/1; Escherichia coli , 0.25/1; Klebsiella pneumoniae , 2/4; Enterobacter cloacae 1/4; and Stenotrophomonas maltophilia 2/8. CF-370 furthermore demonstrated bactericidal activity, activity in serum, a low propensity for resistance, anti-biofilm activity, and synergy with antibiotics. In the pneumonia model, CF-370 alone decreased bacterial densities in lungs, kidneys, and spleen versus vehicle control, and demonstrated significantly increased efficacy when combined with meropenem (vs either agent alone). Conclusions CF-370 is the first engineered lysin described with potent broad-spectrum in vitro activity against multiple clinically relevant gram-negative pathogens, as well as potent in vivo efficacy in an animal model of severe invasive multisystem infection. [ABSTRACT FROM AUTHOR]

The accelerated rise in antimicrobial resistance (AMR) poses a significant global health risk, necessitating the exploration of alternative strategies to combat pathogenic infections. Biofilm-related infections that are unresponsive to standard antibiotics often require the use of higher-order antimicrobials with toxic side effects and the potential to disrupt the microbiome. Probiotic therapy, with its diverse benefits and inherent safety, is emerging as a promising approach to prevent and treat various infections, and as an alternative to antibiotic therapy. In this study, we isolated novel probiotic bacteria from the gut of domestic goats (Capra hircus) and evaluated their antimicrobial and anti-biofilm activities against the 'ESKAPE' group of pathogens. We performed comprehensive microbiological, biochemical, and molecular characterizations, including analysis of the 16S-rRNA gene V1-V3 region and the 16S-23S ISR region, on 20 caprine gut-derived lactic acid bacteria (LAB). Among these, six selected Lactobacillus isolates demonstrated substantial biofilm formation under anaerobic conditions and exhibited robust cell surface hydrophobicity and autoaggregation, and epithelial cell adhesion properties highlighting their superior enteric colonization capability. Notably, these Lactobacillus isolates exhibited broad-spectrum growth inhibitory and anti-biofilm properties against 'ESKAPE' pathogens. Additionally, the Lactobacillus isolates were susceptible to antibiotics listed by the European Food Safety Authority (EFSA) within the prescribed Minimum Inhibitory Concentration limits, suggesting their safety as feed additives. The remarkable probiotic characteristics exhibited by the caprine gut-derived Lactobacillus isolates in this study strongly endorse their potential as compelling alternatives to antibiotics and direct-fed microbial (DFM) feed supplements in the livestock industry, addressing the escalating need for antibiotic-free animal products. [ABSTRACT FROM AUTHOR]

Far-ultraviolet C light, with a wavelength of 200–230 nm, has demonstrated broad-spectrum germicidal efficacy. However, due to increased interest in its use as an alternative antimicrobial, further knowledge about its fundamental bactericidal efficacy is required. This study had two objectives. Firstly, it investigated experimentally the Far-UVC dose–response of common bacteria suspended at various cell densities in transparent buffer, ensuring no influence from photosensitive suspending media. Increasing doses of Far-UVC were delivered to Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in PBS at 101, 102, 103, 105 and 107 CFU·mL−1, with surviving colony-forming units enumerated (n ≥ 3). Secondly, through a systematised literature review, this work sought to explore the impact of genus/species, Gram type, cell form, cell density and irradiance on dose–response. The screening of 483 publications was performed with 25 included in the study. Data for 30 species were collated, analysed and compared with the experimental results. Overall, Gram-positive species showed greater resilience to Far-UVC than Gram-negative; some inter-species and inter-genera differences in resilience were identified; endospores were more resilient than vegetative cells; the results suggested that inactivation efficiency may decrease as cell density increases; and no significant correlation was identified between irradiance and bactericidal dose effect. In conclusion, this study has shown Far-UVC light to be an effective decontamination tool against a vast range of bacterial vegetative cells and endospores. [ABSTRACT FROM AUTHOR]

Background: Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. Results: Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. Conclusions: Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy. [ABSTRACT FROM AUTHOR]

The species included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the genus Enterobacter) have a high capacity to develop antimicrobial resistance (AMR), a health problem that is already among the leading causes of death and could kill 10 million people a year by 2050. The generation of new potentially therapeutic molecules has been insufficient to combat the AMR "crisis", and the World Health Organization (WHO) has stated that it will seek to promote the development of rapid diagnostic strategies. The physicochemical properties of metallic nanoparticles (MNPs) have made it possible to design biosensors capable of identifying low concentrations of ESKAPE bacteria in the short term; other systems identify antimicrobial susceptibility, and some have been designed with dual activity in situ (bacterial detection and antimicrobial activity), which suggests that, in the near future, multifunctional biosensors could exist based on MNPs capable of quickly identifying bacterial pathogens in clinical niches might become commercially available. This review focuses on the use of MNP-based systems for the rapid and accurate identification of clinically important bacterial pathogens, exhibiting the necessity for exhaustive research to achieve these objectives. This review focuses on the use of metal nanoparticle-based systems for the rapid and accurate identification of clinically important bacterial pathogens. [ABSTRACT FROM AUTHOR]

ABSTRACT Amid persistent concerns over microbial foodborne illnesses and escalating antibiotic resistance, we introduce “NP,” a novel and effective broad-spectrum natural antimicrobial product derived from the filtered culture broth of Aspergillus oryzae grown in a food-grade liquid medium. NP demonstrates potent bactericidal activity against a range of food-borne and ESKPAE pathogens, including Staphylococcus aureus (including eight distinct drug-resistant methicillin-resistant Staphylococcus aureus strains), Listeria monocytogenes, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Escherichia coli (including O157:H7) with minimal inhibitory strength ranging from 25% to 100%. In addition, NP exhibits robust antifungal activity against several human pathogenic fungi including Aspergillus fumigatus, Candida albicans, and the prevalent food spoilage mold Penicillium species, arresting spore germination and vegetative cell growth. Mechanistically, NP disrupts the structural integrity of bacterial and fungal cell membranes, increasing membrane permeability and leading to cell death. Furthermore, genome-wide expression analyses of A. fumigatus vegetative cells exposed to NP reveal the downregulation of genes associated with the liveness of the fungal cells including ergosterol biosynthesis, cell wall maintenance, and development, with network analysis highlighting NP’s impact on various metabolic pathways. Notably, NP is presumed safe and thermally stable, holding promise for addressing foodborne illnesses and drug-resistant infections through the development and widespread application of a new generation of antimicrobials.IMPORTANCEThe development of NP, a potent broad-spectrum antimicrobial, is a significant breakthrough in the ongoing challenge against microbial foodborne illnesses and the growing threat of antibiotic resistance. This food-grade culture broth of Aspergillus oryzae demonstrates exceptional broad-spectrum efficacy against a variety of harmful bacteria and fungi, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus and prevalent food spoilage molds. NP exhibits strong bactericidal activity against various foodborne and ESKAPE pathogens, and strong antifungal activity against Penicillium species, Aspergillus fumigatus, and Candida albicans. The potent bactericidal and antifungal properties of NP are a result of its ability to disrupt microbial cell membranes leading to increased permeability. Furthermore, the genome-wide impact of NP on fungal gene expression and metabolic pathways underscores its comprehensive antimicrobial action, leading to transcriptomic and metabolic changes associated with cell death in A. fumigatus.

Spices have been utilized over the centuries for culinary and medicinal purposes. Their usage mostly differed as per the different geographical regions and their ethnicity. While they can be of the same class, they might differ in their flavour, aroma, and therapeutic potential. Herein we have explored the widely used south, east, and southeast Asian spices of the Apiaceae family namely cumin (Cumminum cyminum) and fennel (Foeniculum vulgare) for their therapeutic potential as natural sources of drug repurposing against the infections caused by the multidrug-resistant ESKAPE pathogens. Essentially, crude extracts of fractions in a series of solvents were prepared and assessed for their preliminary antibacterial potential. All these fractions were analysed through GC-MS and the overlapping constituents were further explored. A stepwise approach of virtual screening followed by pharmacological assessment was taken bioinformatically to reveal the potential top rankers having the druggability against selected virulent proteins from the ESKAPE pathogens. Further molecular dynamics simulation affirmed the finding of top-ranking compounds with their antibacterial potentials. Earlier known as anticancer agents, these compounds hold the promise for drug repurposing from natural sources like spices against the multidrug-resistant ESKAPE pathogens. [ABSTRACT FROM AUTHOR]

Due to its increased safety over ultraviolet light, there is interest in the development of antimicrobial violet-blue light technologies for infection control applications. To ensure compatibility with exposed materials and tissue, the light irradiances and dose regimes used must be suitable for the target application. This study investigates the antimicrobial dose responses and germicidal efficiency of 405 nm violet-blue light when applied at a range of irradiance levels, for inactivation of surface-seeded and suspended bacteria. Bacteria were seeded onto agar surfaces (101–108 CFUplate−1) or suspended in PBS (103–109 CFUmL−1) and exposed to increasing doses of 405-nm light (≤ 288 Jcm−2) using various irradiances (0.5–150 mWcm−2), with susceptibility at equivalent light doses compared. Bacterial reductions ≥ 96% were demonstrated in all cases for lower irradiance (≤ 5 mWcm−2) exposures. Comparisons indicated, on a per unit dose basis, that significantly lower doses were required for significant reductions of all species when exposed at lower irradiances: 3–30 Jcm−2/0.5 mWcm−2 compared to 9–75 Jcm−2/50 mWcm−2 for low cell density (102 CFUplate−1) surface exposures and 22.5 Jcm−2/5 mWcm−2 compared to 67.5 Jcm−2/150 mWcm−2 for low density (103 CFUmL−1) liquid exposures (P ≤ 0.05). Similar patterns were observed at higher densities, excluding S. aureus exposed at 109 CFUmL−1, suggesting bacterial density at predictable levels has minimal influence on decontamination efficacy. This study provides fundamental evidence of the greater energy efficacy of 405-nm light for inactivation of clinically-significant pathogens when lower irradiances are employed, further supporting its relevance for practical decontamination applications. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii have emerged as major clinical threats owing to the increasing prevalence of ventilator-associated pneumonia caused by multidrug-resistant or extensively drug-resistant strains. The present study aimed to assess the antibacterial effects and efficacy of LL-37 fragment GF-17D3 and synthetic Scolopendin A2 peptides against resistant clinical strains in vitro and in vivo models. P. aeruginosa, S. aureus, and A. baumannii were isolated from clinical infections. Their antibiotic resistance and minimum inhibitory concentration were assessed. LL-37 fragment GF-17D3 peptide was selected from available databases. Scolopendin A2 peptide's 6th amino acid (proline) was substituted with lysine and peptides and MICs were determined. The biofilm inhibitory activity was quantified at sub MIC concentrations. Synergetic effects of Scolopendin A2 and imipenem were assessed by checkerboard. After mice nasal infection with P. aeruginosa, peptides LD50 was determined. Isolates harbored complete resistance toward the majority of antibiotics and MIC values ranged between 1 and > 512 µg/ml. The majority of isolates exhibited strong biofilm activity. Synthetic peptides showed lower MIC values than antibiotic agents and the lowest MIC values were obtained for synthetic peptides in combination with antibiotics. The Synergisms effect of Scolopendin A2 with imipenem was also determined. Scolopendin A2 was found to have antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 64 µg/ml, 8 µg/ml, and 16 µg/ml, respectively, and LL37 showed antibacterial efficacy against P. aeruginosa, S. aureus, and A. baumannii with MIC 128 µg/ml, 32 µg/ml, and 32 µg/ml, respectively. Both AMPs decreased biofilms by ≥ 96% at 1 × MIC. The biofilm inhibitory activity was measured at sub MIC concentrations of the peptides and the results demonstrated that Scolopendin A2 exhibited anti-biofilm activity at 1/4 × MIC and 1/2 × MIC concentrations was 47.9 to 63.8%, although LL37 among 1/4 × MIC and 1/2 × MIC concentrations was 21.3 to 49.6% against three pathogens. The combination of Scolopendin A2 and antibiotics demonstrated synergistic activity-resistant strains with FIC values ≤ 0.5 for three pathogens, while LL37 and antibiotics showed synergistic activity FIC values ≤ 0.5 for only P. aeruginosa. Infection model Scolopendin A2 with Imipenem (2 × MIC) was efficacious in vivo, with a 100% survival rate following treatment at 2 × MIC after 120 h. The mRNA expression of biofilm-related genes was decreased for both peptides. Synthesis Scolopendin A2 decreased the expression of biofilm formation genes compared to the control group. Synthetic Scolopendin A2 exhibits antimicrobial activity without causing toxicity on the human epithelial cell line. Based on our findings, it seems that synthetic Scolopendin A2 is an appropriate antimicrobial source. That could be a promising option in combination with antibiotics for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant bacteria. Nevertheless, additional experiments are required to assess another potential of this novel AMP. [ABSTRACT FROM AUTHOR]

The improper use and abuse of antibiotics have led to an increase in multidrugresistant (MDR) bacteria resulting in a failure of standard antibiotic therapies. To date, this phenomenon represents a leading public health threat of the 21st century which requires alternative strategies to fight infections such as the identification of new molecules active against MDR strains. In the last 20 years, natural extracts with biological activities attracted scientific interest. Following the One Health Approach, natural by-products represent a sustainable and promising alternative solution. Consistently, the aim of the present study was to evaluate the antimicrobial activity of hydro-alcoholic pomegranate peel extract (PPE) against MDR microorganisms belonging to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. "ESKAPE" group pathogens. Through semiquantitative and quantitative methods, the PPE showed effective antimicrobial activity against Gram-positive and Gram-negative MDR bacteria. The kinetics of bactericidal action of PPE highlighted that microbial death was achieved in a time- and dose-dependent manner. High concentrations of PPE exhibited antioxidant activity, providing a protective effect on cellular systems and red blood cell membranes. Finally, we report, for the first time, a significant intracellular antibacterial property of PPE as highlighted by its bactericidal action against the staphylococcal reference strain and its bacteriostatic effect against clinical resistant strain in the HeLa cell line. In conclusion, due to its characterized content of polyphenolic compounds and antioxidant activity strength, the PPE could be considered as a therapeutic agent alone or in conjunction with standard antibiotics against challenging infections caused by ESKAPE pathogens. [ABSTRACT FROM AUTHOR]

Mild‐heat photothermal antibacterial therapy avoids heat‐induced damage to normal tissues but causes bacterial tolerance. The use of photothermal therapy in synergy with chemodynamic therapy is expected to address this issue. Herein, two pseudo‐conjugated polymers PM123 with photothermal units and PFc with ferrocene (Fc) units are designed to co‐assemble with DSPE‐mPEG2000 into nanoparticle NPM123/Fc. NPM123/Fc under 1064 nm laser irradiation (NPM123/Fc+NIR‐II) generates mild heat and additionally more toxic ∙OH from endogenous H2O2, displaying a strong synergistic photothermal and chemodynamic effect. NPM123/Fc+NIR‐II gives >90% inhibition rates against MDR ESKAPE pathogens in vitro. Metabolomics analysis unveils that NPM123/Fc+NIR‐II induces bacterial metabolic dysregulation including inhibited nucleic acid synthesis, disordered energy metabolism, enhanced oxidative stress, and elevated DNA damage. Further, NPM123/Fc+NIR‐II possesses >90% bacteriostatic rates at infected wounds in mice, resulting in almost full recovery of infected wounds. Immunodetection and transcriptomics assays disclose that the therapeutic effect is mainly dependent on the inhibition of inflammatory reactions and the promotion of wound healing. What is more, thioketal bonds in NPM123/Fc are susceptible to ROS, making it degradable with highly favorable biosafety in vitro and in vivo. NPM123/Fc+NIR‐II with a unique synergistic antibacterial strategy would be much less prone to select bacterial resistance and represent a promising antibiotics‐alternative anti‐infective measure. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Klebsiella pneumoniae (Cr-Kpn) is becoming a growing public health problem through the failure of adequate treatment. This study's objectives are to describe the sources of Cr-Kpn in our hospital over 22 months, associating factors with the outcome of Cr-Kpn-positive patients, especially those with NDM+OXA-48-like (New Delhi Metallo-β-Lactamase and oxacillinase-48), and the effectiveness of the treatments used. Methods: A retrospective observational cohort study including all hospitalized patients with Cr-Kpn isolates. We reported data as percentages and identified independent predictors for mortality over hospital time through multivariate analysis. Results: The main type of carbapenemases identified were NDM+OXA-48-like (49.4%). The statistical analysis identified that diabetes and co-infections with the Gram-negative, non-urinary sites of infection were factors of unfavorable evolution. The Cox regression model identified factors associated with a poor outcome: ICU admission (HR of 2.38), previous medical wards transition (HR of 4.69), and carbapenemase type NDM (HR of 5.98). We did not find the superiority of an antibiotic regimen, especially in the case of NDM+OXA-48-like. Conclusions: The increase in the incidence of Cr-Kpn infections, especially with NDM+OXA-48-like pathogens, requires a paradigm shift in both the treatment of infected patients and the control of the spread of these pathogens, which calls for a change in public health policy regarding the use of antibiotics and the pursuit of a One Health approach. [ABSTRACT FROM AUTHOR]

Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4′-phosphopantetheine (PNS) yielding 3′-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1 M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82 Å and c = 200.37 Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a β/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10−4 M for ATP which is significantly lower than the corresponding values reported for PPAT from other species. [ABSTRACT FROM AUTHOR]

The silver nanoparticles (AgNPs) were produced by employing a biogenic loom and tested for antipathogenic assets against multi-drug-resistant (MDR) ESKAPE bacteria. Biogenically synthesized AgNPs were characterized adopting various high-throughput techniques such as UHRTEM, SEM with EDX, DLS, TGA-DTA, and XRD and spectroscopic analysis showed polydispersion of nanoparticles. In this context, AgNPs with the attribute of spherical-shaped nanoparticles with an average size of 26 nm were successfully synthesized utilizing bacterial supernatant. The antipathogenic activities of AgNPs were assessed against 11 strains of MDR ESKAPE bacteria including Enterococcus faecium; methicillin-resistant Staphylococcus aureus; Klebsiella pneumonia; Acinetobacter baumannii; Pseudomonas aeruginosa; Enterobacter aerogenes; and Enterobacter species. The exposure of biogenic AgNPs in a well diffusion assay showed all the growth inhibitions of ESKAPE bacteria at 200 μg/ml after 18 h of incubation. Growth kinetics demonstrated maximum killing at 60 μg/ml after 4 h of completion. The highest biofilm depletions were found at 100 μg/ml in adhesion assay. Live/dead assays showed effective killing of the ESKAPE bacteria at 10 μg/ml in pre-existing biofilms. The effective inhibitory concentrations of AgNPs were investigated ranging from 10 to 200 μg/ml. The selected pathogens found sensitive to AgNPs are statistically significant (P < 0.05) than that of cefotaxime/AgNO3. Consequently, a broad spectrum of antimicrobial potentials of AgNPs can be alternative to conventional antimicrobial agents for future medicine. [ABSTRACT FROM AUTHOR]

IntroductionBacterial infection and biofilm formation contribute to impaired healing in chronic diabetic wounds. Staphylococcus aureus and Pseudomonas aeruginosa are found in human diabetic wound biofilms. They may develop antibiotic resistance, increasing the urgency for alternative or complementary therapies. Diabetic wound healing may be improved with the use of biomedically engineered scaffolds, which can also serve as delivery systems for antibacterial compounds. Manuka honey is a potent antibacterial and wound care agent due to its high osmolarity, low pH, and constituents (such as methylglyoxal). Honey exhibits bacteriostatic and bactericidal effects, modulates the expression of biofilm forming genes, and restores antibiotic susceptibility in previously drug resistant pathogens.MethodsIn this study, we created a dermal regeneration template (DRT) composed of polycaprolactone-gelatin (PCL-gelatin) and Manuka honey to retain honey in the wound and also provide a scaffold for tissue regeneration.Results and discussionSoluble Manuka honey inhibited the planktonic and biofilm growth of both S. aureus (UWH3) and P. aeruginosa (PA14) co-cultures. Manuka honey embedded PCL-gelatin scaffolds did not exhibit bacteriostatic or bactericidal effects on cocultures of UHW3 and PA14; however, they promoted the expression of AgrA, a gene associated with dispersal of S. aureus biofilms.

IntroductionThis study investigated the interaction with membrane mimetic systems (LUVs), bacterial membranes, the CD spectra, and the bactericidal activity of two designed trematocine mutants, named Trem-HK and Trem-HSK. Mutants were constructed from the scaffold of Trematocine (Trem), a natural 22-amino acid AMP from the Antarctic fish Trematomus bernacchii, aiming to increase their positive charge.MethodsThe selectivity of the designed AMPs towards bacterial membranes was improved compared to Trematocine, verified by their interaction with different LUVs and their membranolytic activity. Additionally, their α-helical conformation was not influenced by the amino acid substitutions. Our findings revealed a significant enhancement in antibacterial efficacy against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae family) pathogens for both Trem-HK and Trem-HSK.ResultsFirstly, we showed that the selectivity of the two new designed AMPs towards bacterial membranes was greatly improved compared to Trematocine, verifying their interaction with different LUVs and their membranolytic activity. We determined that their α-helical conformation was not influenced by the amino acid substitutions. We characterized the tested bacterial collection for resistance traits to different classes of antibiotics. The minimum inhibitory and bactericidal concentration (MIC and MBC) values of the ESKAPE collection were reduced by up to 80% compared to Trematocine. The bactericidal concentrations of Trematocine mutants showed important membranolytic action, evident by scanning electron microscopy, on all tested species. We further evaluated the cytotoxicity and hemolytic activity of the mutants. At 2.5 μM concentration, both mutants demonstrated low cytotoxicity and hemolysis, indicating selectivity towards bacterial cells. However, these effects increased at higher concentrations.DiscussionAssessment of in vivo toxicity using the Galleria mellonella model revealed no adverse effects in larvae treated with both mutants, even at concentrations up to 20 times higher than the lowest MIC observed for Acinetobacter baumannii, suggesting a high potential safety profile for the mutants. This study highlights the significant improvement in antibacterial efficacy achieved by increasing the positive charge of Trem-HK and Trem-HSK. This improvement was reached at the cost of reduced biocompatibility. Further research is necessary to optimize the balance between efficacy and safety for these promising AMPs.

The accelerated rise in antimicrobial resistance (AMR) poses a significant global health risk, necessitating the exploration of alternative strategies to combat pathogenic infections. Biofilm-related infections that are unresponsive to standard antibiotics often require the use of higher-order antimicrobials with toxic side effects and the potential to disrupt the microbiome. Probiotic therapy, with its diverse benefits and inherent safety, is emerging as a promising approach to prevent and treat various infections, and as an alternative to antibiotic therapy. In this study, we isolated novel probiotic bacteria from the gut of domestic goats (Capra hircus) and evaluated their antimicrobial and anti-biofilm activities against the ‘ESKAPE’ group of pathogens. We performed comprehensive microbiological, biochemical, and molecular characterizations, including analysis of the 16S-rRNA gene V1-V3 region and the 16S-23S ISR region, on 20 caprine gut-derived lactic acid bacteria (LAB). Among these, six selected Lactobacillus isolates demonstrated substantial biofilm formation under anaerobic conditions and exhibited robust cell surface hydrophobicity and autoaggregation, and epithelial cell adhesion properties highlighting their superior enteric colonization capability. Notably, these Lactobacillus isolates exhibited broad-spectrum growth inhibitory and anti-biofilm properties against ‘ESKAPE’ pathogens. Additionally, the Lactobacillus isolates were susceptible to antibiotics listed by the European Food Safety Authority (EFSA) within the prescribed Minimum Inhibitory Concentration limits, suggesting their safety as feed additives. The remarkable probiotic characteristics exhibited by the caprine gut-derived Lactobacillus isolates in this study strongly endorse their potential as compelling alternatives to antibiotics and direct-fed microbial (DFM) feed supplements in the livestock industry, addressing the escalating need for antibiotic-free animal products.

Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

Background: The persistence of antibiotic resistance has incited a strong interest in the discovery of agents with novel antimicrobial mechanisms. The direct killing of multidrug-resistant bacteria by cationic antimicrobial peptides (AMPs) underscores their importance in the fight against infections associated with antibiotic resistance. Despite a vast body of AMP literature demonstrating a plurality in structural classes, AMP engineering has been largely skewed toward peptides with idealized amphipathic helices (H-amphipathic). In contrast to helical amphipathicity, we designed a series of peptides that display the amphipathic motifs in the primary structure. We previously developed a rational framework for designing AMP libraries of H-amphipathic peptides consisting of Arg, Trp, and Val (H-RWV, with a confirmed helicity up to 88% in the presence of membrane lipids) tested against the most common MDR organisms. Methods: In this study, we re-engineered one of the series of the H-RWV peptides (8, 10, 12, 14, and 16 residues in length) to display the amphipathicity in the primary structure by side-by-side (linear) alignment of the cationic and hydrophobic residues into the 2 separate linear amphipathic (L-amphipathic) motifs. We compared the 2 series of peptides for antibacterial activity, red blood cell (RBC) lysis, killing and membrane-perturbation properties. Results: The L-RWV peptides achieved the highest antibacterial activity at a minimum length of 12 residues (L-RWV12, minimum optimal length or MOL) with the lowest mean MIC of 3–4 µM, whereas the MOL for the H-RWV series was reached at 16 residues (H-RWV16). Overall, H-RWV16 displayed the lowest mean MIC at 2 µM but higher levels of RBC lysis (25–30%), while the L-RWV series displayed minor RBC lytic effects at the test concentrations. Interestingly, when the S. aureus strain SA719 was chosen because of its susceptibility to most of the peptides, none of the L-RWV peptides demonstrated a high level of membrane perturbation determined by propidium iodide incorporation measured by flow cytometry, with

Background: Antimicrobial resistance is a global health problem, due to morbidity, mortality, and healthcare costs. The misuse of antimicrobials is the main cause of antimicrobial resistance. The aim of this study was to report antimicrobial resistance and antibiotic consumption in a secondary care hospital in Mexico. Methods: Within a cross-sectional study, antimicrobial resistance data on ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and antibiotic consumption from 2020 to 2022 were collected. Antimicrobial resistance was reported based on percentages of resistance and consumption was analyzed using the defined daily dose (DDD)/100 bed days and the AWaRe (Access, Surveillance, Reservation) antibiotic group. Results: Antibiotic consumption in 2020, 2021 and 2022 was 330, 175 and 175 DDD/100 beds day, respectively. The rate of ceftriaxone resistance in E. coli (n = 526) and K. pneumoniae (n = 80) was 76% and 69%, respectively, the rate of carbapenem resistance in A. baumannii (n = 168) and P. aeruginosa (n = 108) was 92% and 52%, respectively; the rate of oxacillin resistance in S. aureus (n = 208) was 27%; and the rate of vancomycin resistance in E. faecium (n = 68) was 47%. Conclusion: The reported results are congruent with global estimates of antibiotic resistance and consumption, providing an overview that could generate actions for antimicrobial optimization at the local and regional levels. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic-resistant bacteria, particularly the most hazardous pathogens, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE)-pathogens pose a significant threat to global health. Current antimicrobial therapies, including those targeting biofilms, have shown limited effectiveness against these superbugs. Nanoparticles, specifically silver nanoparticles (AgNPs), have emerged as a promising alternative for combating bacterial infections. In this study, two types of AgNPs with different physic-chemical properties were evaluated for their antimicrobial and antibiofilm activities against clinical ESKAPE strains. Two types of silver nanoparticles were assessed: spherical silver nanoparticles (AgNPs-1) and cubic-shaped silver nanoparticles (AgNPs-2). AgNPs-2, characterized by a cubic shape and higher surface-area-to-volume ratio, exhibited superior antimicrobial activity compared to spherical AgNPs-1. Both types of AgNPs demonstrated the ability to inhibit biofilm formation and disrupt established biofilms, leading to membrane damage and reduced viability of the bacteria. These findings highlight the potential of AgNPs as effective antibacterial agents against ESKAPE pathogens, emphasizing the importance of nanoparticle characteristics in determining their antimicrobial properties. Further research is warranted to explore the underlying mechanisms and optimize nanoparticle-based therapies for the management of infections caused by antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

Healthcare-associated infections (HAIs) represent a significant global health concern, contributing to millions of cases annually. The rise of antibiotic-resistant bacteria, notably the ESKAPE pathogens, has heightened concerns regarding treatment efficacy and patient outcomes. Balneotherapy offers treatments for a range of dermatological, respiratory, and musculoskeletal conditions using mineral water or therapeutic peloids. This study aimed to evaluate the risk of infections associated with spa procedures by identifying and characterizing bacterial strains (resistance and virulence features) isolated from therapeutic natural samples usually used in balneotherapy. The bacterial strains isolated from waters and peloids were predominantly Gram-positive (64%), most belonging to the Bacillus genus. In sulfurous therapeutic mineral waters (TMWs) (with > 1mg/l titratable S), Serratia fonticola, Staphylococcus sp., Photobacterium damselae, Aeromonas salmonicida, Pseudomonas stutzeri were identified, while in carbonated TMWs (with = 1000 mg/l CO2), Lelliottia amnigena, Pseudomonas aeruginosa, Enterobacter amnigenus, Serratia fonticola, Bacillus cereus were present. The microbiota of sapropelic peloids (with > 10% organic matter) or mineral peloids (with < 10% organic matter) but sulfuric type (> 0.15 g% FeS) included strains of Bacillus spp., Klebsiella oxytoca, Citrobacter freundii, Rautella teriigena, sometimes associated with fecal pollution indicator microorganisms (E. coli, Enterococcus spp.). Despite limited expression of resistance and virulence factors in opportunistic isolated strains, their presence represents a risk, especially in elderly patients with chronic diseases and immunodeficiency. The transfer of genetic factors in dense microbial communities, particularly in muds, amplifies this concern. In conclusion, the therapeutic use of mineral waters and mud necessitates monitoring their microbiological status to ensure their health-promoting quality and beneficial effects. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most prevalent biological entities in the biosphere. However, limitations in both medical relevance and sequencing technologies have led to a systematic underestimation of the genetic diversity within phages. This underrepresentation not only creates a significant gap in our understanding of phage roles across diverse biosystems but also introduces biases in computational models reliant on these data for training and testing. In this study, we focused on publicly available genomes of bacteriophages infecting high-priority ESKAPE pathogens to show the extent and impact of this underrepresentation. First, we demonstrate a stark underrepresentation of ESKAPE phage genomes within the public genome and protein databases. Next, a pangenome analysis of these ESKAPE phages reveals extensive sharing of core genes among phages infecting the same host. Furthermore, genome analyses and clustering highlight close nucleotide-level relationships among the ESKAPE phages, raising concerns about the limited diversity within current public databases. Lastly, we uncover a scarcity of unique lytic phages and phage proteins with antimicrobial activities against ESKAPE pathogens. This comprehensive analysis of the ESKAPE phages underscores the severity of underrepresentation and its potential implications. This lack of diversity in phage genomes may restrict the resurgence of phage therapy and cause biased outcomes in data-driven computational models due to incomplete and unbalanced biological datasets.

Antimicrobial resistance of pathogenic bacteria to currently used antibiotics killed an estimated 1.27 million people in 2019 and it is believed that this toll will increase to 10 million per year by 2050. This remains one of the most urgent threats to human health and the hunt for novel antimicrobial natural products (NPs) from microorganisms is essential in efforts to combat this threat, but recent efforts have been unsuccessful. The investigation of extreme ecological niches, such as hot springs, has gained recent interest due to their unique ecosystems, increasing the chance for novel antimicrobial NP discovery. The Roman Baths (Bath) is the UK’s only hot spring and it remains unexplored in terms of microbial diversity. In this study, samples of water, biofilm and sediment were taken from the King’s Spring and the Great Bath, and 16 S rRNA metagenomic sequencing was performed for bacterial and archaeal community exploration. Additionally, samples were used for culture-dependent isolation of antimicrobial NP producing isolates. At the phylum level, data from water samples revealed a shift from an archaeal dominant community in the King’s Spring (Crenarchaeota, Aenigmarchaeota and Nanoarchaeota) to bacteria in the Great Bath (Cyanobacteria and Proteobacteria). Archaea were still present in the biofilm and sediment of the hot spring but consistently the bacterial phyla Cyanobacteria, Bacteroidota, Proteobacteria and Nitrospirota were dominant at both sites. The antimicrobial NP producing candidate phyla Actinobacteria and Myxococcota were found in all samples. From initial isolation experiments, 297 isolates were purified, of which 15 showed broad spectrum activity against human pathogens, though interestingly these were not from target genera in the Actinobacteria. From these data, there is clear potential for novel NP discovery from the Roman Baths, as has been demonstrated from other thermal hot springs globally.

Globally, antimicrobial (AMR) or multi-drug resistance (MDR) constitutes a current health challenge that is predicted to cause increased infections rates with adverse socioeconomic impacts through increase in healthcare costs. In addition, the group of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. (ESKAPE) pathogens cause debilitating infections (community and nosocomial) and are classed as priority 1 AMR pathogens. This systematic report therefore aimed at providing detailed coverage of new targets for novel antimicrobials development against MDR ESKAPE pathogens to mitigate future AMR spread and improve current public health indices. The prevalent ESKAPE bacterial group show high resistance to quinolones, lactams, cephalosporins, carbapenems and other antibiotic groups, and ability to form biofilms linked to various quorum sensing systems (QSSs) that boost their virulence. These QS pathways have become viable targets in drug design efforts for new antimicrobials development. Also, since antibiotics discovery and development has waned in the past decade, the emergence of advanced computational modelling technologies in drug design, repurposing and development efforts may yet bridge the gap. As such, in this work we provided a comprehensive and systematic overview using relevant, included data and findings on ESKAPE pathogens, their QSSs to target for novel antimicrobial agents’ development, the contributions of computational tools at the heart of novel antimicrobial advancements and their roles in bioprospecting and developing novel ‘druggable’ candidates and therapies with anti-biofilm, and anti-quorum sensing activities to mitigate AMR, biofilm and QS-related pathogenicity factors.

Abstract Mild‐heat photothermal antibacterial therapy avoids heat‐induced damage to normal tissues but causes bacterial tolerance. The use of photothermal therapy in synergy with chemodynamic therapy is expected to address this issue. Herein, two pseudo‐conjugated polymers PM123 with photothermal units and PFc with ferrocene (Fc) units are designed to co‐assemble with DSPE‐mPEG2000 into nanoparticle NPM123/Fc. NPM123/Fc under 1064 nm laser irradiation (NPM123/Fc+NIR‐II) generates mild heat and additionally more toxic ∙OH from endogenous H2O2, displaying a strong synergistic photothermal and chemodynamic effect. NPM123/Fc+NIR‐II gives >90% inhibition rates against MDR ESKAPE pathogens in vitro. Metabolomics analysis unveils that NPM123/Fc+NIR‐II induces bacterial metabolic dysregulation including inhibited nucleic acid synthesis, disordered energy metabolism, enhanced oxidative stress, and elevated DNA damage. Further, NPM123/Fc+NIR‐II possesses >90% bacteriostatic rates at infected wounds in mice, resulting in almost full recovery of infected wounds. Immunodetection and transcriptomics assays disclose that the therapeutic effect is mainly dependent on the inhibition of inflammatory reactions and the promotion of wound healing. What is more, thioketal bonds in NPM123/Fc are susceptible to ROS, making it degradable with highly favorable biosafety in vitro and in vivo. NPM123/Fc+NIR‐II with a unique synergistic antibacterial strategy would be much less prone to select bacterial resistance and represent a promising antibiotics‐alternative anti‐infective measure.

The improper use and abuse of antibiotics have led to an increase in multidrug-resistant (MDR) bacteria resulting in a failure of standard antibiotic therapies. To date, this phenomenon represents a leading public health threat of the 21st century which requires alternative strategies to fight infections such as the identification of new molecules active against MDR strains. In the last 20 years, natural extracts with biological activities attracted scientific interest. Following the One Health Approach, natural by-products represent a sustainable and promising alternative solution. Consistently, the aim of the present study was to evaluate the antimicrobial activity of hydro-alcoholic pomegranate peel extract (PPE) against MDR microorganisms belonging to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. “ESKAPE” group pathogens. Through semiquantitative and quantitative methods, the PPE showed effective antimicrobial activity against Gram-positive and Gram-negative MDR bacteria. The kinetics of bactericidal action of PPE highlighted that microbial death was achieved in a time- and dose-dependent manner. High concentrations of PPE exhibited antioxidant activity, providing a protective effect on cellular systems and red blood cell membranes. Finally, we report, for the first time, a significant intracellular antibacterial property of PPE as highlighted by its bactericidal action against the staphylococcal reference strain and its bacteriostatic effect against clinical resistant strain in the HeLa cell line. In conclusion, due to its characterized content of polyphenolic compounds and antioxidant activity strength, the PPE could be considered as a therapeutic agent alone or in conjunction with standard antibiotics against challenging infections caused by ESKAPE pathogens.

In recent decades, antimicrobial resistance has become a major global public health problem. A distinct category of pathogens, known by the acronym ESKAPE, has become of particular concern because of their ability to escape the effects of available antimicrobial treatments. This acronym represents a select group of pathogenic bacteria, some of which are highly resistant to antibiotics and can cause severe and potentially fatal infections. The term "ESKAPE" is an acronym derived from the initials of these bacteria and includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. Antimicrobial resistance in these pathogens is fuelled by several factors, including overuse and misuse of antibiotics, natural selection and transfer of resistance genes between bact. To meet this challenge, the global community is engaged in multiple efforts, including research and development of new antibiotics, improving practices in the use of these drugs and promoting antimicrobial research and education. In the fight against pathogens and their antimicrobial resistance, we face a constant challenge in contemporary medicine, with significant implications for healthcare delivery and public health.

[Display omitted] Isosteric replacement of the oxadiazole ring by amide bond in the structure of new non-β-lactam antibiotics led to compounds with higher activity against Gram-positive pathogens of ESKAPE panel. A series of 17 compounds were synthesized by acylation of 4-(4-fluorophenoxy)aniline with various amino acids. The spirocyclic derivative with 6-methylsulfonyl-2,6-diazaspiro[3.4]octane moiety showed excellent minimum inhibitory concentrations of 0.093–0.75 μg ml−1 against a number of methicillin-resistant Staphylococcus aureus strains. [ABSTRACT FROM AUTHOR]

Far-ultraviolet C light, with a wavelength of 200–230 nm, has demonstrated broad-spectrum germicidal efficacy. However, due to increased interest in its use as an alternative antimicrobial, further knowledge about its fundamental bactericidal efficacy is required. This study had two objectives. Firstly, it investigated experimentally the Far-UVC dose–response of common bacteria suspended at various cell densities in transparent buffer, ensuring no influence from photosensitive suspending media. Increasing doses of Far-UVC were delivered to Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in PBS at 101, 102, 103, 105 and 107 CFU·mL−1, with surviving colony-forming units enumerated (n ≥ 3). Secondly, through a systematised literature review, this work sought to explore the impact of genus/species, Gram type, cell form, cell density and irradiance on dose–response. The screening of 483 publications was performed with 25 included in the study. Data for 30 species were collated, analysed and compared with the experimental results. Overall, Gram-positive species showed greater resilience to Far-UVC than Gram-negative; some inter-species and inter-genera differences in resilience were identified; endospores were more resilient than vegetative cells; the results suggested that inactivation efficiency may decrease as cell density increases; and no significant correlation was identified between irradiance and bactericidal dose effect. In conclusion, this study has shown Far-UVC light to be an effective decontamination tool against a vast range of bacterial vegetative cells and endospores.

IntroductionAntibiotic-resistant Acinetobacter baumannii is a very important nosocomial pathogen worldwide. Thousands of studies have been conducted about this pathogen. However, there has not been any attempt to use all this information to highlight the research trends concerning this pathogen.MethodsHere we use unsupervised learning and natural language processing (NLP), two areas of Artificial Intelligence, to analyse the most extensive database of articles created (5,500+ articles, from 851 different journals, published over 3 decades).ResultsK-means clustering found 113 theme clusters and these were defined with representative terms automatically obtained with topic modelling, summarising different research areas. The biggest clusters, all with over 100 articles, are biased toward multidrug resistance, carbapenem resistance, clinical treatment, and nosocomial infections. However, we also found that some research areas, such as ecology and non-human infections, have received very little attention. This approach allowed us to study research themes over time unveiling those of recent interest, such as the use of Cefiderocol (a recently approved antibiotic) against A. baumannii.DiscussionIn a broader context, our results show that unsupervised learning, NLP and topic modelling can be used to describe and analyse the research themes for important infectious diseases. This strategy should be very useful to analyse other ESKAPE pathogens or any other pathogens relevant to Public Health.

The synthesis of a new class of effective antibiotics and antimicrobial agents with less toxicity is highly desirable due to bacterial resistance to antibiotics has increased dramatically. In this context, molecules that embedding a spiro moiety are attractive from a medicinal chemistry point of view as these spiro heterocycles show a vital part in the development of delivery systems for antimicrobial therapies. In the present study, the synthesis and antimicrobial evaluation of structurally attractive complex hybrid heterocycles comprising spriooxindolopyrrolidine and indole heterocycles was attained in quantitative yields by cycloaddition strategy. The new class of spirocompouds were unequivocally assigned through spectroscopic analysis and the antimicrobial efficacy were assessed against six microbial pathogens. Among them, compound 4a, bearing chlorine substituted derivative showed significant activity against tested ESKAPE pathogens. The maximum zone of inhibition observed against ESKAPE microbial pathogens causing infectious disease ranged from 6.75 ± 0.40 to 19.75 ± 1.15 mm, with MIC values ranging between 16.00 to > 256.00 µg/ml .

The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics. Hence, there is a need to search for new sources of antibiotics that either exhibit novel structures or express a new mechanism of action. The lichen Usnea, with its wide range of unique, biologically potent secondary metabolites, may solve this problem. In this study, Usnea species were collected in the Northern Philippines, identified through combined morphological and biochemical characterization, and tested for antimicrobial activities against the multidrug-resistant ESKAPE pathogens, i.e., Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae, two standard antibiotic-sensitive test bacteria, and a yeast. A total of 46 lichen specimens were collected and later identified as Usnea baileyi (10), U. diffracta (10), U. glabrata (12), U. longissima (4), and U. rubicunda (10). The results show that the crude extracts of the Usnea species exhibited promising in vitro inhibitory activities against standard antibiotic-sensitive (E. faecalis ATCC 29212) and multidrug-resistant (methicillin-resistant S. aureus and E. faecalis) Gram-positive bacteria. Additionally, lichen compounds of representative specimens per species were identified and profiled using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The detection of lichen acids (LA) via HPLC showed the presence of 24 peaks of lichen acids. TLC-bioautography identified the bioactive lichen acids as alectronic acid, connorstictic acid, consalazinic acid, diffractaic acid, echinocarpic acid, erythrin acid, galbinic acid, hypoconstictic acid, hyposalazinic acid, hypostictic acid, lobaric acid, menegazzaic acid, micareic acid, pannarin, salazinic acid, stictic acid, and usnic acid. Our study highlighted the wide spectrum of opportunities for using lichens for the discovery of potential antimicrobial agents. [ABSTRACT FROM AUTHOR]

Among the various mechanisms that bacteria use to develop antibiotic resistance, the multiple expression of β-lactamases is particularly problematic, threatening public health and increasing patient mortality rates. Even if a combination therapy—in which a β-lactamase inhibitor is administered together with a β-lactam antibiotic—has proven effective against serine-β-lactamases, there are no currently approved metallo-β-lactamase inhibitors. Herein, we demonstrate that quercetin and its analogs are promising starting points for the further development of safe and effective metallo-β-lactamase inhibitors. Through a combined computational and in vitro approach, taxifolin was found to inhibit VIM-2 expressing P. aeruginosa cell proliferation at <4 μg/mL as part of a triple combination with amoxicillin and clavulanate. Furthermore, we tested this combination in mice with abrasive skin infections. Together, these results demonstrate that flavonol compounds, such as taxifolin, may be developed into effective metallo-β-lactamase inhibitors. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial peptides (CAMPs) are powerful molecules with antimicrobial, antibiofilm and endotoxin-scavenging activities. These properties make CAMPs very attractive drugs in the face of the rapid increase in multidrug-resistant (MDR) pathogens, but they are limited by their susceptibility to proteolytic degradation. An intriguing solution to this issue could be the development of functional mimics of CAMPs with structures that enable the evasion of proteases. Peptoids (N-substituted glycine oligomers) are an important class of peptidomimetics with interesting benefits: easy synthetic access, intrinsic proteolytic stability and promising bioactivities. Here, we report the characterization of P13#1, a 13-residue peptoid specifically designed to mimic cathelicidins, the best-known and most widespread family of CAMPs. P13#1 showed all the biological activities typically associated with cathelicidins: bactericidal activity over a wide spectrum of strains, including several ESKAPE pathogens; the ability to act in combination with different classes of conventional antibiotics; antibiofilm activity against preformed biofilms of Pseudomonas aeruginosa, comparable to that of human cathelicidin LL-37; limited toxicity; and an ability to inhibit LPS-induced proinflammatory effects which is comparable to that of "the last resource" antibiotic colistin. We further studied the interaction of P13#1 with SDS, LPSs and bacterial cells by using a fluorescent version of P13#1. Finally, in a subcutaneous infection mouse model, it showed antimicrobial and anti-inflammatory activities comparable to ampicillin and gentamicin without apparent toxicity. The collected data indicate that P13#1 is an excellent candidate for the formulation of new antimicrobial therapies. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Klebsiella pneumoniae (Cr-Kpn) is becoming a growing public health problem through the failure of adequate treatment. This study’s objectives are to describe the sources of Cr-Kpn in our hospital over 22 months, associating factors with the outcome of Cr-Kpn-positive patients, especially those with NDM+OXA-48-like (New Delhi Metallo-β-Lactamase and oxacillinase-48), and the effectiveness of the treatments used. Methods: A retrospective observational cohort study including all hospitalized patients with Cr-Kpn isolates. We reported data as percentages and identified independent predictors for mortality over hospital time through multivariate analysis. Results: The main type of carbapenemases identified were NDM+OXA-48-like (49.4%). The statistical analysis identified that diabetes and co-infections with the Gram-negative, non-urinary sites of infection were factors of unfavorable evolution. The Cox regression model identified factors associated with a poor outcome: ICU admission (HR of 2.38), previous medical wards transition (HR of 4.69), and carbapenemase type NDM (HR of 5.98). We did not find the superiority of an antibiotic regimen, especially in the case of NDM+OXA-48-like. Conclusions: The increase in the incidence of Cr-Kpn infections, especially with NDM+OXA-48-like pathogens, requires a paradigm shift in both the treatment of infected patients and the control of the spread of these pathogens, which calls for a change in public health policy regarding the use of antibiotics and the pursuit of a One Health approach.

The ESKAPE pathogens are a group of bacteria that are a leading cause of health-care associated infections and are known to be agents of chronic, biofilm-mediated infections. These chronic bacterial infections often respond poorly to antibiotics and in some cases may require surgical intervention in order to cure the infection. As biofilms are often the critical mediator of a chronic infection, it is essential to develop therapies that target bacteria within the biofilm state. Herein, we report the development of a rapid, 96-well plate-based assay that employs conditions specific for each species to optimize biofilm production and allow for easy identification of differences in biofilm mass after treatment with anti-biofilm candidates. We used these ESKAPE-specific biofilm assays to test our previously identified Salmonella anti-biofilm small molecule compounds, JG-1 and M4, for anti-biofilm activity. The results demonstrated that JG-1 and M4 have anti-biofilm activity against Enterobacter spp., S. aureus, E. faecium, P. aeruginosa, and A. baumannii. In addition, we identified that M4 has significant antimicrobial activity against S. aureus and E. faecium at concentrations >10 μM (X μg/mL). These findings support the claim that JG-1 and M4 have broad-spectrum anti-biofilm activity, while M4 has antimicrobial activity against the Gram-positive members of the ESKAPE pathogens. Thus, these compounds have the potential to have a significant impact on treating multiple types of commonly encountered biofilm-mediated infections.

The burden of Hospital care-associated infections (HCAIs) is becoming a global concern. This is compounded by the emergence of virulent and high-risk bacterial strains such as “ESKAPE” pathogens – (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), especially within Intensive care units (ICUs) that house high-risk and immunocompromised patients. In this review, we discuss the contributions of AMR pathogens to the increasing burden of HCAIs and provide insights into AMR mechanisms, with a particular focus on last-resort antibiotics like polymyxins. We extensively discuss how structural modifications of surface-membrane lipopolysaccharides and cationic interactions influence and inform AMR, and subsequent severity of HCAIs. We highlight some bacterial phenotypic survival mechanisms against polymyxins. Lastly, we discuss the emergence of plasmid-mediated resistance as a phenomenon making mitigation of AMR difficult, especially within the ICUs. This review provides a balanced perspective on the burden of HCAIs, associated pathogens, implication of AMR and factors influencing emerging AMR mechanisms.

ABSTRACT Biosynthetic gene clusters (BGCs) are a subset of consecutive genes present within a variety of organisms to produce specialized metabolites (SMs). These SMs are becoming a cornerstone to produce multiple medications including antibacterial and anticancer agents. Natural products (NPs) also play a pivotal role in enhancing the virulence of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), which represent a global health threat. We aimed to sequence and computationally analyze the BGCs present in 66 strains pertaining to three different ESKAPE pathogenic species: 21 A. baumannii, 28 K. pneumoniae, and 17 P. aeruginosa strains recovered from clinical settings in Egypt. DNA was extracted using QIAamp DNA Mini kit and Illumina NextSeq 550 was used for whole-genome sequencing. The sequences were quality-filtered by fastp and assembled by Unicycler. BGCs were detected by antiSMASH, BAGEL, GECCO, and PRISM, and aligned using Clinker. The highest abundance of BGCs was detected in P. aeruginosa (590), then K. pneumoniae (146) and the least in A. baumannii strains (133). P. aeruginosa isolates shared mostly the non-ribosomal peptide synthase (NRPS) type, K. pneumoniae isolates shared the ribosomally synthesized and post-translationally modified peptide-like (RiPP-like) type, while A. baumannii isolates shared the siderophore type. Most of the isolates harbored non-ribosomal peptide (NRP) BGCs with few K. pneumoniae isolates encoding polyketide BGCs. Sactipeptides and bottromycin BGCs were the most frequently detected RiPP clusters. We hypothesize that each species’ BGC signature confers its virulence. Future experiments will link the detected clusters with their species and determine whether the encoded SMs are produced and cause their virulence. IMPORTANCE Our study analyzes the biosynthetic gene clusters (BGCs) present in 66 assemblies from clinical ESKAPE pathogen isolates pertaining to Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa strains. We report their sequencing and assembly followed by the analysis of their BGCs using several bioinformatics tools. We then focused on the most abundant BGC type in each species and we discussed their potential roles in the virulence of each species. This study is pivotal to further build on its experimental work that deciphers the role in virulence, possible antibacterial effects, and characterization of the encoded specialized metabolites (SMs). The study highlights the importance of studying the “harmful” BGCs and understanding the pathogenicity and virulence of those species, as well as possible benefits if the SMs were used as antibacterial agents. This could be the first study of its kind from Egypt and would shed light on BGCs from ESKAPE pathogens from Egypt.

A series of eight 5-nitrofuran-tagged oxazolyl tetrahydropyrazolopyridines (THPPs) has been prepared in six stages with excellent regioselectivity. The testing of these compounds against pathogens of the ESKAPE panel showed a good activity of lead compound 1-(2-methoxyethyl)-5-(5-nitro-2-furoyl)-3-(1,3-oxazol-5-yl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c] pyridine (13g), which is superior to nitrofurantoin. These results confirmed the benefit of combining a THPP scaffold with a nitrofuran warhead. Certain structure–activity relationships were established in the course of this study which were rationalized by the induced-fit docking experiments in silico. [ABSTRACT FROM AUTHOR]