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.

Investigation of Peptidoglycan-Associated Lipoprotein of Acinetobacter baumannii and Its Interaction with Fibronectin To Find Its Therapeutic Potential.

Acinetobacter baumannii causes hospital-acquired infections and is responsible for high mortality and morbidity. The interaction of this bacterium with the host is critical in bacterial pathogenesis and infection. Here, we report the interaction of peptidoglycan-associated lipoprotein (PAL) of A. baumannii with host fibronectin (FN) to find its therapeutic potential. The proteome of A. baumannii was explored in the host-pathogen interaction database to filter out the PAL of the bacterial outer membrane that interacts with the host's FN protein. This interaction was confirmed experimentally using purified recombinant PAL and pure FN protein. To investigate the pleiotropic role of PAL protein, different biochemical assays using wild-type PAL and PAL mutants were performed. The result showed that PAL mediates bacterial pathogenesis, adherence, and invasion in host pulmonary epithelial cells and has a role in the biofilm formation, bacterial motility, and membrane integrity of bacteria. All of the results suggest that PAL's interaction with FN plays a vital role in host-cell interaction. In addition, the PAL protein also interacts with Toll-like receptor 2 and MARCO receptor, which suggests the role of PAL protein in innate immune responses. We have also investigated the therapeutic potential of this protein for vaccine and therapeutic design. Using reverse vaccinology, PAL's potential epitopes were filtered out that exhibit binding potential with host major histocompatibility complex class I (MHC-I), MHC-II, and B cells, suggesting that PAL protein is a potential vaccine target. The immune simulation showed that PAL protein could elevate innate and adaptive immune response with the generation of memory cells and would have subsequent potential to eliminate bacterial infection. Therefore, the present study highlights the interaction ability of a novel host-pathogen interacting partner (PAL-FN) and uncovers its therapeutic potential to combat infection caused by A. baumannii.

Therapeutic strategies against potential antibiofilm targets of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is the causative agent of various hospital-acquired infections. Biofilm formation is one of the various antimicrobial resistance (AMR) strategies and is associated with high mortality and morbidity. Hence, it is essential to review the potential antibiofilm targets in A. baumannii and come up with different strategies to combat these potential targets. This review covers different pathways involved in the regulation of biofilm formation in A. baumannii like quorum sensing (QS), cyclic-di-GMP signaling, two-component system (TCS), outer-membrane protein (ompA), and biofilm-associated protein (BAP). A newly discovered mechanism of electrical signaling-mediated biofilm formation and contact-dependent biofilm modulation has also been discussed. As biofilm formation and its maintenance in A. baumannii is facilitated by these potential targets, the detailed study of these targets and pathways can bring light to different therapeutic strategies such as anti-biofilm peptides, natural and synthetic molecule inhibitors, QS molecule degrading enzymes, and other strategies. These strategies may help in suppressing the lethality of biofilm-mediated infections. Targeting essential proteins/targets which are crucial for biofilm formation and regulation may render new therapeutic strategies that can aid in combating biofilm, thus reducing the recalcitrant infections and morbidity associated with the biofilm of A. baumannii.

Antipersister strategies against stress induced bacterial persistence.

The increase in antibiotic non-responsive bacteria is the leading concern in current research oriented to eliminate pathogens. Nowadays, the excess use of antibiotics without specifically understanding the potentiality of killing pathogens and bacterial survival patterns has helped bacteria emerge indefatigably. Bacteria use various mechanisms such as resistance, persistence, and tolerance to ensure survival. Among these, persistence is a mechanism by which bacteria reside in their dormant state, bypassing the effects of treatments, making it crucial for bacterial survival. Persistent bacterial cells arise from the normal bacterial population as a slow-growing subset of bacteria with no metabolic flux. This behavior renders it to survive for a longer duration and at higher concentrations of antibiotics. They are one of the underlying causes of recurrence of bacterial infections. The present article explains the detailed molecular mechanisms and strategies of bacterial persistence, including the toxin-antitoxin modules, DNA damage, the formation of inactive ribosomal complexes, (p)ppGpp network, antibiotic-induced persistence, which are triggered by drug-induced stress. The article also comprehensively covers the epigenetic memory of persistence in bacteria, and anti-persistent therapeutics like antimicrobial molecules, synthetic peptides, acyldepsipeptide antibiotics, and endolysin therapy to reduce persister cell formation and control their frequency. These strategies could be utilized in combating the pathogenic bacteria undergoing persistence.

Efflux pumps in multidrug-resistant Acinetobacter baumannii: Current status and challenges in the discovery of efflux pumps inhibitors.

Acinetobacter baumannii is an ESKAPE pathogen known to cause fatal nosocomial infections. With the surge of multidrug resistance (MDR) in the bacterial system, effective treatment measures have become very limited. The MDR in A. baumannii is contributed by various factors out of which efflux pumps have gained major attention due to their broad substrate specificity and wide distribution among bacterial species. The efflux pumps are involved in the MDR as well as contribute to other physiological processes in bacteria, therefore, it is critically important to inhibit efflux pumps in order to combat emerging resistance. The present review provides insight about the different efflux pump systems in A. baumannii and their role in multidrug resistance. A major focus has been put on the different strategies and alternate therapeutics to inhibit the efflux system. This includes use of different efflux pump inhibitors-natural, synthetic or combinatorial therapy. The use of phage therapy and nanoparticles for inhibiting efflux pumps have also been discussed here. Moreover, the present review provides the knowledge of barriers in development of efflux pump inhibitors (EPIs) and their approval for commercialization. Here, different prospectives have been discussed to improve the therapeutic development process and make it more compatible for clinical use.

Subtractive proteomic analysis of antigenic extracellular proteins and design a multi-epitope vaccine against Staphylococcus aureus.

Staphylococcus aureus is a versatile Gram's positive bacterium that can reside as an asymptomatic colonizer, which can cause a wide range of skin, soft-tissue, and nosocomial infections. A vaccine against multi-drug resistant S. aureus, therefore, is urgently needed. Subtractive proteomics and reverse vaccinology are newly emerging techniques to design multiepitope-based vaccines. The analysis of 7290 proteomes (sensitive and resistant strains), five potent nonhuman homologous vaccine targets [(UNIPORT ID Q2FZL3 (Staphopain B), Q2G2R8 (Staphopain A), Q2FWP0 (uncharacterized leukocidin-like protein 1), Q2G1S6 (uncharacterized protein), and Q2FWV3 (Staphylokinase, putative)] were selected. These proteins were absent in the gut microbiome, which further enhances the significance of these proteins in vaccine design. These five virulence-associated proteins mainly have a role in the invasion mechanism in the host phagocyte cells. MHC I, MHC II, and B cell epitopes were identified in these five proteins. Finalized epitopes were examined by different online servers to screen suitable epitopes for multi-epitope based vaccine design. Shortlisted antigenic and nonallergenic associated epitopes were joined with linkers to design 30 variants (VSA1-VSA30) of multi-epitope vaccine conjugates. The antigenicity and allergenicity of all the 30 vaccine constructs were identified, and VSA30 was found to have the highest antigenicity and lowest allergenicity, and hence was selected for further study. Accordingly, VSA30 was docked with different HLA allelic variants, and the best-docked complex (VSA30-1SYS) was further analyzed by molecular dynamics simulation (MDS). The MDS result confirms the interaction of VSA30 with MHC (HLA-allelic variant). Thus, the final vaccine construct was in silico cloned in the pET28a vector for suitable expression in a heterologous system. Therefore, the designed vaccine construct VSA-30 can be developed as an appropriate vaccine to target S. aureus infection. VSA-30 still needs experimental validation to assure the antigenic and immunogenic properties.

Comparative mechanism based study on disinfectants against multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii has emerged as a hospital-acquired pathogen and has spread in the hospital settings, leading to enhanced nosocomial outbreaks associated with high death rates. Therefore, the aim of the current study is to determine the effective concentration of disinfectants like sodium hypochlorite, hydrogen peroxide, and chlorine dioxide, against multidrug-resistant (MDR) strains of A. baumannii. In this study, we have investigated the effect of disinfectants on different MDR strains i.e. RS307, RS6694, RS7434, RS10953, RS122, and sensitive strain ATCC-19606 of A. baumannii, via differential growth curves analysis, disc diffusion assay, estimation of reactive oxygen species (ROS), lipid peroxidation, and protein carbonylation. All the results collectively showed that 1% sodium hypochlorite (P value < 0.0027), 2.5% hydrogen peroxide (P value = 0.0032), and 10 mM (P value = 0.017) chlorine dioxide significantly inhibit the growth of MDR strains of A. baumannii. A significant increase in the ROS generations, altered lipid peroxidation, and a decrease in protein carbonylation was also observed after treatment with disinfectants, which confirmed its ROS-dependent damage mechanism. These disinfectants also enhance the membrane leakage of reducing sugar, protein, and DNA. The current study highlights and recommends the use of 2.5% hydrogen peroxide to control the MDR strains of A. baumannii in the hospital setup. Therefore, the present results will help in selecting concentrations of different disinfectants for regular use in hospital setups to eradicate the multidrug-resistant A. baumannii from the hospital setup.

Effect of secondary metabolite of Actinidia deliciosa on the biofilm and extra-cellular matrix components of Acinetobacter baumannii.

Acinetobacter baumannii, opportunistic nosocomial pathogen, increases gradually in the clinical setup. The high level of resistance mechanisms acquired by these bacteria makes their eradication difficult and biofilm formation is one of them. Biofilm comprises of closely packed bacterial population crowded together by extra-cellular matrix (ECM). ECM contains bacterial secreted polymers such as exopolysaccharides (EPS), proteins and extracellular-DNA (e-DNA) and rarely amyloidogenic proteins. Biofilm offers protection of underlying bacterial population against chemotherapeutic agents and host immune system. Therefore, present efforts are focused to find a novel therapeutic that targets biofilm-associated infections. Plants are used as a natural therapeutic for numerous ailments. In order to find an alternative of the available antibacterial drugs, we have focused on the natural herbal active compounds. In this study, we have extracted active compounds from various medicinal plants and screened its anti-biofilm activity against carbapenem resistant strain of A. baumannii. Results showed that polar extract of kiwi (Actinidia deliciosa) and clove (Syzygium aromaticum) exhibit effective anti-biofilm activity. These two plants were also used for their phytochemical screening and TLC profiling to find out the constituting secondary metabolites. Actinidia deliciosa extract contains an alkaloid (sanquinarine) as well as a flavonoid (hydroxyflavone). Anti-biofilm effect of this extract on the ECM of A. baumannii showed that it reduces EPS, protein and eDNA contents in the ECM. Proteins of ECM have also shown to form amyloid like structure, which was evident from its interaction with the Congo Red. CFU counting after Actinidia deliciosa extract treatment also supported the results. Therefore, it can be concluded that polar extract of A. deliciosa can be used to find suitable alternative therapeutic to control biofilm formation by carbapenem resistant strain of Acinetobacter baumannii.

Interaction and simulation studies suggest the possible molecular targets of intrinsically disordered amyloidogenic antimicrobial peptides in Acinetobacter baumannii.

Acinetobacter baumannii is one of the causing agents of nosocomial infections. A wide range of antibiotics fails to work against these pathogens. Hence, there is an urgent requirement to develop other therapeutics to solve this problem. Antimicrobial peptides (AMPs) are a diverse group of naturally occurring peptides that have the ability to kill diverse groups of microorganisms. The major challenge of using AMPs as therapeutics is their unstable nature and the fact that most of their molecular targets are still unknown. In this study, we have selected intrinsically disordered and amyloidogenic AMPs, showing activity against A. baumannii, that is, Bactenecin, Cath BF, Citropin 1.1, DP7, NA-CATH, Tachyplesin, and WAM-1. To identify the probable target of these AMPs in A. baumannii, calculation of docking score, binding energy, dissociation constant, and molecular dynamics analysis was performed with selected seventeen possible molecular targets. The result showed that the most probable molecular targets of most of the intrinsically disordered amyloidogenic AMPs were UDP-N-acetylenol-pyruvoyl-glucosamine reductase (MurB), followed by 33-36 kDa outer membrane protein (Omp 33-36), UDP-N-acetylmuramoyl-l-alanyl-d-glutamate-2,6-diaminopimelate ligase (MurE), and porin Subfamily Protein (PorinSubF). Further, molecular dynamics analysis concluded that the target of antimicrobial peptide Bactenecin is MurB of A. baumannii, and identified other molecular targets of selected AMPs. Additionally, the oligomerization capacity of the selected AMPs was also investigated, and it was shown that the selected AMPs form oligomeric states, and interact with their molecular targets in that state. Experimental validation using purified AMPs and molecular targets needs to be done to confirm the interaction.Communicated by Ramaswamy H. Sarma.

Assessment of potassium ion channel during electric signalling in biofilm formation of Acinetobacter baumannii for finding antibiofilm molecule.

Acinetobacter baumannii is an opportunistic ESKAPE pathogen which causes nosocomial infections and can produce biofilms that act as resistant determinants. The role of quorum sensing (chemical signaling) in biofilm establishment has already been studied extensively, but the existence of electrochemical signaling during biofilm formation by A. baumannii has not yet been investigated. The current study evaluated the presence of electrical signaling, types of ion channels involved, and their role in biofilm formation using spectroscopic and microbiological methods. The findings suggest that the potassium ion channel has a significant role in the electrical signaling during the biofilm formation by A. baumannii. Further, in-silico screening, molecular mechanics, and molecular dynamic simulation studies identify a potential lead, ZINC12496555(a specific inhibitor), which targets the potassium ion channel protein of A. baumannii. Mutational analysis of the interacting residues showed alterations in the unfolding rate of this protein after the selected mutation, which shows its role in the stability of this protein. It was also observed that identified lead has high antibiofilm activity, no human off-targets, and non-cytotoxicity to cell lines. Thus, identified lead against the potassium channel of A baumannii may be used as an effective therapeutic for the treatment of A. baumannii infections after further experimental validation.

Interaction of RecA mediated SOS response with bacterial persistence, biofilm formation, and host response.

Antibiotics have a primary mode of actions, and most of them have a common secondary mode of action via reactive species (ROS and RNS) mediated DNA damage. Bacteria have been able to tolerate this DNA damage by SOS (Save-Our-Soul) response. RecA is the universal essential key protein of the DNA damage mediated SOS repair in various bacteria including ESKAPE pathogens. In addition, antibiotics also triggers activation of various other bacterial mechanisms such as biofilm formation, host dependent responses, persister subpopulation formation. These supporting the survival of bacteria in unfriendly natural conditions i.e. antibiotic presence. This review highlights the detailed mechanism of RecA mediated SOS response as well as role of RecA-LexA interaction in SOS response. The review also focuses on inter-connection between DNA damage repair pathway (like SOS response) with other survival mechanisms of bacteria such as host mediated RecA induction, persister-SOS interplay, and biofilm-SOS interplay. This understanding of inter-connection of SOS response with different other survival mechanisms will prove beneficial in targeting the SOS response for prevention and development of therapeutics against recalcitrant bacterial infections. The review also covers the significance of RecA as a promising potent therapeutic target for hindering bacterial SOS response in prevailing successful treatments of bacterial infections and enhancing the conventional antibiotic efficiency.

Potentiate the activity of current antibiotics by naringin dihydrochalcone targeting the AdeABC efflux pump of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is an ESKAPE pathogen responsible for severe nosocomial infections. Among all the mechanisms contributing to multidrug resistance, efflux pumps have gained significant attention due to their widespread distribution among bacterial species and broad substrate specificity. This study has investigated the diverse roles of efflux pumps present in carbapenem-resistant A. baumannii (CRAB) and screen an efflux pump inhibitor. The result showed the presence of AdeABC, AdeFGH, AdeIJK, and AbeM efflux pumps in CRAB, and experimental studies using gene mutants demonstrated the significant role of AdeABC in carbapenem resistance, biofilm formation, surface motility, pathogenesis, bacterial adherence, and invasion to the host cells. The structure-based ligand screening, molecular mechanics, molecular dynamics simulation, and experimental validation using efflux pump mutants and antibiotic accumulation assay identified naringin dihydrochalcone (NDC) as the lead against AdeB. This lead was selected as a capping agent for silver nanoparticles. The NDC-capped silver nanoparticles (NDC-AgNPs) were characterized by UV-spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and scanning electron microscopy (SEM). The investigated molecular mechanism showed that the NDC-AgNPs possessed multiple mechanisms of action. In addition to efflux inhibitory activity, it also generates reactive oxygen and nitrogen species as well as causes change in the electrochemical gradient in CRAB. The proton gradient is important for the function of AdeABC; hence altering the electrochemical gradient also disrupts its efflux activity. Moreover, A. baumannii did not develop any resistance against NDC-AgNPs till several generations which were investigated. The NDC-AgNPs were also found to be effective against carbapenem-resistant clinical isolates of A. baumannii. Therefore, the present study provided an insight into the efflux pump mediated carbapenem resistance and possible inhibitor NDC-AgNPs to combat AdeABC efflux pump mediated resistance.

Immunoinformatic approach to design a multiepitope vaccine targeting non-mutational hotspot regions of structural and non-structural proteins of the SARS CoV2.

BACKGROUND: The rapid Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV2) outbreak caused severe pandemic infection worldwide. The high mortality and morbidity rate of SARS CoV2 is due to the unavailability of vaccination and mutation in this virus. The present article aims to design a potential vaccine construct VTC3 targeting the non-mutational region of structural and non-structural proteins of SARS CoV2. METHODS: In this study, vaccines were designed using subtractive proteomics and reverse vaccinology. To target the virus adhesion and evasion, 10 different structural and non-structural proteins have been selected. Shortlisted proteins have been screened for B cell, T cell and IFN gamma interacting epitopes. 3D structure of vaccine construct was modeled and evaluated for its physicochemical properties, immunogenicity, allergenicity, toxicity and antigenicity. The finalized construct was implemented for docking and molecular dynamics simulation (MDS) with different toll-like receptors (TLRs) and human leukocyte antigen (HLA). The binding energy and dissociation construct of the vaccine with HLA and TLR was also calculated. Mutational sensitivity profiling of the designed vaccine was performed, and mutations were reconfirmed from the experimental database. Antibody production, clonal selection, antigen processing, immune response and memory generation in host cells after injection of the vaccine was also monitored using immune simulation. RESULTS: Subtractive proteomics identified seven (structural and non-structural) proteins of this virus that have a role in cell adhesion and infection. The different epitopes were predicted, and only extracellular epitopes were selected that do not have similarity and cross-reactivity with the host cell. Finalized epitopes of all proteins with minimum allergenicity and toxicity were joined using linkers to designed different vaccine constructs. Docking different constructs with different TLRs and HLA demonstrated a stable and reliable binding affinity of VTC3 with the TLRs and HLAs. MDS analysis further confirms the interaction of VTC3 with HLA and TLR1/2 complex. The VTC3 has a favorable binding affinity and dissociation constant with HLA and TLR. The VTC3 does not have similarities with the human microbiome, and most of the interacting residues of VTC3 do not have mutations. The immune simulation result showed that VTC3 induces a strong immune response. The present study designs a multiepitope vaccine targeting the non-mutational region of structural and non-structural proteins of the SARS CoV2 using an immunoinformatic approach, which needs to be experimentally validated.

Design of novel hybrid secondary metabolite targets to diguanylate cyclase of Acinetobacter baumannii.

Biofilm formation in bacteria is a resistance determinant and is positively regulated by cyclic diguanylate signaling. This signaling is a near universal signaling, and c-di-GMP produced by diguanylate cyclase (DGC) in this signaling is involved in different bacterial behaviors. The present study aims to find a plant-based novel hybrid therapeutic agent that can target the DGC of Acinetobacter baumannii. In this study, we have tried to design a hybrid molecule from the anti-biofilm plant secondary metabolites and screened its binding with the DGC of A. baumannii. The modeled and validated DGC was used to identify the active site and docking grid. Designed hybrid compounds were analysed for their interaction with the active site residues of DGC of A. baumannii. Further, the binding free energies of the docked complexes obtained from the Generalized Born model and Solvent Accessibility (MMGBSA) were analysed. The results indicated that VR-QEg-180 has a predicted high binding affinity with enzyme DGC as compared to other hybrids, parent secondary metabolites and positive control. Molecular dynamics simulation (MDS) analysis confirmed the interaction of VR-QEg-180 with DGC of the A. baumannii. The designed lead has favorable ADMET properties, has no human off-targets and has no predicted cytotoxicity in cell lines. Therefore, the designed hybrid molecule (VR-QEg-180) targeting the DGC of A. baumannii may play a very significant role in controlling this pathogen.

Therapeutic strategies against autophagic escape by pathogenic bacteria.

Growing multidrug-resistant (MDR) strains of various infectious bacterial species are hindering research aiming to eliminate such infections. During a bacterial infection, the host response eliminates the pathogen via fusion of the endocytic vesicles with lysosomes, called xenophagy. However, MDR bacteria have evolved strategies to escape xenophagy. In this review, we propose novel therapeutics for overcoming such escape, including chimeric antibiotics, nanoformulations for the induction of autophagy in infected cells, and small interfering (si)RNA-mediated silencing of genes to inhibit the host-pathogen interaction. We also discuss the role of combinations of antibiotics showing synergy, the administrative routes of differentially capped nanoparticles (NPs), and the use of different types of nanoformulations for eliminating pathogenic bacteria from the host.

Rational targeting of Wzb phosphatase and Wzc kinase interaction inhibits extracellular polysaccharides synthesis and biofilm formation in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic nosocomial pathogen, and responsible for high mortality and morbidity. Biofilm formation is one of the resistance determinants, where extracellular polysaccharide (EPS) is an essential component. EPS synthesis and its export is regulated by the bacterial Wza-Wzb-Wzc system. Wzc exhibits auto-phosphorylation protein tyrosine kinase activity, while Wzb is a protein tyrosine phosphatase. Wzb mediates dephosphorylation of Wzc. Dephosphorylated Wzc is required for the export of the EPS through porin Wza-Wzc complex. It shows that the interaction of Wzb with Wzc is critical for the export of EPS. Therefore, if the Wzb-Wzc interaction is inhibited, then it might hinder the EPS transport and diminish the biofilm formation. In this study, we have modelled the Wzb, and Wzc proteins and further validated using PSVS, ProSA, RAMPAGE, and PDBsum. The modelled proteins were used for protein-protein docking. The docked protein-protein complex was minimized by Schrodinger software using OPLS_2005 force field. The binding site of the minimized Wzb-Wzc complex was identified by Sitemap. The high throughput virtual screening identified Labetalol hydrochloride and 4-{1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino] propyl} phenol from FDA-approved drug library based on their interaction at the interface of Wzb-Wzc complex. The inhibitor-protein complex was further undergone molecular mechanics analysis using Generalized Born model and Solvent Accessibility (MMGBSA) to estimate the binding free energies. The lead was also used to generate the pharmacophore model and screening the molecule with antimicrobial scaffold. The identified lead was experimentally validated for its effect on EPS quantity and biofilm formation by A. baumannii. Wzb-Wzc interaction is essential for biofilm and EPS export; hence, the identified lead might be useful to regulate the biofilm formation by A. baumannii.

Assessment of Molecular Mechanism of Gallate-Polyvinylpyrrolidone-Capped Hybrid Silver Nanoparticles against Carbapenem-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic nosocomial pathogen and causes bacteremia, urinary tract infections, meningitis, and pneumonia. The emergence of drug-resistant strain makes most of the current antibiotics ineffective. It is high time to screen some therapeutics against drug-resistant strains. Plant-based medicines have recently emerged as one of the important therapeutic choices. Therefore, in the present study, we have screened the metabolites of Phyllanthus emblica, Ocimum tenuiflorum, and Murraya koenigii for their antibacterial effect against carabapenem-resistant strain (RS-307) of A. baumannii. The result showed that the methanolic extract of P. emblica inhibits the growth of RS-307. The composition of this extract was determined using phytochemical screening and nuclear magnetic resonance (1D and 2D-NMR). The mechanism of action of the plant extract was validated by estimating reactive oxygen species (ROS), lipid peroxidation, protein carbonylation, and membrane damage. The result showed that treatment with this extract showed a significant elevation in the production of ROS generations, lipid peroxidation, and protein carbonylation. This confirms that plant extract treatment confirmed ROS-dependent membrane damage mechanism. The NMR result showed the presence of ethyl gallate, ellagic acid, chebulagic acid, quercetin, flavonoid, and alkaloid. To validate the antimicrobial activity of the secondary metabolite (i.e., gallic acid), we synthesized gallate-polyvinylpyrrolidone-capped hybrid silver nanoparticles (G-PVP-AgNPs) and characterized using Fourier transform infrared spectroscopy (FTIR). G-PVP-AgNPs showed good antimicrobial activity against RS-307, and its mechanism of action was investigated using fluorescence and transmission electron microscopy and FTIR that confirmed ROS-dependent killing mechanism. Therefore, the present study highlighted and recommended the use of G-PVP-AgNPs as suitable therapeutics against carbapenem-resistant A. baumannii.

Prioritization of potential vaccine targets using comparative proteomics and designing of the chimeric multi-epitope vaccine against Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa is one of the worldwide health problems involved in elevated mortality and morbidity. Therefore, it is important to find a therapeutic for this pathogen. In the present study, we have designed a chimeric vaccine against P. aeruginosa with the help of comparative proteomics and reverse vaccinology approaches. Using comparative subtractive proteomic analysis of 1,191 proteomes of P. aeruginosa, a total of twenty unique non-redundant proteomes were selected. In these proteomes, fifteen outer membrane proteins (OMPs) of P. aeruginosa were selected based on the basis of hydrophilicity, non-secretory nature, low transmembrane helix (<1), essentiality, virulence, pathway association, antigenic, and protein-protein network analysis. Reverse vaccinology approach was used to identify antigenic and immunogenic MHC class I, MHC class II and B cell epitopes present in the selected OMPs that can enhance T cell and B cell mediated immunogenicity. The selected epitopes were shortlisted based on their allergenicity, toxicity potentials, solubility, and hydrophilicity analysis. Immunogenic peptides were used to design a multi-epitope vaccine construct. Immune-modulating adjuvants and PADRE (Pan HLA-DR epitopes) sequence were added with epitopes sequence to enhance the immunogenicity. All the epitopes, adjuvants and PADRE sequence were joined by linkers. The designed vaccine constructs (VT1, VT2, VT3, and VT4) were analyzed by their physiochemical properties using different tools. Selected chimeric vaccine constructs (VT1, VT3, and VT4) were further shortlisted by their docking score with different HLA alleles. The final selected VT4 construct was docked with TLR4/MD2 complex and confirmed by molecular dynamics simulation studies. The final vaccine VT-4 construct was in-silico cloned in pET28a. Therefore, the designed construct VT4 may be studied to control the interaction of P. aeruginosa with host and infection caused by P. aeruginosa.

Proteomic analysis of iron-regulated membrane proteins identify FhuE receptor as a target to inhibit siderophore-mediated iron acquisition in Acinetobacter baumannii.

Survival of the Acinetobacter baumannii inside host requires different micronutrients such as iron, but their bioavailability is limited because of nutritional immunity created by host. A. baumannii has to develop mechanisms to acquire nutrient iron during infection. The present study is an attempt to identify membrane proteins involved in iron sequestration mechanism of A. baumannii using two-dimensional electrophoresis and LC-MS/MS analysis. The identified iron-regulated membrane protein (IRMP) of A. baumannii was used for its interaction studies with different siderophores, and designing of the inhibitor against A. baumannii targeting this IRMP. Membrane proteomic results identified over-expression of four membrane proteins (Fhu-E receptor, ferric-acinetobactin receptor, ferrienterochelin receptor, and ferric siderophore receptor) under iron-limited condition. A. baumannii produces siderophores that have good interaction with the FhuE receptor. Result also showed that FhuE receptor has interaction with siderophores produced by other bacteria. Interaction of FhuE receptor and siderophores helps in iron sequestration and survival of Acinetobacter under nutritional immunity imposed by the host. Hence it becomes essential to find a potential inhibitor for the FhuE receptor that can inhibit the survival of A. baumannii in the host. In-silico screening, and molecular mechanics studies identified ZINC03794794 and ZINC01530652 as a likely lead to design inhibitor against the FhuE receptor of A. baumannii. The designed inhibitor is experimentally validated for its antibacterial activity on the A. baumannii. Therefore, designed inhibitor interferes with the iron acquisition mechanism of Acinetobacter hence may prove useful for preventing infection caused by A. baumannii by limiting nutrient availability.

Molecular mechanism of antimicrobial activity of chlorhexidine against carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes hospital-acquired infections, especially in those with impaired immune function. Biocides or disinfectants are widely used antibacterial agents used to eradicate the effect of A. baumannii on inanimate objects and health care environments. In the current study, the antimicrobial activity of chlorhexidine has been investigated against carbapenem-resistant (RS-307, RS-7434, RS-6694, and RS-122), and sensitive (ATCC-19606 and RS-10953) strains of A. baumannii. We have determined growth kinetics, antimicrobial susceptibility, ROS production, lipid peroxidation, cell viability using flow cytometry assay (FACS), and membrane integrity by scanning electron microscope (SEM). The effect of chlorhexidine on the bacterial membrane has also been investigated using Fourier transform infrared (FTIR) spectroscopy. The present study showed that 32µg/ml chlorhexidine treatment results in the decreased bacterial growth, CFU count and cell viability. The antibacterial activity of chlorhexidine is due to the elevated ROS production and higher lipid peroxidation. These biochemical changes result in the membrane damage and alteration in the membrane proteins, phospholipids, carbohydrates, nucleic acids as evident from the FTIR and SEM data. Therefore, chlorhexidine has the potential to be used in the hospital setups to remove the spread of A. baumannii.