Emergence of persister cells in Staphylococcus aureus: calculated or fortuitous move?

A stable but reversible phenotype switch from normal to persister state is advantageous to the intracellular pathogens to cause recurrent infections and to evade the host immune system. Staphylococcus aureus is a versatile opportunistic pathogen known to cause chronic infections with significant mortality. One of the notable features is the ability to switch to a per-sisters cell, which is found in planktonic and biofilm states. This phenotypic switch is always an open question to explore the hidden fundamental science that coheres with a calculated or fortuitous move. Toxin-antitoxin modules, nutrient stress, and an erroneous translation-enabled state of dormancy entail this persistent behaviour in S. aureus. It is paramount to get a clear picture of why the cell chooses to enter a persistent condition, as it would decide the course of treatment. Analyzing the exit from a persistent state to an active state and the subsequent repercussion of this transition is essential to determine its role in chronic infections. This review attempts to provide a constructed argument discussing the most widely accepted mechanisms and identifying the various attributes of persistence.

Exploring the anti-caries properties of baicalin against Streptococcus mutans: an in vitro study.

Plant-derived molecules are excellent alternatives to antibiotics as anti-infective agents owing to their minimal cytotoxicity. Herein, the anti-infective property of the hydroxyflavone baicalin, was investigated against biofilms of the key dental caries pathogen Streptococcus mutans. Baicalin inhibited sucrose-dependent biofilm formation at a concentration of 500 µg ml-1 without affecting bacterial growth. It significantly inhibited acid production for an extended period of 8 h. Microscopic analysis revealed a 6-fold reduction in the number of adhered cells with baicalin treatment. Transcriptomic analysis of the mid-log phase and biofilm cells showed marked downregulation of the virulence genes required for biofilm formation and acid production. This study sheds significant new light on the potential for baicalin to be developed into an anti-caries agent.

A polymer-based anti-quorum catheter coating to challenge MDR Staphylococcus aureus: in vivo and in vitro approaches.

BACKGROUND: MDR Staphylococcus aureus is a major aetiological agent of catheter-associated infections. A quorum sensing targeted drug development approach proves to be an effective alternative strategy to combat such infections. METHODS: Intravenous catheters were coated with polymethacrylate copolymers loaded with the antivirulent compound 2-[(methylamino)methyl]phenol (2MAMP). The in vitro drug release profile and kinetics were established. The anti-biofilm effect of the coated catheters was tested against clinical isolates of MDR S. aureus. The in vivo studies were carried out using adult male Wistar rats by implanting coated catheters in subcutaneous pockets. Histopathological analysis was done to understand the immunological reactions induced by 2MAMP. RESULTS: A uniform catheter coating of thickness 0.1 mm was achieved with linear sustained release of 2MAMP for 6 h. The coating formulation was cytocompatible. The in vitro and in vivo anti-adherence studies showed reduced bacterial accumulation in coated catheters after 48 h. The histopathological results confirmed that the coated catheter did not bring about any adverse inflammatory response. CONCLUSIONS: The developed anti-quorum-coated catheter that is non-toxic and biocompatible has the potential to be used in other medical devices, thereby preventing catheter-associated infections.

Mitigating candidiasis with acarbose by targeting Candida albicans α-glucosidase: in-silico, in-vitro and transcriptomic approaches.

Biofilm-associated candidiasis poses a significant challenge in clinical settings due to the limited effectiveness of existing antifungal treatments. The challenges include increased pathogen virulence, multi-drug resistance, and inadequate penetration of antimicrobials into biofilm structures. One potential solution to this problem involves the development of novel drugs that can modulate fungal virulence and biofilm formation, which is essential for pathogenesis. Resistance in Candida albicans is initiated by morphological changes from yeast to hyphal form. This transition triggers a series of events such as cell wall elongation, increased adhesion, invasion of host tissues, pathogenicity, biofilm formation, and the initiation of an immune response. The cell wall is a critical interface for interactions with host cells, primarily through various cell wall proteins, particularly mannoproteins. Thus, cell wall proteins and enzymes are considered potential antifungal targets. In this regard, we explored α-glucosidase as our potential target which plays a crucial role in processing mannoproteins. Previous studies have shown that inhibition of α-glucosidase leads to defects in cell wall integrity, reduced adhesion, diminished secretion of hydrolytic enzymes, alterations in immune recognition, and reduced pathogenicity. Since α-glucosidase, primarily converts carbohydrates, our study focuses on FDA-approved carbohydrate mimic drugs (Glycomimetics) with well-documented applications in various biological contexts. Through virtual screening of 114 FDA-approved carbohydrate-based drugs, a pseudo-sugar Acarbose, emerged as a top hit. Acarbose is known for its pharmacological potential in managing type 2 diabetes mellitus by targeting α-glucosidase. Our preliminary investigations indicate that Acarbose effectively inhibits C. albicans biofilm formation, reduces virulence, impairs morphological switching, and hinders the adhesion and invasion of host cells, all at very low concentrations in the nanomolar range. Furthermore, transcriptomic analysis reveals the mechanism of action of Acarbose, highlighting its role in targeting α-glucosidase.

Revisiting ESKAPE Pathogens: virulence, resistance, and combating strategies focusing on quorum sensing.

The human-bacterial association is long-known and well-established in terms of both augmentations of human health and attenuation. However, the growing incidents of nosocomial infections caused by the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) call for a much deeper understanding of these organisms. Adopting a holistic approach that includes the science of infection and the recent advancements in preventing and treating infections is imperative in designing novel intervention strategies against ESKAPE pathogens. In this regard, this review captures the ingenious strategies commissioned by these master players, which are teamed up against the defenses of the human team, that are equally, if not more, versatile and potent through an analogy. We have taken a basketball match as our analogy, dividing the human and bacterial species into two teams playing with the ball of health. Through this analogy, we make the concept of infectious biology more accessible.

Design, dynamic docking, synthesis, and in vitro validation of a novel DNA gyrase B inhibitor.

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate-resistant Staphylococcus aureus (VRSA) are among the WHO's high priority pathogens. Among these two, MRSA is the most globally documented pathogen that necessitates the pressing demand for new classes of anti-MRSA drugs. Bacterial gyrase targeted therapeutics are unique strategies to overcome cross-resistance as they are present only in bacteria and absent in higher eukaryotes. The GyrB subunit is essential for the catalytic functions of the bacterial enzyme DNA Gyrase, thereby constituting a promising druggable target. The current study performed a structure-based virtual screening to designing GyrB target-specific candidate molecules. The de novo ligand design of novel hit molecules was performed using a rhodanine scaffold. Through a systematic in silico screening process, the hit molecules were screened for their synthetic accessibility, drug-likeness and pharmacokinetics properties in addition to its target specific interactions. Of the 374 hit molecules obtained through de novo ligand design, qsl-304 emerged as the most promising ligand. The molecular dynamic simulation studies confirmed the stable interaction between the key residues and qsl-304. qsl-304 was synthesized through a one-step chemical synthesis procedure, and the in vitro activity was proven, with an IC50 of 31.23 µg/mL against the novobiocin resistant clinical isolate, Staphylococcus aureus sa-P2003. Further studies on time-kill kinetics showed the bacteriostatic nature with the diminished recurrence of resistance. The on-target gyrB inhibition further proved the efficacy of qsl-304.Communicated by Ramaswamy H. Sarma.

Hidden agenda of Enterococcus faecalis lifestyle transition: planktonic to sessile state.

Enterococcus faecalis, a human gastrointestinal tract commensal, is known to cause nosocomial infections. Interestingly, the pathogen's host colonization and persistent infections are possibly linked to its lifestyle changes from planktonic to sessile state. Also, the multidrug resistance and survival fitness acquired in the sessile stage of E. faecalis has challenged treatment regimes. This situation exists because of the critical role played by several root genes and their molecular branches, which are part of quorum sensing, aggregation substance, surface adhesions, stress-related response and sex pheromones in the sessile state. It is therefore imperative to decode the hidden agenda of E. faecalis and understand the significant factors influencing biofilm formation. This would, in turn, augment the development of novel strategies to tackle E. faecalis infections.

Photoluminescence-Based Bioassay With Cysteamine-Capped TiO2 Nanoparticles for the Selective Recognition of N-Acyl Homoserine Lactones.

Currently available diagnostic procedures for infections are laborious and time-consuming, resulting in a substantial financial burden by increasing morbidity, increased costs of hospitalization, and mortality. Therefore, innovative approaches to design diagnostic biomarkers are imperative to assist in the rapid and sensitive diagnosis of microbial infections. Acyl homoserine lactones (AHLs) are ubiquitous bacterial signaling molecules that are found to be significantly upregulated in infected sites. In this pioneering work, we have developed a simple photoluminescence-based assay using cysteamine-capped titanium oxide (TiO2) nanoparticles for AHL detection. The PL intensity variation of the oxygen defect state of TiO2 was used for the biosensing measurements. The bioassays were validated using two well-studied AHL molecules (C4-HSL and 3-oxo-C12 HSL) of an important human pathogen, Pseudomonas aeruginosa. The developed system has a maximum relative response of 98%. Furthermore, the efficacy of the system in simulated host urine using an artificial urine medium showed a linear detection range of 10-160 nM. Also, we confirmed the relative response and specificity of the system in detecting AHLs produced by P. aeruginosa in a temporal manner.

Baicalein Inhibits Streptococcus mutans Biofilms and Dental Caries-Related Virulence Phenotypes.

Dental caries, the most common oral disease, is a major public healthcare burden and affects more than three billion people worldwide. The contemporary understanding of the need for a healthy microbiome and the emergence of antimicrobial resistance has resulted in an urgent need to identify compounds that curb the virulence of pathobionts without microbial killing. Through this study, we have demonstrated for the first time that 5,6,7-trihydroxyflavone (Baicalein) significantly downregulates crucial caries-related virulence phenotypes in Streptococcus mutans. Baicalein significantly inhibited biofilm formation by Streptococcus mutans UA159 (MBIC50 = 200 µM), without significant growth inhibition. Notably, these concentrations of baicalein did not affect the commensal S. gordonii. Strikingly, baicalein significantly reduced cell surface hydrophobicity, autoaggregation and acid production by S. mutans. Mechanistic studies (qRT-PCR) showed downregulation of various genes regulating biofilm formation, surface attachment, quorum sensing, acid production and competence. Finally, we demonstrate the potential translational value of baicalein by reporting synergistic interaction with fluoride against S. mutans biofilms.

Development of an Antibiotic Resistance Breaker to Resensitize Drug-Resistant Staphylococcus aureus: In Silico and In Vitro Approach.

Efflux pumps are one of the predominant microbial resistant mechanisms leading to the development of multidrug resistance. In Staphylococcus aureus, overexpression of NorA protein enables the efflux of antibiotics belonging to the class of fluoroquinolones and, thus, makes S. aureus resistant. Hence, NorA efflux pumps are being extensively exploited as the potential drug target to evade bacterial resistance and resensitize bacteria to the existing antibiotics. Although several molecules are reported to inhibit NorA efflux pump effectively, boronic acid derivatives were shown to have promising NorA efflux pump inhibition. In this regard, the current study exploits 6-(3-phenylpropoxy)pyridine-3-boronic acid to further improve the activity and reduce cytotoxicity using the bioisostere approach, a classical medicinal chemistry concept. Using the SWISS-Bioisostere online tool, from the parent compound, 42 compounds were obtained upon the replacement of the boronic acid. The 42 compounds were docked with modeled NorA protein, and key molecular interactions of the prominent compounds were assessed. The top hit compounds were further analyzed for their drug-like properties using ADMET studies. The identified potent lead, 5-nitro-2-(3-phenylpropoxy)pyridine (5-NPPP), was synthesized, and in vitro efficacy studies have been proven to show enhanced efflux inhibition, thus acting as a potent antibiotic breaker to resensitize S. aureus without elucidating any cytotoxic effect to the host Hep-G2 cell lines.

A photoluminescence biosensor for the detection of N-acyl homoserine lactone using cysteamine functionalized ZnO nanoparticles for the early diagnosis of urinary tract infections.

A urinary tract infection (UTI) is a recurrent infection that requires timely diagnosis and appropriate treatment. Conventional urinalysis methods are laborious and time-consuming, and lack sensitivity and specificity. In this context, photoluminescence (PL)-based biosensors have gained more attention due to their fast response time, and enhanced sensitivity and specificity. In relation to this, a PL-based biosensor was developed using ZnO nanoparticles obtained via a microwave-assisted process functionalized with cysteamine (ZnO-Cys) to detect the quorum sensing signalling molecules of Gram-negative bacteria, N-acyl-homoserine lactones (AHLs). These AHLs are involved in bacterial communication and are responsible for activating virulence and pathogenicity. Biosensing measurements based on PL intensity variations corresponding to the O2 acceptor defect level of ZnO with reference to ZnO-Cys were considered. A maximum sensitivity of 97% was achieved in the detection of AHL. The linear detection range of the developed biosensor was 10-120 nM in artificial urine media (AUM). The effect of pH on the sensitivity of the biosensor in AUM was also investigated and reported. The developed sensor was validated using the AHLs produced by Pseudomonas aeruginosa (MCC3101) in real-time analysis, which further confirmed the overall specificity and sensitivity.

Reciprocal Cooperation of Type A Procyanidin and Nitrofurantoin Against Multi-Drug Resistant (MDR) UPEC: A pH-Dependent Study.

Uropathogenic Escherichia coli (UPEC) accounts for the majority of complicated and uncomplicated urinary tract infections. The use of phytomolecules in the treatment of UTI is fast gaining attention. The current report identifies a multidrug-resistant strain (QSLUPEC7), which is a strong biofilm producer, among the considered clinical isolates. The antimicrobial and antibiofilm activity was evaluated for the phytomolecule, Type A procyanidin (TAP) from Cinnamomum zeylanicum against QSLUPEC7. TAP treatment did not affect the growth of the MDR strain but affected the biofilm formation (~70% inhibition). The confocal microscopic examination reveals the biofilm inhibition and the live cells in the biofilm corroborates the antimicrobial results. Further, the synergy studies of TAP and nitrofurantoin (NIT) were carried out at different pH. TAP acts synergistically with nitrofurantoin at different pH considered. A closer look in the results reveals that at pH 5.8, maximum growth inhibition is recorded. The gene expression analysis shows that TAP alone and in combination with NIT downregulates the major fimbriae adhesins of UPEC. The results conclude that the TAP has an antibiofilm activity against the multidrug-resistant strain of UPEC, without affecting the growth. Also, TAP reciprocally cooperates with nitrofurantoin at different pH by downregulating the adhesins of UPEC.

Photophysical studies on curcumin-sophorolipid nanostructures: applications in quorum quenching and imaging.

Sophorolipid biosurfactants are biodegradable, less toxic and FDA approved. The purified acidic form of sophorolipid is stimuli-responsive with self-assembling properties and used for solubilizing hydrophobic drugs. This study encapsulated curcumin (CU) with acidic sophorolipid (ASL) micelles and analysed using photophysical studies like UV-visible spectroscopy, photoluminescence (PL) spectroscopy and time-correlated single photon counting (TCSPC). TEM images have revealed ellipsoid micelles of approximately 100 nm size and were confirmed by dynamic light scattering. The bacterial fluorescence uptake studies showed the uptake of formed CUASL nanostructures into both Gram-positive and Gram-negative bacteria. They also showed quorum quenching activity against Pseudomonas aeruginosa. The results have demonstrated this system has potential theranostic applications.

Modulating the Global Response Regulator, LuxO of V. cholerae Quorum Sensing System Using a Pyrazine Dicarboxylic Acid Derivative (PDCApy): An Antivirulence Approach.

Vibrio cholerae is a Gram-negative pathogen which causes acute diarrhoeal disease, cholera by the expression of virulence genes through quorum sensing (QS) mechanism. The QS circuit of V. cholerae is controlled by the global quorum regulator, LuxO, which at low cell density (LCD) state produces major virulence factors such as, toxin co-regulated pilus (TCP) and cholera toxin (CT) to mediate infection. On the contrary, at the high cell density (HCD) state the virulent genes are downregulated and the vibrios are detached from the host intestinal epithelial cells, promoted by HapA protease. Hence, targeting the global regulator LuxO would be a promising approach to modulate the QS to curtail V. cholerae pathogenesis. In our earlier studies, LuxO targeted ligand, 2,3 pyrazine dicarboxylic acid (PDCA) and its derivatives having desired pharmacophore properties were chemically synthesized and were shown to have biofilm inhibition as well as synergistic activity with the conventionally used antibiotics. In the present study, the QS modulatory effect of the PDCA derivative with pyrrolidine moiety designated as PDCApy against the V. cholerae virulence gene expression was analyzed at various growth phases. The data significantly showed a several fold reduction in the expression of the genes, tcp and ct whereas the expression of hapR was upregulated at the LCD state. In addition, PDCApy reduced the adhesion and invasion of the vibrios onto the INT407 intestinal cell lines. Collectively, our data suggest that PDCApy could be a potential QS modulator (QSM) for the antivirulence therapeutic approach.

Antimicrobial and Antibiofilm Potential of Acyclic Amines and Diamines against Multi-Drug Resistant Staphylococcus aureus.

Multi-drug resistant Staphylococcus aureus (MDRSA) remains a great challenge despite a decade of research on antimicrobial compounds against their infections. In the present study, various acyclic amines and diamines were chemically synthesized and tested for their antimicrobial as well as antibiofilm activity against MDRSA. Among all the synthesized compounds, an acyclic diamine, (2,2'-((butane-1,4-diylbis(azanediyl)bis(methylene))diphenol) designated as ADM 3, showed better antimicrobial activity (minimum inhibitory concentration at 50 µg/mL) and antibiofilm activity (MBIC50 at 5 µg/mL). In addition, ADM 3 was capable of reducing the virulence factors expression (anti-virulence). Confocal laser scanning microscope analysis of the in vitro tested urinary catheters showed biofilm reduction as well as bacterial killing by ADM 3. On the whole, our data suggest that acyclic diamines, especially ADM 3 can be a potent lead for the further studies in alternative therapeutic approaches.