Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Structural insight into the binding mode of cefotaxime and meropenem to TEM-1, SHV-1, KPC-2, and Amp-C type beta-lactamases.

Antimicrobial resistance is an emerging threat to public health around the world. The study employs computational and biophysical methods to investigate the properties of cefotaxime and meropenem's binding to various beta-lactamases like TEM-1, SHV-1, KPC-2, and Amp-C. The enzyme kinetics of purified proteins revealed an increase in Michaelis constant (Km) value in the presence of meropenem and cefotaxime, indicating a decrease in enzyme affinity for nitrocefin. Proteins interact with meropenem/cefotaxime, causing quenching through complex formation. All proteins have one binding site, and binding constant (Kb) values are 104, indicating strong interaction. The study found that meropenem and cefotaxime had high fitness scores for Amp-C, KPC-2,TEM-1 and SHV-1, with binding energy ranging from -7.4 to -7.8, and hydrogen bonds between them. Molecular Dynamic simulation of protein-ligand complexes revealed cefotaxime-binding proteins have slightly lower Root Mean Square Deviation(RMSD) than meropenem-binding proteins, indicating stable association antibiotics with these proteins.

Inhibitors against New Delhi metallo-betalactamase-1 (NDM-1) and its variants endemic in Indian settings along with the laboratory functional gain mutant of NDM-1.

PURPOSE: The emergence of NDM-1 producing bacteria has become common in both hospital and community settings, but no inhibitor has yet been available for clinical treatment. Hence, demanding the urgent need of NDM-1 inhibitors, we initiated to screen broad spectrum inhibitors against NDM natural variants and laboratory mutant. METHODS: We used docking and molecular dynamics simulations, in silico pharmacokinetic investigations, and density functional theory calculation to characterize molecules. Furthermore, an in vitro study, including MIC, kinetics, and fluorescence study were carried out to confirm the efficacies of the selected compounds. RESULTS: According to the findings of the computational studies, three compounds were effective against NDM variants. Fourfold reduction in MIC of imipenem and meropenem was observed when combined with inhibitors (D2573, D2148, and D63) against blaNDM-1, blaNDM-4, blaNDM-6, and blaNDM-1Q123A, while twofold reduction in MIC of imipenem and meropenem was observed against blaNDM-5 and blaNDM-7. Similarly in the presence of inhibitors (D2573, D2148, and D63) the efficiency of nitrocefin hydrolysis by NDM-4, NDM-6, and Q123A decreases to much more extent as compared to NDM-5 and NDM-7. These results showed that the efficacy of these broad spectrum inhibitors decreases with increasing resistance of NDM variants. CONCLUSION: This is the first time inhibitors were tested against different NDM natural variants which are endemic in Indian settings. Moreover, a functional gain laboratory mutant was also checked for their efficacies. We may propose these molecules for the pre-clinical trial to further translate.

Bacterial abundance and antimicrobial resistance patterns of uropathogens among pregnant women with asymptomatic bacteriuria: Association with glycemic status.

Objectives: Antimicrobial resistance (AMR), a growing global menace, poses a significant threat to maternal and fetal health. Gestational diabetes mellitus (GDM) causes double trouble in pregnancy, increasing the risk of a variety of infectious morbidities while also raising the possible association with AMR. Asymptomatic bacteriuria (ASB) is a common problem in pregnancy, but little research has been done to date explicitly examining the relationship between GDM and ASB and yielded conflicting results. Even fewer studies have specifically examined the relationship between GDM and AMR in women with ASB. Retrieving the most recent information on the disease burden, the range of causative pathogens, their patterns of AMR, and associated risk factors in pregnant women is crucial to stop the exponential rise in AMR in pregnancy and improve maternal and neonatal outcomes of infectious morbidities. Hence, this study was planned to investigate the association between glycemic status and the contemporary bacterial profile, antimicrobial resistance(AMR), and associated variables among pregnant women with ASB. Study design: This prospective, hospital-based, cross-sectional study was conducted among 320 pregnant women; divided into two groups, GDM and non-GDM. Data regarding sociodemographic and clinical characteristics were collected using a structured questionnaire. Clean-catch midstream urine samples were investigated for the presence of significant bacterial uropathogens and their AMR pattern was determined using recommended culture methods. Results: We found ASB in 46.25% of study participants with significantly higher occurrence in the GDM group. Dominant isolates were Escherichia coli followed by Klebsiella pneumoniae. AMR was noted in 51.35% and multidrug resistance(MDR) in 23.65% of isolates. Overall AMR, MDR and higher degrees of AMR were higher among uropathogens isolated from the GDM group as compared to the non GDM group, although the difference was not statistically significant. Conclusion: The high occurrence of ASB in pregnancy along with substantially high AMR in this study suggests the need for effective infection control and stewardship programmes. By defining the association of ASB and AMR with hyperglycemia, our study calls for the exploitation of this potential association in halting the pandemic of AMR and in improving the management of infectious morbidities, thus in-turn alleviating their undesired maternal and infant outcomes.

Mechanistic Approach of Effective Combination of Antibiotics Against Clinical Bacterial Strains Having New Delhi Metallo-Beta-Lactamase Variants and Functional Gain Laboratory Mutant.

Antimicrobial resistance has emerged as a serious issue for physicians and health-care workers treating infections that could lead to the next pandemic. One of the key resistance mechanisms is beta-lactamases. Although several beta-lactamase inhibitors in combination with antibiotics have been created and are being utilized in clinical settings, resistance to these formulations has also been evolving in the bacterial population due to their distinct targets. In this study we used effective combination of antibiotic as an approach to inhibit multidrug resistance bacteria. We used four combinations and checked its efficacy against NDM (New Delhi Metallo-beta-lactamase) variants and functional gain laboratory mutant by employing FICI, enzyme kinetics, fluorescence and computational biology approaches (Docking and Molecular Dynamics Simulation). FICI values of all the combinations were either less than 0.5 or equal to 0.5. Binding features acquired by spectroscopic techniques showed important interaction and complex formation between drugs and enzymes with decreased ksv and kq values. In steady-state kinetics, a reduction in hydrolytic efficiency of enzymes was shown by cooperative binding behaviour when they were treated with different drugs. We have also tested functional gain laboratory mutant developed in our lab, keeping in view that if in future upcoming variants of this kind be emerged then these mutants could also be subsided by combinational therapy. This study identifies three other combinations better than fluoroquinolones effective against NDM variants and laboratory mutant.

Repurposing FDA approved drug molecules against A B C classes of ß-lactamases: a computational biology and molecular dynamics simulations study.

ß-lactamase are the main resistance factor for ß-lactam antibiotics in Gram-negative bacteria. Since ß-lactam antibiotics are being utilised as an antimicrobial agents extensively for the past 70 years, a large number of ß-lactam-inactivating ß-lactamases have been produced by bacteria. Here, we employed a structure-based drug discovery approach to identify and assess the efficacy of a potential medication that might block the ß-lactamases which hydrolyse antibiotics. The FDA-approved medications were subjected to virtual screening, molecular docking, molecular dynamics simulations, density functional theory, and covalent docking against the ß-lactamases. We identified diosmin, hidrosmin, monoxuritin and solasulfone as ß-lactamase inhibitors which are authorised for therapeutic use in humans. These medications interact in a remarkable variety of non-covalent ways with the conserved residues in the substrate-binding pocket of the ß-lactamases. Diosmin has been identified as an inhibitor that binds covalently to the NDM-1 a class B metallo-betalactamase. After experimental validation and clinical demonstration, this study offers adequate evidence for the therapeutic use of these drugs for controlling multidrug resistance.Communicated by Ramaswamy H. Sarma.

Studies on the interaction of 2,4-dibromophenol with human hemoglobin using multi-spectroscopic, molecular docking and molecular dynamics techniques.

2,4-Dibromophenol (DBP) has several industrial applications, including as a wood preservative and flame retardant. This study investigated the interaction between DBP and human hemoglobin (Hb) using spectroscopic, molecular docking and molecular dynamic techniques. The UV-visible spectra showed ground-state complex formation between DBP and Hb. Fluorescence studies revealed that DBP binding caused significant quenching of Hb fluorescence by the static quenching mechanism. The binding of DBP to Hb is a spontaneous process that involves van der Waals forces and hydrogen bonds. There is one DBP binding site on each Hb molecule that is located at the α1ß2 interface of Hb. DBP binding did not alter the microenvironment of tyrosine and tryptophan residues in Hb. Circular dichroism studies revealed that DBP increased the α-helical content of Hb. The intrinsic esterase activity of Hb was inhibited by DBP in a concentration-dependent manner. Molecular docking showed that DBP binds to Hb via hydrogen bonds, hydrophobic, van der Waals and π-π interactions. Molecular dynamics simulation confirmed that the Hb-DBP complex is stable. Overall, the results of this study clearly show that DBP induces structural changes and interferes with the function of Hb. This can have important implications for human health.Communicated by Ramaswamy H. Sarma.

Updates on the Virulence Factors Produced by Multidrug-Resistant Enterobacterales and Strategies to Control Their Infections.

The Enterobacterales order is a massive group of Gram-negative bacteria comprised of pathogenic and nonpathogenic members, including beneficial commensal gut microbiota. The pathogenic members produce several pathogenic or virulence factors that enhance their pathogenic properties and increase the severity of the infection. The members of Enterobacterales can also develop resistance against the common antimicrobial agents, a phenomenon called antimicrobial resistance (AMR). Many pathogenic Enterobacterales members are known to possess antimicrobial resistance. This review discusses the virulence factors, pathogenicity, and infections caused by multidrug-resistant Enterobacterales, especially E. coli and some other bacterial species sharing similarities with the Enterobacterales members. We also discuss both conventional and modern approaches used to combat the infections caused by them. Understanding the virulence factors produced by the pathogenic bacteria will help develop novel strategies and methods to treat infections caused by them.

Current Update on New Delhi Metallo-ß-lactamase (NDM) Variants: New Challenges in the Journey of Evolution.

New Delhi Metallo-ß-lactamase is an enzyme produced by gram-negative bacteria which has become one of the global concerns for physicians to treating the infection. These Metallo- ß-lactamase are capable of catalyzing the hydrolysis of almost all ß-lactam antibiotics, endangering infection treatment. Substitution of single or multiple amino acids results in new NDM variants. Forty NDM variants have been identified in different bacterial strains across the globe. In this review, we focused on the structural insight of all NDM variants including the type of amino acid residues and their position of substitution, country of origin, and type of bacteria carrying these resistant markers. We also discussed the carbapenemase activity and stability of enzymes that helps to design potent inhibitors to combat drug-resistant infections.

Efficacy of Novel Combinations of Antibiotics against Multidrug-Resistant-New Delhi Metallo-Beta-Lactamase-Producing Strains of Enterobacterales.

The emergence of multidrug-resistance (MDR)-New Delhi metallo-beta-lactamase (NDM)-producing microorganisms-has become a serious concern for treating such infections. Therefore, we investigated the effective antimicrobial combinations against multidrug-resistant New Delhi metallo-beta-lactamase-producing strains of Enterobacterales. The tests were carried out using the 2D(two-dimensional) checkerboard method. Of 7 antimicrobials, i.e., doripenem (DRP), streptomycin (STR), cefoxitin (FOX), imipenem (IPM), cefotaxime (CTX), meropenem (MER), and gentamicin (GEN), 19 different combinations were used, and out of them, three combinations showed synergistic effects against 31 highly drug-resistant strains carrying blaNDM and other associated resistance markers. Changes in the minimum inhibitory concentration (MIC) values were interpreted using the test fractional inhibitory concentration index (FIC Index). The FIC Index values of these combinations were found in the range of 0.1562 to 0.5, which shows synergy, whereas no synergism was observed in the remaining antimicrobial combinations. We conclude that these antibiotic combinations can be analyzed in in vivo and pharmacological studies to establish an effective therapeutic approach.

It's all relative: analyzing microbiome compositions, its significance, pathogenesis and microbiota derived biofilms: Challenges and opportunities for disease intervention.

Concept of microorganisms has largely been perceived from their pathogenic view point. Nevertheless, it is being gradually revisited in terms of its significance to human health and now appears to be the most dominant force that shapes the immune system of the human body and also determines an individual's predisposition to diseases. Human inhabits bacterial diversity (which is predominant among all microbial communities in human body) occupying 0.3% of body mass, known as microbiota. On birth, a part of microbiota that child obtains is essentially a mother's legacy. So, the review was initiated with this critical topic of microbiotal inheritance. Since, each body site has distinct physiological specifications; therefore, they contain discrete microbiome composition that has been separately discussed along with dysbiosis-induced pathologies originating in different body organs. Factors affecting microbiome composition and may cause dysbiosis like antibiotics, delivery, feeding method etc. as well as the strategies that immune system adopts to prevent dysbiosis have been highlighted. We also tried to bring into attention the topic of dysbiosis induced biofilms, that enables cohort to survive stresses, evolve, disseminate and infection resurgence that is still in dormancy. Eventually, we put spotlight on microbiome significance in medical therapeutics. We didn't merely confine article to gut microbiota, that is being studied more extensively. Numerous community forms at diverse body sites are inter-related, and being exposed to awfully variable perturbations appear to be challenging to evaluate perturbation risks holistically. All aspects have been elaborately discussed to achieve a global depiction of human microbiota in order to meet urgent necessity for protocol standardisation. Demonstrates that environmental challenges (antibiotic use, alterations in diet, stress, smoking etc.) might cause dysbiosis i.e. transition of healthy microbiome composition to the one in which pathogenic microorganisms become more abundant, and eventually results in an infected state.

Domestic LED bulb induced photodynamic effect of Toluidine Blue O-embedded silicone catheters against urinary tract infection.

BACKGROUND: Novel combination of Toluidine Blue O (TBO) embedded silicone catheter with domestic/household LED bulb has a potential in clinical infection such as prevention of multi drug resistant catheter-associated urinary tract infections (CAUTIs) through photodynamic therapy. MATERIAL AND METHODS: Preliminarily, TBO was entrapped into silicone catheter by swell-encapsulation-shrink method. Further, in vitro study was carried out to check the antimicrobial photodynamic efficacy of TBO with domestic/household LED light. Antibiofilm activity was evaluated by scanning electron microscopy. RESULTS: The results showed that these modified TBO embedded silicone catheters showed significant antimicrobial and antibiofilm activity against vancomycin resistant Staphylococcus aureus VRSA. Small piece (1 cm) of TBO-embedded silicone catheter (700 µM) showed 6 log10 reduction in the viable count when exposed for only 5 min of domestic/household LED bulb, while 1 cm piece of 500 µM and 700 µM concentration of TBO-embedded catheter eradicated all bacterial load when exposed to 15 min of light. Segment of medical grade TBO-embedded silicone catheters were used to carry out investigation of reactive oxygen species generation mainly singlet oxygen that contributes to type II phototoxicity. CONCLUSION: These modified catheter provides cost effective, easy to manage and less time consuming therapy to eliminate CAUTIs.

Farm to table: colistin resistance hitchhiking through food.

Colistin is a high priority, last-resort antibiotic recklessly used in livestock and poultry farms. It is used as an antibiotic for treating multi-drug resistant Gram-negative bacterial infections as well as a growth promoter in poultry and animal farms. The sub-therapeutic doses of colistin exert a selection pressure on bacteria leading to the emergence of colistin resistance in the environment. Colistin resistance gene, mcr are mostly plasmid-mediated, amplifying the horizontal gene transfer. Food products such as chicken, meat, pork etc. disseminate colistin resistance to humans through zoonotic transfer. The antimicrobial residues used in livestock and poultry often leaches to soil and water through faeces. This review highlights the recent status of colistin use in food-producing animals, its association with colistin resistance adversely affecting public health. The underlying mechanism of colistin resistance has been explored. The prohibition of over-the-counter colistin sales and as growth promoters for animals and broilers has exhibited effective stewardship of colistin resistance in several countries.

Metagenomic Insight into Microbiome and Antibiotic Resistance Genes of High Clinical Concern in Urban and Rural Hospital Wastewater of Northern India Origin: a Major Reservoir of Antimicrobial Resistance.

India is one of the largest consumers and producers of antibiotics and a hot spot for the emergence and proliferation of antimicrobial resistance genes (ARGs). Indian hospital wastewater (HWW) accumulates ARGs from source hospitals and often merges with urban wastewater, with the potential for environmental and human contamination. Despite its putative clinical importance, there is a lack of high-resolution resistome profiling of Indian hospital wastewater, with most studies either relying on conventional PCR-biased techniques or being limited to one city. In this study, we comprehensively analyzed antibiotic resistomes of wastewater from six Indian hospitals distributed in rural and urban areas of northern India through shotgun metagenomics. Our study revealed the predominance of ARGs against aminoglycoside, macrolide, carbapenem, trimethoprim, and sulfonamide antibiotics in all the samples through both read-based analysis and assembly-based analysis. We detected the mobile colistin resistance gene mcr-5.1 for the first time in Indian hospital sewage. blaNDM-1 was present in 4 out of 6 samples and was carried by Pseudomonas aeruginosa in HWW-2, Klebsiella pneumoniae in HWW-4 and HWW-6, and Acinetobacter baumanii in HWW-5. Most ARGs were plasmid-mediated and hosted by Proteobacteria. We identified virulence factors and transposable elements flanking the ARGs, highlighting the role of horizontal gene transmission of ARGs. IMPORTANCE There is a paucity of research on detailed antibiotic resistome and microbiome diversity of Indian hospital wastewater. This study reports the predominance of clinically concerning ARGs such as the beta-lactamases blaNDM and blaOXA and the colistin resistance gene mcr and their association with the microbiome in six different Indian hospital wastewaters of both urban and rural origin. The abundance of plasmid-mediated ARGs and virulence factors calls for urgent AMR crisis management. The lack of proper wastewater management strategies meeting international standards and open drainage systems further complicates the problem of containing the ARGs at these hospitals. This metagenomic study presents the current AMR profile propagating in hospital settings in India and can be used as a reference for future surveillance and risk management of ARGs in Indian hospitals.

mltG gene deletion mitigated virulence potential of Streptococcus mutans: An in-vitro, ex-situ and in-vivo study.

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall, which is essential for cell integrity and intrinsic biogenesis pathways; hence, the cell wall is a potential target for several antibiotics. Among several lytic transglycosylases (LTs), the mltG gene plays a crucial role in the synthesis of peripheral PG. It localises the re-modelled PGs for septum formation and cleavage across the bacterial cell wall during daughter cells separation. However, the role of mltG gene in bacterial virulence, particularly in Gram-positive bacteria during dentine biofilm and caries development, has remained unexplored. Hence, we exploited Gram-positive Streptococcus mutans cells for the very first time to construct a mltG knock-out bacterial strain, e.g., ΔmltG S. mutans. Systematic comparative investigations revealed that doubling time (Td), survival, enzymatic efficiencies, pH tolerance, bio-synthesise of lipid, proteins and DNA, biofilm formation and dentine lesions were significantly (p < 0.001) compromised in case of ΔmltG S. mutans than wild type strain. The qRT-PCR based gene expression profiling revealed that transcriptional expression of critically important genes involved in biofilm, metabolism, and stress response were dysregulated in the mutant. Besides, an incredible reduction in dentine caries development was found in the molar teeth of Wistar rats and also in human extracted teeth. Concisely, these trends obtained evidently advocated the fact that the deletion of mltG gene can be a potential target to impair the S. mutans virulence through severe growth retardation, thereby reducing the virulence potential of S. mutans.

Artificial intelligence as a smart approach to develop antimicrobial drug molecules: A paradigm to combat drug-resistant infections.

Antimicrobial resistance (AMR) is a silent pandemic with the third highest global mortality. The antibiotic development pipeline is scarce even though AMR has escalated uncontrollably. Artificial intelligence (AI) is a revolutionary approach, accelerating drug discovery because of its fast pace, cost efficiency, lower labor requirements, and fewer chances of failure. AI has been used to discover several beta-lactamase inhibitors and antibiotic alternatives from antimicrobial peptides (AMPs), nonribosomal peptides, bacteriocins, and marine natural products. The significant recent increase in the use of AI platforms by pharmaceutical companies could result in the discovery of efficient antibiotic alternatives with lower chances of resistance generation.

Orally fed EGCG coronate food released TiO2 and enhanced penetrability into body organs via gut.

Owing to unique nano-scale properties, TiO2-NPs (T-NPs) are employed as food-quality enhancers in >900 processed food products. Whereas, epigallocatechin-3-gallate (EGCG), a green tea polyphenol is consumed in traditional brewed tea, globally. Taken together, we aimed to investigate whether human gastric-acid digested T-NPs and complex tea catechins yield ionic species (Ti4+, Ti3+ etc.) and active EGCG forms to meet favourable conditions for in vivo bio-genesis of EGCG-coronated TiO2-NPs (ET-NPs) in human gut. Secondly, compared to bare-surface micro and nano-scale TiO2, i.e., T-MPs and T-NPs, respectively, how EGCG coronation on ET-NPs in the gut facilitates the modulation of intrinsic propensity of internalization of TiO2 species into bacteria, body-organs, and gut-microbiota (GM), and immune system. ET-NPs were synthesized in non-toxic aqueous solution at varied pH (3-10) and characterised by state-of-the-arts for crystallinity, surface-charge, EGCG-encapsulation, stability, size, composition and morphology. Besides, flow-cytometry (FCM), TEM, EDS, histopathology, RT-PCR, 16S-rRNA metagenomics and ELISA were also performed to assess the size and surface dependent activities of ET-NPs, T-NPs and T-MPs vis-a-vis planktonic bacteria, biofilm, GM bacterial communities and animal's organs. Electron-microscopic, NMR, FTIR, DLS, XRD and EDS confirmed the EGCG coronation, dispersity, size-stability of ET-NPs, crystallinity and elemental composition of ET-NPs-8 and T-NPs. Besides, FCM, RT-PCR, 16S-rRNA metagenomics, histopathology, SEM and EDS analyses exhibited that EGCG coronation in ET-NPs-8 enhanced the penetration into body organs (i.e., liver and kidney etc.) and metabolically active bacterial communities of GM.

Functional Amyloids in Pseudomonas aeruginosa Are Essential for the Proteome Modulation That Leads to Pathoadaptation in Pulmonary Niches.

Persistence and survival of Pseudomonas aeruginosa in chronic lung infections is closely linked to the biofilm lifestyle. One biofilm component, functional amyloid of P. aeruginosa (Fap), imparts structural adaptations for biofilms; however, the role of Fap in pathogenesis is still unclear. Conservation of the fap operon encoding Fap and P. aeruginosa being an opportunistic pathogen of lung infections prompted us to explore its role in lung infection. We found that Fap is essential for establishment of lung infection in rats, as its genetic exclusion led to mild focal infection with quick resolution. Moreover, without an underlying cystic fibrosis (CF) genetic disorder, overexpression of Fap reproduced the CF pathotype. The molecular basis of Fap-mediated pulmonary adaptation was explored through surface-associated proteomics in vitro. Differential proteomics positively associated Fap expression with activation of known proteins related to pulmonary pathoadaptation, attachment, and biofilm fitness. The aggregative bacterial phenotype in the pulmonary niche correlated with Fap-influenced activation of biofilm sustainability regulators and stress response regulators that favored persistence-mediated establishment of pulmonary infection. Fap overexpression upregulated proteins that are abundant in the proteome of P. aeruginosa in colonizing CF lungs. Planktonic lifestyle, defects in anaerobic pathway, and neutrophilic evasion were key factors in the absence of Fap that impaired establishment of infection. We concluded that Fap is essential for cellular equilibration to establish pulmonary infection. Amyloid-induced bacterial aggregation subverted the immune response, leading to chronic infection by collaterally damaging tissue and reinforcing bacterial persistence. IMPORTANCE Pseudomonas aeruginosa is inextricably linked with chronic lung infections. In this study, the well-conserved Fap operon was found to be essential for pathoadaptation in pulmonary infection in a rat lung model. Moreover, the presence of Fap increased pathogenesis and biofilm sustainability by modulating bacterial physiology. Hence, a pathoadaptive role of Fap in pulmonary infections can be exploited for clinical application by targeting amyloids. Furthermore, genetic conservation and extracellular exposure of Fap make it a commendable target for such interventions.

Chemically synthesised flavone and coumarin based isoxazole derivatives as broad spectrum inhibitors of serine ß-lactamases and metallo-ß-lactamases: a computational, biophysical and biochemical study.

The ß-lactam antibiotics are the most effective medicines for treating bacterial infections. Resistance to them, particularly through the production of ß-lactamases, which can hydrolyse all kinds of ß-lactams, poses a threat to their continued use. The synthesised flavone and coumarin based isoxazole derivatives have the potential to be used as broad-spectrum inhibitors of the mechanistically different serine-(SBL) and metallo-ß-lactamases (MBL). The synthesised compounds were discovered as potent ß-lactamase inhibitors using molecular docking and in silico pharmacokinetic analysis. We studied the binding of chemically synthesised inhibitors to clinically significant ß-lactamases of class A, B, and C using biophysical and biochemical approaches, and computational analyses. These molecules follow Lipinski's rule of five and have acceptable solubility, permeability, and oral bioavailability. These molecules were found to be non-toxic and non-carcinogenic. MIC results suggest that these molecules restore the antibiotic efficacy against class A, B, and C ß-lactamases. Kinetics data showed that these molecules reduce the catalytic efficiency of clinically relevant class A, B, and C ß-lactamases. Fluorescence study showed significant interaction between these flavone-/coumarin-based isoxazole derivatives and class A/B/ C ß-lactamases. This study showed promising effect of these new generation compounds as broad spectrum ß-lactamase inhibitors of both SBLs and MBLs.Communicated by Ramaswamy H. Sarma.