The Engineered Lysin CF-370 Is Active Against Antibiotic-Resistant Gram-Negative Pathogens In Vitro and Synergizes With Meropenem in Experimental Pseudomonas aeruginosa Pneumonia.

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.

Siderophore-mediated iron acquisition by Klebsiella pneumoniae.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1. IMPORTANCE: Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

Identification and Characterization of CC-AMP1-like and CC-AMP2-like Peptides in Capsicum spp.

Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants (Capsicum chinense x frutescens), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum. In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.

Investigation of cross-opsonic effect leads to the discovery of PPIase-domain containing protein vaccine candidate to prevent infections by Gram-positive ESKAPE pathogens.

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.

Antibiotic resistance: a global crisis, problems and solutions.

Healthy state is priority in today's world which can be achieved using effective medicines. But due to overuse and misuse of antibiotics, a menace of resistance has increased in pathogenic microbes. World Health Organization (WHO) has announced ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) as the top priority pathogens as these have developed resistance against certain antibiotics. To combat such a global issue, it is utmost important to identify novel therapeutic strategies/agents as an alternate to such antibiotics. To name certain antibiotic adjuvants including: inhibitors of beta-lactamase, efflux pumps and permeabilizers for outer membrane can potentially solve the antibiotic resistance problems. In this regard, inhibitors of lytic domain of lytic transglycosylases provide a novel way to not only act as an alternate to antibiotics but also capable of restoring the efficiency of previously resistant antibiotics. Further, use of bacteriophages is another promising strategy to deal with antibiotic resistant pathogens. Taking in consideration the alternatives of antibiotics, a green synthesis nanoparticle-based therapy exemplifies a good option to combat microbial resistance. As horizontal gene transfer (HGT) in bacteria facilitates the evolution of new resistance strains, therefore identifying the mechanism of resistance and development of inhibitors against it can be a novel approach to combat such problems. In our perspective, host-directed therapy (HDT) represents another promising strategy in combating antimicrobial resistance (AMR). This approach involves targeting specific factors within host cells that pathogens rely on for their survival, either through replication or persistence. As many new drugs are under clinical trials it is advisable that more clinical data and antimicrobial stewardship programs should be conducted to fully assess the clinical efficacy and safety of new therapeutic agents.

Escaping the ESKAPE pathogens: A review on antibiofilm potential of nanoparticles.

ESKAPE pathogens, a notorious consortium comprising Enterococcusfaecium, Staphylococcusaureus, Klebsiellapneumoniae, Acinetobacterbaumannii, Pseudomonasaeruginosa, and Enterobacter species, pose formidable challenges in healthcare settings due to their multidrug-resistant nature. The increasing global cases of antimicrobial-resistant ESKAPE pathogens are closely related to their remarkable ability to form biofilms. Thus, understanding the unique mechanisms of antimicrobial resistance of ESKAPE pathogens and the innate resilience of biofilms against traditional antimicrobial agents is important for developing innovative strategies to establish effective control methods against them. This review offers a thorough analysis of biofilm dynamics, with a focus on the general mechanisms of biofilm formation, the significant contribution of persister cells in the resistance mechanisms, and the recurrence of biofilms in comparison to planktonic cells. Additionally, this review highlights the potential strategies of nanoparticles for managing biofilms in the ESKAPE group of pathogens. Nanoparticles, with their unique physicochemical properties, provide promising opportunities for disrupting biofilm structures and improving antimicrobial effectiveness. The review has explored interactions between nanoparticles and biofilms, covering a range of nanoparticle types such as metal, metal-oxide, surface-modified, and functionalized nanoparticles, along with organic nanoparticles and nanomaterials. The additional focus of this review also encompasses green synthesis techniques of nanoparticles that involve plant extract and supernatants from bacterial and fungal cultures as reducing agents. Furthermore, the use of nanocomposites and nano emulsions in biofilm management of ESKAPE is also discussed. To conclude, the review addresses the current obstacles and future outlooks in nanoparticle-based biofilm management, stressing the necessity for further research and development to fully exploit the potential of nanoparticles in addressing biofilm-related challenges.

Discovery of novel dihydronaphthalene-imidazole ligands as potential inhibitors of Staphylococcus aureus multidrug resistant NorA efflux pump: A combination of experimental and in silico molecular docking studies.

Overexpression of the efflux pump is a predominant mechanism by which bacteria show antimicrobial resistance (AMR) and leads to the global emergence of multidrug resistance (MDR). In this work, the inhibitory potential of library of dihydronapthyl scaffold-based imidazole derivatives having structural resemblances with some known efflux pump inhibitors (EPI) were designed, synthesized and evaluated against efflux pump inhibitor against overexpressing bacterial strains to study the synergistic effect of compounds and antibiotics. Out of 15 compounds, four compounds (Dz-1, Dz-3, Dz-7, and Dz-8) were found to be highly active. DZ-3 modulated the MIC of ciprofloxacin, erythromycin, and tetracycline by 128-fold each against 1199B, XU212 and RN4220 strains of S. aureus respectively. DZ-3 also potentiated tetracycline by 64-fold in E. coli AG100 strain. DZ-7 modulated the MIC of both tetracycline and erythromycin 128-fold each in S. aureus XU212 and S. aureus RN4220 strains. DZ-1 and DZ-8 showed the moderate reduction in MIC of tetracycline in E. coli AG100 only by 16-fold and 8-fold, respectively. DZ-3 was found to be the potential inhibitor of NorA as determined by ethidium bromide efflux inhibition and accumulation studies employing NorA overexpressing strain SA-1199B. DZ-3 displayed EPI activity at non-cytotoxic concentration to human cells and did not possess any antibacterial activity. Furthermore, molecular docking studies of DZ-3 was carried out in order to understand the possible binding sites of DZ-3 with the active site of the protein. These studies indicate that dihydronaphthalene scaffolds could serve as valuable cores for the development of promising EPIs.

Bacteriophage therapy against ESKAPE bacterial pathogens: Current status, strategies, challenges, and future scope.

The ESKAPE pathogens are the primary threat due to their constant spread of drug resistance worldwide. These pathogens are also regarded as opportunistic pathogens and could potentially cause nosocomial infections. Most of the ESKAPE pathogens have developed resistance to almost all the antibiotics that are used against them. Therefore, to deal with antimicrobial resistance, there is an urgent requirement for alternative non-antibiotic strategies to combat this rising issue of drug-resistant organisms. One of the promising alternatives to this scenario is implementing bacteriophage therapy. This under-explored mode of treatment in modern medicine has posed several concerns, such as preferable phages for the treatment, impact on the microbiome (or gut microflora), dose optimisation, safety, etc. The review will cover a rationale for phage therapy, clinical challenges, and propose phage therapy as an effective therapeutic against bacterial coinfections during pandemics. This review also addresses the expected uncertainties for administering the phage as a treatment against the ESKAPE pathogens and the advantages of using lytic phage over temperate, the immune response to phages, and phages in combinational therapies. The interaction between bacteria and bacteriophages in humans and countless animal models can also be used to design novel and futuristic therapeutics like personalised medicine or bacteriophages as anti-biofilm agents. Hence, this review explores different aspects of phage therapy and its potential to emerge as a frontline therapy against the ESKAPE bacterial pathogen.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :ß3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :ß3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑßγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits ß and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal ß-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

Enhanced antimicrobial efficacy and energy efficiency of low irradiance 405-nm light for bacterial decontamination.

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.

Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59 Å resolution.

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 ß/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.

Rosin as a Natural Alternative for the effective disinfection of ESKAPE Pathogens and Clostridioides difficile spores.

AIM: Hospital-acquired infections (HAIs) caused by antimicrobial-resistant ESKAPE pathogens are a significant concern for the healthcare industry, with an estimated cost of up to ${\$}$45 billion per year in the US alone. Clostridioides difficile is an additional opportunistic pathogen that also poses a serious threat to immunocompromised patients in hospitals. Infections caused by these pathogens lead to increased hospital stays and repeated readmission, resulting in a significant economic burden. Disinfectants and sporicidals are essential to reduce the risk of these pathogens in hospitals, but commercially available products can have a number of disadvantages including inefficacy, long contact times, short shelf lives, and operator health hazards. In this study we evaluated the effectiveness of Rosin (a natural substance secreted by coniferous trees as a defence mechanism against wounds in tree bark) and its commercial derivative Rosetax-21 as disinfectants and sporicidal against the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and spore preparations from Clostridioides difficile. METHODS AND RESULTS: Both Rosin and Rosetax-21 were tested under simulated clean and dirty conditions (with BSA) against the ESKAPE pathogens, and C. difficile spore preparations. In clean conditions, Rosin (5% weight/volume: w/v) demonstrated significant efficacy against five of the ESKAPE pathogens, with A. baumannii and E. faecium being the most susceptible, and K. pneumoniae the most resistant, showing only a one-log reduction after a 5 min treatment. However, in dirty conditions, all pathogens including K. pneumoniae exhibited at least a 3-log reduction to Rosin within 5 min. Rosetax-21 (5% w/v) was found to be less effective than Rosin in clean conditions, a trend that was exacerbated in the presence of BSA. Additionally, both Rosin and Rosetax-21 at 2.5% (w/v) achieved complete eradication of C. difficile spores when combined with 0.5% glutaraldehyde, though their standalone sporicidal activity was limited. CONCLUSIONS: The findings from this study highlight the potential of Rosin and Rosetax-21 as both bactericidal and sporicidal disinfectants, with their efficacy varying based on the conditions and the pathogens tested. This presents an avenue for the development of novel healthcare disinfection strategies, especially against HAIs caused by antimicrobial-resistant ESKAPE pathogens and C. difficile.

Antimicrobial resistance landscape in a metropolitan city context using open drain wastewater-based metagenomic analysis.

One Health concept recognizes the inextricable interactions of diverse ecosystems and their subsequent effect on human, animal and plant health. Antimicrobial resistance (AMR) is a major One Health concern and is predicted to cause catastrophes if appropriate measures are not implemented. To understand the AMR landscape in a south Indian metropolitan city, metagenomic analysis of open drains was performed. The data suggests that in January 2022, macrolide class of antibiotics contributed the highest resistance of 40.1% in the city, followed by aminoglycoside- 24.4%, tetracycline- 11.3% and lincosamide- 6.7%. The 'mutations in the 23S rRNA gene conferring resistance to macrolide antibiotics' were the major contributor of resistance with a prevalence of 39.7%, followed by '16s rRNA with mutation conferring resistance to aminoglycoside antibiotics'- 22.2%, '16S rRNA with mutation conferring resistance to tetracycline derivatives'- 9.2%, and '23S rRNA with mutation conferring resistance to lincosamide antibiotics'- 6.7%. The most prevalent antimicrobial resistance gene (ARG) 'mutations in the 23S rRNA gene conferring resistance to macrolide antibiotics' was present in multiple pathogens including Escherichia coli, Campylobacter jejuni, Acinetobacter baumannii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Klebsiella pneumoniae and Helicobacter pylori. Most of the geographical locations in the city showed a similar landscape for AMR. Considering human mobility and anthropogenic activities, such an AMR landscape could be common across other regions too. The data indicates that pathogens are evolving and acquiring antibiotic resistance genes to evade antibiotics of multiple major drug classes in diverse hosts. The outcomes of the study are relevant not only in understanding the resistance landscape at a broader level but are also important for identifying the resistant drug classes, the mechanisms of gaining resistance and for developing new drugs that target specific pathways. This kind of surveillance protocol can be extended to regions in other developing countries to assess and combat the problem of antimicrobial resistance.

ESKAPE pathogens in pediatric cardiac surgery patients: 5-year microbiological monitoring in a tertiary hospital in Kazakhstan.

Hospital acquired infections greatly affect recovery and survival in pediatric surgical patients. We evaluated prevalence and antimicrobial resistance of ESKAPE pathogens in neonates and infants subjected to cardiac surgery in a tertiary hospital in Central Kazakhstan between 2019 and 2023 (2,278 patients) using routine methods of microbiological detection. ESKAPE pathogens were found in 1,899 out of 2,957 samples (Staphylococcus aureus - 35.3%, Klebsiella pneumoniae - 27.8%, Acinetobacter baumannii - 14.5%, Pseudomonas aeruginosa - 12.4%, Enterobacter sp. - 8.8%, Enterococcus faecium - 1.2%). The total prevalence of ESKAPE increased significantly from 45.1 to 76.9% (P = 0.005) during the study period. The resistance significantly increased in methicillin-resistant S. aureus (MRSA, from 13.7 to 41.9%, P = 0.041) but decreased in carbapenem-resistant P. aeruginosa (from 64.3 to 37.7%, P = 0.037) and carbapenem-resistant A. baumannii (from 48.5 to 19.1%, P = 0.039). Gradual but non-significant changes were shown in third-generation cephalosporin resistant K. pneumoniae (from 63.6 to 45.2%) and carbapenem-resistant K. pneumoniae (from 0 to 8.3%). The relative prevalence of ESKAPE pathogens steadily increased in our pediatric cardiac surgery patients in 2019-2023. The most frequent were S. aureus, K. pneumoniae, and A. baumannii, with dramatically increasing tendencies for MRSA. Our results highlight the necessity for a well-designed infection control strategy and constant microbiological monitoring in pediatric cardiac surgery departments.

The therapeutic potential of indole hybrids, dimers, and trimers against drug-resistant ESKAPE pathogens.

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter (ESKAPE) species as causative agents are characterized by increased levels of resistance toward multiple classes of first-line as well as last-resort antibiotics and represent serious global health concerns, creating a critical need for the development of novel antibacterials with therapeutic potential against drug-resistant ESKAPE species. Indole derivatives with structural and mechanistic diversity demonstrated broad-spectrum antibacterial activity against various clinically important pathogens including drug-resistant ESKAPE. Moreover, several indole-based agents that are exemplified by creatmycin have already been used in clinics or under clinical trials for the treatment of bacterial infections, demonstrating that indole derivatives hold great promise for the development of novel antibacterials. This review is an endeavor to highlight the current scenario of indole hybrids, dimers, and trimers with therapeutic potential against drug-resistant ESKAPE pathogens, covering articles published from 2020 to the present, to open new avenues for the exploration of novel antidrug-resistant ESKAPE candidates.

Molecular Periphery Design Allows Control of the New Nitrofurans Antimicrobial Selectivity.

A series of 13 new 3-substituted 5-(5-nitro-2-furyl)-1,2,4-oxadiazoles was synthesized from different aminonitriles. All compounds were screened in the disc diffusion test at a 100 µg/mL concentration to determine the bacterial growth inhibition zone presence and diameter, and then the minimum inhibitory concentrations (MICs) were determined for the most active compounds by serial dilution. The compounds showed antibacterial activity against ESKAPE bacteria, predominantly suppressing the growth of 5 species out of the panel. Some compounds had similar or lower MICs against ESKAPE pathogens compared to ciprofloxacin, nitrofurantoin, and furazidin. In particular, 3-azetidin-3-yl-5-(5-nitro-2-furyl)-1,2,4-oxadiazole (2h) inhibited S. aureus at a concentration lower than all comparators. Compound 2e (5-(5-nitro-2-furyl)-3-[4-(pyrrolidin-3-yloxy)phenyl]-1,2,4-oxadiazole) was active against Gram-positive ESKAPE pathogens as well as M. tuberculosis. Differences in the molecular periphery led to high selectivity for the compounds. The induced-fit docking (IFD) modeling technique was applied to in silico research. Molecular docking results indicated the targeting of compounds against various nitrofuran-associated biological targets.

Porcine model of a complicated skin and soft tissue infection caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa poses a significant threat to both immunocompetent and immunocompromised individuals, often resulting in life-threatening infections. With increasing antimicrobial resistance, novel therapeutic strategies are urgently needed. Although animal models are crucial for preclinical studies, limited data are available for porcine models, more specifically for P. aeruginosa complicated skin and soft tissue infections (cSSTIs). This study presents a novel porcine model inducing and sustaining cSSTI for 14 days. Six pigs (120 wounds) were used for the development of infections, and within this group, two pigs (40 wounds) were used to evaluate the progression of the cSSTI infection. The model demonstrated bacterial loads of more than 107 CFU/gram of tissue or higher. The cSSTI fully developed within three days and remained well above these levels until day 14 post-infection. Due to the immunocompetence of this model, all the immunological processes associated with the response to the presence of infection and the wound healing process are preserved.

The Capsule Increases Susceptibility to Last-Resort Polymyxins, but Not to Other Antibiotics, in Klebsiella pneumoniae.

The extracellular capsule is a virulence factor present in many facultative pathogens, but its role in antimicrobial resistance remains controversial. To shed light on this debate, we tested six antibiotics on four Klebsiella pneumoniae species complex strains. Noncapsulated strains exhibited increased tolerance to polymyxins, but not to other antibiotics, as measured using the MIC. Our results urge caution on the use of therapeutic agents that target the capsule and may result in selection for its inactivation.

PlyKp104, a Novel Phage Lysin for the Treatment of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Other Gram-Negative ESKAPE Pathogens.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

Safe and easy in vitro evaluation of tmRNA-SmpB-mediated trans-translation from ESKAPE pathogenic bacteria.

In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.