Bacterial Multidrug Resistance Research Articles

Anti-Staphy Peptides Rationally Designed from Cry10Aa Bacterial Protein.

Bacterial infections pose a significant threat to human health, constituting a major challenge for healthcare systems. Antibiotic resistance is particularly concerning in the context of treating staphylococcal infections. In addressing this challenge, antimicrobial peptides (AMPs), characterized by their hydrophobic and cationic properties, unique mechanism of action, and remarkable bactericidal and immunomodulatory capabilities, emerge as promising alternatives to conventional antibiotics for tackling bacterial multidrug resistance. This study focuses on the Cry10Aa protein as a template for generating AMPs due to its membrane-penetrating ability. Leveraging the Joker algorithm, six peptide variants were derived from α-helix 3 of Cry10Aa, known for its interaction with lipid bilayers. In vitro, antimicrobial assays determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) required for inhibiting the growth of Staphylococcus aureus, Escherichia coli, Acinetobacter baummanii, Enterobacter cloacae, Enterococcus facallis, Klebsiella pneumonia, and Pseudomonas aeruginosa. Time-kill kinetics were performed using the parental peptide AMPCry10Aa, as well as AMPCry10Aa_1 and AMPCry10Aa_5, against E. coli ATCC, S. aureus 111 and S. aureus ATCC strains showing that AMPCry10Aa_1 and AMPCry10Aa_5 peptides can completely reduce the initial bacterial load with less than 2 h of incubation. AMPCry10Aa_1 and AMPCry 10Aa_5 present stability in human serum and activity maintenance up to 37 °C. Cytotoxicity assays, conducted using the MTT method, revealed that all of the tested peptides exhibited cell viability >50% (IC50). The study also encompassed evaluations of the structure and physical-chemical properties. The three-dimensional structures of AMPCry10Aa and AMPCry10Aa_5 were determined through nuclear magnetic resonance (NMR) spectroscopy, indicating the adoption of α-helical segments. Electron paramagnetic resonance (EPR) spectroscopy elucidated the mechanism of action, demonstrating that AMPCry10Aa_5 enters the outer membranes of E. coli and S. aureus, causing substantial increases in lipid fluidity, while AMPCry10Aa slightly increases lipid fluidity in E. coli. In conclusion, the results obtained underscore the potential of Cry10Aa as a source for developing antimicrobial peptides as alternatives to conventional antibiotics, offering a promising avenue in the battle against antibiotic resistance.

#1776 Distinct changes in gut microbiota of patients with kidney graft rejection

Abstract Background and Aims Kidney graft rejection still represents the major cause of graft loss in kidney transplant recipients. Of growing interest is the bidirectional relationship between gut microbiome and immune system suggesting that gut microbiota can affect allograft outcome. Method In this cross-sectional case control study, we characterized the gut microbial profile of adult renal transplant recipients with and without graft rejection in order to define a cohort-specific microbial fingerprint through 16S rRNA gene sequencing. We used very strict inclusion and exclusion criteria to address confounder of microbiota composition. Results Different relative abundances in several gut microbial taxa were detectable in control patients compared to patients with kidney allograft rejection. Alpha diversity was lower in the rejection group and beta diversity revealed dissimilarity between patients with and without kidney graft rejection (P < .01). When the rejection group was stratified according to different types of allograft rejection, major changes were identified between patients with chronic T cell mediated rejection (cTCMR) and controls. Changes in alpha diversity within the gut microbiome were related to the probability of cTCMR (P < .05). Kidney transplant patients without rejection showed significant enrichment of rather anti-inflammatory taxa whereas in the rejection group bacteria well known for their role in chronic inflammation were increased. For example, ASV 362 belonging to the genus Bacteroides and ASV 312 belonging to Tanerrelaceae were enriched in no rejection (P < .001 and P < .01) whereas ASV 365 was enriched in patients with allograft rejection (P = .04). Looking at metagenomic functions, a higher abundance of genes coding for enzymes involved in bacterial multidrug resistance and processing of short chain fatty acids was found in patients without rejection but an increase in enzymes involved in NADPH production was seen in patients with allograft rejection. Conclusion A distinct microbial fingerprint of patients with allograft rejection might serve as non-invasive biomarker in the future.

Development of Inhibitory Compounds for Metallo-beta-lactamase through Computational Design and Crystallographic Analysis.

Metallo-β-lactamases (MBL) deactivate β-lactam antibiotics through a catalytic reaction caused by two zinc ions at the active center. Since MBLs deteriorate a wide range of antibiotics, they are dangerous factors for bacterial multidrug resistance. In this work, organic synthesis, computational design, and crystal structure analysis were performed to obtain potent MBL inhibitors based on a previously identified hit compound. The hit compound comprised 3,4-dihydro-2(1H)-quinolinone linked with a phenyl-ether-methyl group via a thiazole ring. In the first step, the thiazole ring was replaced with a tertiary amine to avoid the planar structure. In the second step, we virtually modified the compound by keeping the quinolinone backbone. Every modified compound was bound to a kind of MBL, imipenemase-1 (IMP-1), and the binding pose was optimized by a molecular mechanics calculation. The binding scores were evaluated for the respective optimized binding poses. Given the predicted binding poses and calculated binding scores, candidate compounds were determined for organic syntheses. The inhibitory activities of the synthesized compounds were measured by an in vitro assay for two kinds of MBLs, IMP-1 and New Delhi metallo-β-lactamase (NDM-1). A quinolinone connected with an amine bound with methyl-phenyl-ether-propyl and cyclohexyl-ethyl showed a 50% inhibitory concentration of 4.8 μM. An X-ray crystal analysis clarified the binding structure of a synthesized compound to IMP-1. The δ-lactam ring of quinolinone was hydrolyzed, and the generated carboxyl group was coordinated with zinc ions. The findings on the chemical structure and binding pose are expected to be a base for developing MBL inhibitors.

Extending the Potency and Lifespan of Antibiotics: Inhibitors of Gram-Negative Bacterial Efflux Pumps.

Efflux is a natural process found in all prokaryotic and eukaryotic cells that removes a diverse range of substrates from inside to outside. Many antibiotics are substrates of bacterial efflux pumps, and modifications to the structure or overexpression of efflux pumps are an important resistance mechanism utilized by many multidrug-resistant bacteria. Therefore, chemical inhibition of bacterial efflux to revitalize existing antibiotics has been considered a promising approach for antimicrobial chemotherapy over two decades, and various strategies have been employed. In this review, we provide an overview of bacterial multidrug resistance (MDR) efflux pumps, of which the resistance nodulation division (RND) efflux pumps are considered the most clinically relevant in Gram-negative bacteria, and describe over 50 efflux inhibitors that target such systems. Although numerous efflux inhibitors have been identified to date, none have progressed into clinical use because of formulation, toxicity, and pharmacokinetic issues or a narrow spectrum of inhibition. For these reasons, the development of efflux inhibitors has been considered a difficult and complex area of research, and few active preclinical studies on efflux inhibitors are in progress. However, recently developed tools, including but not limited to computational tools including molecular docking models, offer hope that further research on efflux inhibitors can be a platform for research and development of new bacterial efflux inhibitors.

Nature-Inspired Micro/Nano-Structured Antibacterial Surfaces.

The problem of bacterial resistance has become more and more common with improvements in health care. Worryingly, the misuse of antibiotics leads to an increase in bacterial multidrug resistance and the development of new antibiotics has virtually stalled. These challenges have prompted the need to combat bacterial infections with the use of radically different approaches. Taking lessons from the exciting properties of micro-/nano-natural-patterned surfaces, which can destroy cellular integrity, the construction of artificial surfaces to mimic natural functions provides new opportunities for the innovation and development of biomedicine. Due to the diversity of natural surfaces, functional surfaces inspired by natural surfaces have a wide range of applications in healthcare. Nature-inspired surface structures have emerged as an effective and durable strategy to prevent bacterial infection, opening a new way to alleviate the problem of bacterial drug resistance. The present situation of bactericidal and antifouling surfaces with natural and biomimetic micro-/nano-structures is briefly reviewed. In addition, these innovative nature-inspired methods are used to manufacture a variety of artificial surfaces to achieve extraordinary antibacterial properties. In particular, the physical antibacterial effect of nature-inspired surfaces and the functional mechanisms of chemical groups, small molecules, and ions are discussed, as well as the wide current and future applications of artificial biomimetic micro-/nano-surfaces. Current challenges and future development directions are also discussed at the end. In the future, controlling the use of micro-/nano-structures and their subsequent functions will lead to biomimetic surfaces offering great potential applications in biomedicine.

Etiology of bacterial pneumonia and multi-drug resistance pattern among pneumonia suspected patients in Ethiopia: a systematic review and meta-analysis

BackgroundBacterial pneumonia can affect all age groups, but people with weakened immune systems, young children, and the elderly are at a higher risk. Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa are the most common causative agents of pneumonia, and they have developed high MDR in recent decades in Ethiopia. This systematic review and meta-analysis aimed to determine the pooled prevalence of bacterial pneumonia and multidrug resistance in Ethiopia.MethodsThe articles were searched extensively in the electronic databases and grey literature using entry terms or phrases. Studies meeting the eligibility criteria were extracted in MS Excel and exported for statistical analysis into STATA version 14 software. The pooled prevalence of bacterial pneumonia and multidrug resistance were calculated using a random-effects model. Heterogeneity was assessed by using the I2 value. Publication bias was assessed using a funnel plot and Egger’s test. A sensitivity analysis was done to assess the impact of a single study on the pooled effect size.ResultOf the 651 studies identified, 87 were eligible for qualitative analysis, of which 11 were included in the meta-analysis consisting of 1154 isolates. The individual studies reported prevalence of bacterial pneumonia ranging from 6.19 to 46.3%. In this systematic review and metanalysis, the pooled prevalence of bacterial pneumonia in Ethiopia was 37.17% (95% CI 25.72–46.62), with substantial heterogeneity (I2 = 98.4%, p < 0.001) across the studies. The pooled prevalence of multidrug resistance in bacteria isolated from patients with pneumonia in Ethiopia was 67.73% (95% CI: 57.05–78.40). The most commonly isolated bacteria was Klebsiella pneumoniae, with pooled prevalence of 21.97% (95% CI 16.11–27.83), followed by Streptococcus pneumoniae, with pooled prevalence of 17.02% (95% CI 9.19–24.86), respectively.ConclusionThe pooled prevalence of bacterial isolates from bacterial pneumonia and their multidrug resistance were high among Ethiopian population. The initial empirical treatment of these patients remains challenging because of the strikingly high prevalence of antimicrobial resistance.

Injectable Phage-Loaded Microparticles Effectively Release Phages to Kill Methicillin-Resistant Staphylococcus aureus.

The increasing prevalence of bacterial multidrug antibiotic resistance has led to a serious threat to public health, emphasizing the urgent need for alternative antibacterial therapeutics. Lytic phages, a class of viruses that selectively infect and kill bacteria, offer promising potential as alternatives to antibiotics. However, injectable carriers with a desired release profile remain to be developed to deliver them to infection sites. To address this challenge, phage-loaded microparticles (Phage-MPs) have been developed to deliver phages to the infection site and release phages for an optimal therapeutic effect. The Phage-MPs are synthesized by allowing phages to be electrostatically attached onto the porous polyethylenimine-modified silk fibroin microparticles (SF-MPs). The high specific surface area of SF-MPs allows them to efficiently load phages, reaching about 1.25 × 1010 pfu per mg of microparticles. The Phage-MPs could release phages in a controlled manner to achieve potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Unlike the diffuse biodistribution of free phages post-intraperitoneal injection, Phage-MPs could continuously release phages to effectively boost the local phage concentration at the bacterial infection site after they are intraperitoneally injected into an abdominal MRSA-infected mouse model. In a mouse abdominal MRSA infection model, Phage-MPs significantly reduce the bacterial load in major organs, achieving an efficient therapeutic effect. Furthermore, Phage-MPs demonstrate outstanding biocompatibility both in vitro and in vivo. Overall, our research lays the foundation for a new generation of phage-based therapies to combat antibiotic-resistant bacterial infections.

Structural insights into the Clp protein degradation machinery.

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Molecular determinant deciphering of MIC-guided RND efflux substrates in E. coli

Antimicrobial resistance poses an urgent and formidable global public health threat. The escalation of bacterial multidrug resistance to antibiotics has the potential to become a leading cause of global mortality if there is no substantial improvement in antimicrobial development and therapy protocols. In light of this, it is imperative to identify the molecular determinants responsible for the reduced antibiotic activity associated with RND efflux pumps. This comprehensive study meticulously examines Minimum Inhibitory Concentration (MIC) data obtained from in vitro tests for various antibiotic families and non-active dye compounds, sourced from diverse literature references. The primary focus of this study is to assess the susceptibility of these agents to efflux-resistant Escherichia coli strains, integrating both MIC data and relevant physicochemical properties. The central objective is to unveil the specific substituents that significantly influence the uptake process mediated by the AcrAB-TolC efflux system. This exploration seeks to reveal the consequences of these substituents on pharmacodynamic responses, providing valuable insights into Structure-Activity Relationships. It is noteworthy that this analysis represents a pioneering effort, with prospective implications for RND efflux pump-producing strains. Ultimately, deciphering efflux markers is crucial to effectively mitigate the emergence of specific resistance and to better monitor the role of this primary resistance mechanism in Gram-negative bacteria, particularly as observed in clinical antibiotic therapy practice.

Distinct Changes in Gut Microbiota of Patients With Kidney Graft Rejection.

Kidney graft rejection still represents the major cause of graft loss in kidney transplant recipients. Of growing interest is the bidirectional relationship between gut microbiome and immune system suggesting that gut microbiota can affect allograft outcome. In this cross-sectional case-control study, we characterized the gut microbial profile of adult renal transplant recipients with and without graft rejection to define a cohort-specific microbial fingerprint through 16S ribosomal RNA gene sequencing. We used very strict inclusion and exclusion criteria to address confounder of microbiota composition. Different relative abundances in several gut microbial taxa were detectable in control patients compared with patients with kidney allograft rejection. Alpha diversity was lower in the rejection group and beta diversity revealed dissimilarity between patients with and without kidney graft rejection (P < 0.01). When the rejection group was stratified according to different types of allograft rejection, major changes were identified between patients with chronic T-cellular-mediated rejection and controls. Changes in alpha diversity within the gut microbiome were related to the probability of chronic T-cellular-mediated rejection (P < 0.05). Kidney transplant patients without rejection showed significant enrichment of rather anti-inflammatory taxa whereas in the rejection group bacteria well known for their role in chronic inflammation were increased. For example, amplicon sequence variant (ASV) 362 belonging to the genus Bacteroides and ASV 312 belonging to Tannerellaceae were enriched in no rejection (P < 0.001 and P < 0.01), whereas ASV 365 was enriched in patients with allograft rejection (P = 0.04). Looking at metagenomic functions, a higher abundance of genes coding for enzymes involved in bacterial multidrug resistance and processing of short-chain fatty acids was found in patients without rejection but an increase in enzymes involved in nicotinamide adenine dinucleotide phosphate production was seen in patients with allograft rejection. A distinct microbial fingerprint of patients with allograft rejection might serve as noninvasive biomarker in the future.

Clinical practice guidelines for meropenem therapy in neonatal sepsis (2024)

Meropenem is one of the most widely used special-grade antimicrobial agents in the treatment of neonatal sepsis. However, its irrational use has led to an increasingly severe problem of bacterial multidrug resistance. The guideline was developed following standardized methods and procedures, and provides 12 recommendations specifically addressing 9 clinical issues. The recommendations cover various aspects of meropenem use in neonates, including timing of administration, recommended dosage, extended infusion, monitoring and assessment, antimicrobial adjustment strategies, treatment duration, and treatment strategies for carbapenem-resistant Enterobacteriaceae infections. The aim of the guideline is to provide evidence-based recommendations and guidance for the rational use of meropenem in neonates with sepsis.

Efficacy of outer membrane permeabilization in promoting aromatic isothiocyanates-mediated eradication of multidrug resistant Gram-negative bacteria and bacterial persisters.

Multidrug resistant (MDR) bacteria are recognized to be one of the most important problems in public health. The outer membrane permeability is a critical intrinsic mechanism of bacterial resistance. In addition, bacteria produce a small number of dormant persister cells causing multidrug tolerance that reduces antimicrobial efficacy. This study aimed to evaluate the inhibitory effects of the combination of aromatic isothiocyanates (ITCs) with membrane-active agents on bacterial persisters and MDR Gram-negative bacteria. Our study demonstrated that membrane-active agents, particularly ethylenediaminetetraacetic acid (EDTA) synergistically enhanced the inhibitory activity of aromatic benzyl ITC and phenethyl ITC against most Gram-negative bacteria strains with fractional inhibitory concentration index values ranging from 0.18 to 0.5 and 0.16 to 0.5, respectively, and contributed to an 8- to 64-fold minimal inhibitory concentration reduction compared with those of aromatic ITCs alone. The EDTA-aromatic ITCs combination effectively reduced the survival rates of tested bacteria and significantly eradicated bacterial persisters (p = 0.033 and 0.037, respectively). The growth kinetics analysis also supported the enhanced inhibitory effect of EDTA-aromatic ITCs combination against tested bacteria. Our results suggested an alternate treatment strategy against Gram-negative bacteria, promoting the entry of aromatic ITCs into bacterial cytoplasm to facilitate bacterial clearance and thus preventing the development of bacterial resistance.

A Wireless, CRISPR-Polymer Dot Electrochemical Sensor for the Diagnosis of Bacterial Pneumonia and Multi-Drug Resistance.

Rapid and accurate diagnosis is crucial for managing the global health threat posed by multidrug-resistant bacterial infections; however, current methods have limitations in either being time-consuming, labor-intensive, or requiring instruments with high costs. Addressing these challenges, we introduce a wireless electrochemical sensor integrating the CRISPR/Cas system with electroconductive polymer dot (PD) nanoparticles to rapidly detect bacterial pathogens from human sputum. To enhance the electroconductive properties, we synthesized copper-ion-immobilized PD (PD-Cu), followed by conjugation of the deactivated Cas9 protein (dCas9) onto PD-Cu-coated Si electrodes to generate the dCas9-PD-Cu sensor. The dCas9-PD-Cu sensor integrated with isothermal amplification can specifically detect target nucleic acids of multidrug-resistant bacteria, such as the antibiotic resistance genes kpc-2 and mecA. The dCas9-PD-Cu sensor exhibits high sensitivity, allowing for the detection of ∼54 femtograms of target nucleic acids, based on measuring the changes in resistivity of the Si electrodes through target capture by dCas9. Furthermore, a wireless sensing platform of the dCas9-PD-Cu sensor was established using a Bluetooth module and a microcontroller unit for detection using a smartphone. We demonstrate the feasibility of the platform in diagnosing multidrug-resistant bacterial pneumonia in patients' sputum samples, achieving 92% accuracy. The current study presents a versatile biosensor platform that can overcome the limitations of conventional diagnostics in the clinic.

Lipid nanoparticles based on natural matrices with activity against multidrug resistant bacterial species.

Lately, the bacterial multidrug resistance has been a reason to public health concerning around world. The development of new pharmacology therapies against infections caused by multidrug-resistant bacteria is urgent. In this work, we developed 10 NLC formulations composed of essential oils (EO), vegetable butter and surfactant. The formulations were evaluated for long-term and thermal cycling stability studies in terms of (particle size, polydispersion index and Zeta potential). In vitro antimicrobial assays were performed using disk diffusion test and by the determination of the minimum inhibitory concentration (MIC) performed with fresh and a year-old NLC. The most promising system and its excipients were structurally characterized through experimental methodologies (FTIR-ATR, DSC and FE-SEM). Finally, this same formulation was studied through nanotoxicity assays on the chicken embryo model, analyzing different parameters, as viability and weight changes of embryos and annexes. All the developed formulations presented long-term physicochemical and thermal stability. The formulation based on cinnamon EO presented in vitro activity against strains of Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from humans and in vivo biocompatibility. Considering these promising results, such system is able to be further tested on in vivo efficacy assays.

Discovery of adjuvants with antibacterial potentiation activity against carbapenemase-producing Enterobacterales based on in silico virtual screening

Bacterial multi-drug resistance has become a concern worldwide, especially after the emergence of carbapenemases. Adjuvants with antibacterial potentiation activity can resensitise drug-resistant strains to carbapenems. However, only a few adjuvants with antibacterial potentiation activity are currently available in clinical practice. Here, we first docked the library containing more than 30,000 small molecules to carbapenemases including Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-5 (NDM-5), through in silico virtual screening to obtain lead compounds against carbapenemase-producing Enterobacterales. Meanwhile, the in vitro antibacterial potentiation assays revealed that ibandronate, azacytidine, ribostamycin sulfate and cidofovir exhibited synergistic or additive activity in the presence of meropenem, with good biocompatibility based on red blood cell hemolysis and cell viability tests. Furthermore, the combination of meropenem and azacytidine showed high efficacy in a mouse sepsis model infected with an NDM-5-producing clinical strain, with a 100% survival rate, decreased bacterial burden and alleviated pathological deterioration. These results suggest that the virtual screening is a promising strategy to identify new antibiotic adjuvants targeting carbapenemase-producing Enterobacterales.

Insights into antibiotic and heavy metal resistance interactions in Escherichia coli isolated from livestock manure and fertilized soil

Heavy metal and antibiotic-resistant bacteria from livestock feces are ecological and public health problems. However, the distribution and relationships of antibiotic resistance genes (ARGs), heavy metal resistance genes (HMRGs), and virulence factors (VFs) and their transmission mechanisms remain unclear. Therefore, we investigated the resistance of Escherichia coli, the prevalence of its ARGs, HMRGs, and VFs, and their transmission mechanisms in livestock fresh feces (FF), composted feces (CF), and fertilized soil (FS). In total, 99.54% (n = 221) and 91.44% (n = 203) of E. coli were resistant to at least one antibiotic and one heavy metal, respectively. Additionally, 72.52% (n = 161) were multi-drug resistant (MDR), of which Cu-resistant E. coli accounted for 72.67% (117/161). More than 99.34% (88/89) of E. coli carried multidrug ARGs, VFs, and the Cu resistance genes cueO and cusABCRFS. The Cu resistance genes cueO and cusABCRFS were mainly located on chromosomes, and cueO and cusF were positively associated with HMRGs, ARGs, and VFs. The Cu resistance genes pcoABCDRS were located on the plasmid pLKYL-P02 flanked by ARGs in PF18C from FF group and on chromosomes flanked by HMRGs in SAXZ1-1 from FS group. These results improved our understanding of bacterial multidrug and heavy metal resistance in the environment.

Efflux Pump Inhibition-Based Screening and Anti-Infective Evaluation of Punica granatum Against Bacterial Pathogens.

Drug efflux pumps contribute to bacterial multidrug resistance (MDR), reducing antibiotic effectiveness and causing treatment failures. Besides their role in MDR, efflux pumps also assist in the transportation of quorum sensing (QS) signal molecules and increased the tolerance of biofilms. Recently, the search for efflux pump inhibitors from natural sources, including anti-infective plants, has gained attention as a potential therapy against drug-resistant bacteria. In this study, 19 traditional Indian medicinal plants were screened for their efflux pump inhibitory activity against Escherichia coli TGI. The promising extract, i.e., Punica granatum was subsequently fractioned in the solvents of increasing polarity. Among them, at sub-MIC active EPI fraction was PGEF (P. granatum ethyl acetate fraction), further investigated for anti-infective potential against Chromobacterium violaceum 12,472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. PGEF was also evaluated for in vivo efficacy in Caenorhabditis elegans model. Major phytocompounds were analyzed by mass spectroscopic techniques. At respective Sub-MIC, PGEF reduced violacein production by 71.14% in C. violaceum 12,472. Moreover, PGEF inhibited pyocyanin (64.72%), pyoverdine (48.17%), protease (51.35%), and swarming motility (44.82%) of P. aeruginosa PAO1. Furthermore, PGEF reduced the production of prodigiosin and exoprotease by 64.73% and 61.80%, respectively. Similarly, at sub-MIC, PGEF inhibited (≥ 50%) biofilm development in all test pathogens. The key phytocompounds detected in active fraction include 5-hydroxymethylfurfural, trans-p-coumaric acid 4- glucoside, (-)-Epicatechin 3'-O-glucuronide, and ellagic acid. Interestingly, PGEF also demonstrated anti-infective efficacy against the PAO1-infected C. elegans test model and highlighting its therapeutic potential as an anti-infective agent to combat drug-resistant problems.

Type I Photosensitizer Targeting Glycans: Overcoming Biofilm Resistance by Inhibiting the Two-Component System, Quorum Sensing, and Multidrug Efflux.

Stubborn biofilm infections pose serious threats to human health due to the persistence, recurrence, and dramatically magnified antibiotic resistance. Photodynamic therapy has emerged as a promising approach to combat biofilm. Nevertheless, how to inhibit the bacterial signal transduction system and the efflux pump to conquer biofilm recurrence and resistance remains a challenging and unaddressed issue. Herein, a boric acid-functionalized lipophilic cationic type I photosensitizer, ACR-DMP, is developed, which efficiently generates •OH to overcome the hypoxic microenvironment and photodynamically eradicates methicillin-resistant Staphylococcus aureus (MRSA) and biofilms. Furthermore, it not only alters membrane potential homeostasis and osmotic pressure balance due to its strong binding ability with plasma membrane but also inhibits quorum sensing and the two-component system, reduces virulence factors, and regulates the activity of the drug efflux pump attributed to the glycan-targeting ability, helping to prevent biofilm recurrence and conquer biofilm resistance. In vivo, ACR-DMP successfully obliterates MRSA biofilms attached to implanted medical catheters, alleviates inflammation, and promotes vascularization, thereby combating infections and accelerating wound healing. This work not only provides an efficient strategy to combat stubborn biofilm infections and bacterial multidrug resistance but also offers systematic guidance for the rational design of next-generation advanced antimicrobial materials.

Real-time, label-free detection and identification of bacteria through non-invasive optical imaging

Currently, traditional and newer molecular and mass spectrometry techniques of identifying bacteria from biological samples requires lengthy sample preparation, growth and labelling/staining assays. Thus, there is a pressing clinical need for an adjunct method that accurately identifies bacteria in real time. Here we report on the evaluation of confocal microscopy for the identification of clinically important and multi-drug resistant (MDR) bacteria in real time, using their intrinsic fluorescence features, i.e., emission spectra and fluorescence lifetime. The results demonstrate that difference in emission spectra and fluorescence lifetimes can be used as a fingerprint for identification of 12 bacterial species and MDR strains in real-time. Photostability or time-traces of bacteria demonstrated that these parameters could be used for tracking and recording without a need for labelling. Further, dilution experiments demonstrated that using intrinsic fluorescence S. aureus, Klebsiella pneumoniae and Escherichia coli bacteria can be detected and identified at clinically relevant concentrations as low as 2 × 102 CFU/mL. This non-invasive, non-labelling optical methodology may serve as the basis for development of a device that would quickly and accurately identify bacteria in biological samples. Thus, this intrinsic fluorescence technique would provide clinicians information, within minutes from sampling, to base accurate and specific treatments for patients.

Green Synthesis of Silver Nanoparticles Using Jacobaea maritima and the Evaluation of Their Antibacterial and Anticancer Activities.

Much attention has been gained on green silver nanoparticles (green-AgNPs) in the medical field due to their remarkable effects against multi-drug resistant (MDR) microorganisms and targeted cancer treatment. In the current study, we demonstrated a simple and environment-friendly (i.e., green) AgNP synthesis utilizing Jacobaea maritima aqueous leaf extract. This leaf is well-known for its medicinal properties and acts as a reducing and stabilizing agent. Nanoparticle preparation with the desired size and shape was controlled by distinct parameters; for instance, temperature, extract concentration of salt, and pH. The characterization of biosynthesized AgNPs was performed by the UV-spectroscopy technique, dynamic light scattering, scanning electron microscopy, X-ray diffraction, and Fourier-transform infrared. The successful formation of AgNPs was confirmed by a surface plasmon resonance at 422 nm using UV-visible spectroscopy and color change observation with a particle size of 37± 10 nm and a zeta potential of -10.9 ± 2.3 mV. SEM further confirmed the spherical size and shape of AgNPs with a size varying from 28 to 52 nm. Antibacterial activity of the AgNPs was confirmed against all Gram-negative and Gram-positive bacterial reference and MDR strains that were used in different inhibitory rates, and the highest effect was on the E-coli reference strain (MIC = 25 μg/mL). The anticancer study of AgNPs exhibited an IC50 of 1.37 μg/mL and 1.98 μg/mL against MCF-7 (breast cancer cells) and A549 (lung cancer cells), respectively. Therefore, this green synthesis of AgNPs could have a potential clinical application, and further in vivo study is required to assess their safety and efficacy.