A low-cost, hands-on module to characterize antimicrobial compounds using an interdisciplinary, biophysical approach.

We have developed a hands-on experimental module that combines biology experiments with a physics-based analytical model in order to characterize antimicrobial compounds. To understand antibiotic resistance, participants perform a disc diffusion assay to test the antimicrobial activity of different compounds and then apply a diffusion-based analytical model to gain insights into the behavior of the active antimicrobial component. In our experience, this module was robust, reproducible, and cost-effective, suggesting that it could be implemented in diverse settings such as undergraduate research, STEM (science, technology, engineering, and math) camps, school programs, and laboratory training workshops. By providing valuable interdisciplinary research experience in science outreach and education initiatives, this module addresses the paucity of structured training or education programs that integrate diverse scientific fields. Its low-cost requirements make it especially suitable for use in resource-limited settings.

Single-cell control of initial spatial structure in biofilm development using laser trapping.

Biofilms are sessile communities of microbes that are spatially structured by an embedding matrix. Biofilm infections are notoriously intractable. This arises, in part, from changes in the bacterial phenotype that result from spatial structure. Understanding these interactions requires methods to control the spatial structure of biofilms. We present a method for growing biofilms from initiating cells whose positions are controlled with single-cell precision using laser trapping. The native growth, motility, and surface adhesion of positioned microbes are preserved, as we show for model organisms Pseudomonas aeruginosa and Staphylococcus aureus. We demonstrate that laser-trapping and placing bacteria on surfaces can reveal the effects of spatial structure on bacterial growth in early biofilm development.

Tools of the Trade: Image Analysis Programs for Confocal Laser-Scanning Microscopy Studies of Biofilms and Considerations for Their Use by Experimental Researchers.

Confocal laser-scanning microscopy (CLSM) is the bedrock of the microscopic visualization of biofilms. Previous applications of CLSM in biofilm studies have largely focused on observations of bacterial or fungal elements of biofilms, often seen as aggregates or mats of cells. However, the field of biofilm research is moving beyond qualitative observations alone, toward the quantitative analysis of the structural and functional features of biofilms, across clinical, environmental, and laboratory conditions. In recent times, several image analysis programs have been developed to extract and quantify biofilm properties from confocal micrographs. These tools not only vary in their scope and relevance to the specific biofilm features under study but also with respect to the user interface, compatibility with operating systems, and raw image requirements. Understanding these considerations is important when selecting tools for quantitative biofilm analysis, including at the initial experimental stages of image acquisition. In this review, we provide an overview of image analysis programs for confocal micrographs of biofilms, with a focus on tool selection and image acquisition parameters that are relevant for experimental researchers to ensure reliability and compatibility with downstream image processing.

Talk To A Scientist: a Framework for a Webinar-Based Outreach Program for Scientists to Engage with K-12 Students.

Science outreach programs that enable real-time interactions between scientists and school-aged children are known to positively impact learning gains and students' perceptions of scientists. To expand K-12 outreach by scientists, it is important to build structured outreach programs which offer scientists well-defined opportunities, while providing school students regular and diverse interactions. We describe Talk To A Scientist, a science outreach platform in India, where scientists use a webinar-based approach to share their research with K-12 students (6 to 16 years). Running weekly for nearly 3 years, Talk To A Scientist has hosted over 100 live interactions, with a wide reach to participants across the country. Here, we outline the framework used to build Talk To A Scientist and discuss key gains, considerations, and challenges in the development of the program. We also suggest potential adaptations with which this framework can serve as a guideline for the implementation of similar K-12 outreach programs across diverse country- and context-specific settings.

Adding a new dimension: Multi-level structure and organization of mixed-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a 4-D wound microenvironment.

Biofilms in wounds typically consist of aggregates of bacteria, most often Pseudomonas aeruginosa and Staphylococcus aureus, in close association with each other and the host microenvironment. Given this, the interplay across host and microbial elements, including the biochemical and nutrient profile of the microenvironment, likely influences the structure and organization of wound biofilms. While clinical studies, in vivo and ex vivo model systems have provided insights into the distribution of P. aeruginosa and S. aureus in wounds, they are limited in their ability to provide a detailed characterization of biofilm structure and organization across the host-microbial interface. On the other hand, biomimetic in vitro systems, such as host cell surfaces and simulant media conditions, albeit reductionist, have been shown to support the co-existence of P. aeruginosa and S. aureus biofilms, with species-dependent localization patterns and interspecies interactions. Therefore, composite in vitro models that bring together key features of the wound microenvironment could provide unprecedented insights into the structure and organization of mixed-species biofilms. We have built a four-dimensional (4-D) wound microenvironment consisting of a 3-D host cell scaffold of co-cultured human epidermal keratinocytes and dermal fibroblasts, and an in vitro wound milieu (IVWM); the IVWM provides the fourth dimension that represents the biochemical and nutrient profile of the wound infection state. We leveraged this 4-D wound microenvironment, in comparison with biofilms in IVWM alone and standard laboratory media, to probe the structure of mixed-species P. aeruginosa and S. aureus biofilms across multiple levels of organization such as aggregate dimensions and biomass thickness, species co-localization and spatial organization within the biomass, overall biomass composition and interspecies interactions. In doing so, the 4-D wound microenvironment platform provides multi-level insights into the structure of mixed-species biofilms, which we incorporate into the current understanding of P. aeruginosa and S. aureus organization in the wound bed.

Of biofilms and beehives: An analogy-based instructional tool to introduce biofilms in school and undergraduate curriculum.

The concept of biofilms and biofilm-based research is largely absent or minimally described in school and undergraduate life science curriculum. While it is well-established that microbes, such as bacteria and fungi, most often exist in multicellular biofilm communities, descriptions in standard biology textbooks continue to focus on the single-celled form of microbial life. We have developed an analogy-based instructional tool to introduce and explain biofilms to school and undergraduate students. The module employs an analogy with beehives, given that biofilms and beehives are both 'superorganism' states, to explain key biofilm features such as development and structure, chemical communication, division of labor and emergent properties. We delivered this analogy-based learning tool to a cohort of 49 students, including middle-to-high school and undergraduate students, and based on participant feedback and learnings, present a formal evaluation of the instructional tool. Further, we outline prerequisites and learning approaches that can enable the delivery of this module in classroom and virtual learning settings, including suggestions for pre-lesson reading, student-centred interactive activities, and specific learning objectives. Taken together, this instructional analogy holds potential to serve as an educational tool to introduce biofilms in school and undergraduate curricula in a relatable and comprehensible manner.

Biofilms with a Dash of Color: A Hands-On Activity for School Students to Build a Biofilm Model and Use It to Understand Antibiotic Tolerance in Biofilms.

It is increasingly recognized that microbes such as bacteria exist in communities or biofilms, both in the environment and human body. However, school biology curricula continue to focus on the free-floating form of bacterial life, with minimal descriptions of biofilms. Consequently, there is a need to introduce biofilms to school students, to not only to develop a fundamental understanding of microbial life but also to highlight the challenges posed by biofilm infections to antibiotic treatment. We have developed a hands-on activity for students to build a biofilm model and use it in comparison with a model of free-living (planktonic) bacteria, to test the role of the extracellular matrix in the antibiotic tolerance of biofilms. The activity uses simple, easy-to-obtain supplies and is designed to be conducted in an in-person or virtual format for elementary and middle school students in the age group of 6 to 13 years. We conducted the activity in virtual mode (via Zoom) for 59 school students across India, and we present feedback and acquired knowledge that could be used to execute and adapt this accessible and engaging science experience.

'Targeting' the search: An upgraded structural and functional repository of antimicrobial peptides for biofilm studies (B-AMP v2.0) with a focus on biofilm protein targets.

Bacterial biofilms, often as multispecies communities, are recalcitrant to conventional antibiotics, making the treatment of biofilm infections a challenge. There is a push towards developing novel anti-biofilm approaches, such as antimicrobial peptides (AMPs), with activity against specific biofilm targets. In previous work, we developed Biofilm-AMP, a structural and functional repository of AMPs for biofilm studies (B-AMP v1.0) with more than 5000 structural models of AMPs and a vast library of AMP annotations to existing biofilm literature. In this study, we present an upgraded version of B-AMP, with a focus on existing and novel bacterial biofilm targets. B-AMP v2.0 hosts a curated collection of 2502 biofilm protein targets across 473 bacterial species, with structural protein models and functional annotations from PDB, UniProt, and PubMed databases. The biofilm targets can be searched for using the name of the source organism, and function and type of protein, and results include designated Target IDs (unique to B-AMP v2.0), UniProt IDs, 3D predicted protein structures, PDBQT files, pre-defined protein functions, and relevant scientific literature. To present an example of the combined applicability of both, the AMP and biofilm target libraries in the repository, we present two case studies. In the first case study, we expand an in silico pipeline to evaluate AMPs against a single biofilm target in the multidrug resistant, bacterial pathogen Corynebacterium striatum, using 3D protein-peptide docking models from previous work and Molecular Dynamics simulations (~1.2µs). In the second case study, we build an in silico pipeline to identify candidate AMPs (using AMPs with both anti-Gram positive and anti-Gram negative activity) against two biofilm targets with a common functional annotation in Pseudomonas aeruginosa and Staphylococcus aureus, widely-encountered bacterial co-pathogens. With its enhanced structural and functional capabilities, B-AMP v2.0 serves as a comprehensive resource for AMP investigations related to biofilm studies. B-AMP v2.0 is freely available at https://b-amp.karishmakaushiklab.com and will be regularly updated with structural models of AMPs and biofilm targets, as well as 3D protein-peptide interaction models for key biofilm-forming pathogens.

Biofilms in Wounds: New Advances in Therapy and in Healing Management.

Biofilms are the major way of life for both bacteria and fungi [...].

Looking beyond the smokescreen: can the oral microbiome be a tool or target in the management of tobacco-associated oral cancer?

A wide range of microbes inhabit the oral cavity, and bacterial and fungal communities most often exist as structured communities or biofilms. The use of tobacco alters the structure of the oral microbiome, including that of potentially malignant lesions, and the altered oral microbiome influences key microenvironmental changes such as chronic inflammation, secretion of carcinogenic toxins, cellular and tissue remodelling and suppression of apoptosis. Given this, it is clear that the bacterial and fungal biofilms in potentially malignant states are likely not passive entities, but could play a critical role in shaping potential malignant and carcinogenic conditions. This holds potential towards leveraging the oral microbiome for the management of tobacco-associated potentially malignant lesions and oral cancer. Here, we explore this line of investigation by reviewing the effects of tobacco in shaping the oral microbiome, and analyse the available evidence in the light of the microbiome of oral potentially malignant and cancerous lesions, and the role of dysbiosis in carcinogenesis. Finally, we discuss possible interventions and approaches using which the oral microbiome could be leveraged towards precision-based oral cancer therapeutics.

Milieu matters: An in vitro wound milieu to recapitulate key features of, and probe new insights into, mixed-species bacterial biofilms.

Bacterial biofilms are a major cause of delayed wound healing. Consequently, the study of wound biofilms, particularly in host-relevant conditions, has gained importance. Most in vitro studies employ refined laboratory media to study biofilms, representing conditions that are not relevant to the infection state. To mimic the wound milieu, in vitro biofilm studies often incorporate serum or plasma in growth conditions, or employ clot or matrix-based biofilm models. While incorporating serum or plasma alone is a minimalistic approach, the more complex in vitro wound models are technically demanding, and poorly compatible with standard biofilm assays. Based on previous reports of clinical wound fluid composition, we have developed an in vitro wound milieu (IVWM) that includes, in addition to serum (to recapitulate wound fluid), matrix elements and biochemical factors. With Luria-Bertani broth and Fetal Bovine Serum (FBS) for comparison, the IVWM was used to study planktonic growth, biofilm features, and interspecies interactions, of common wound pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. We demonstrate that the IVWM recapitulates widely reported in vivo biofilm features such as biomass formation, metabolic activity, increased antibiotic tolerance, 3D structure, and interspecies interactions for monospecies and mixed-species biofilms. Further, the IVWM is simple to formulate, uses laboratory-grade components, and is compatible with standard biofilm assays. Given this, it holds potential as a tractable approach to study wound biofilms under host-relevant conditions.

AMPing Up the Search: A Structural and Functional Repository of Antimicrobial Peptides for Biofilm Studies, and a Case Study of Its Application to Corynebacterium striatum, an Emerging Pathogen.

Antimicrobial peptides (AMPs) have been recognized for their ability to target processes important for biofilm formation. Given the vast array of AMPs, identifying potential anti-biofilm candidates remains a significant challenge, and prompts the need for preliminary in silico investigations prior to extensive in vitro and in vivo studies. We have developed Biofilm-AMP (B-AMP), a curated 3D structural and functional repository of AMPs relevant to biofilm studies. In its current version, B-AMP contains predicted 3D structural models of 5544 AMPs (from the DRAMP database) developed using a suite of molecular modeling tools. The repository supports a user-friendly search, using source, name, DRAMP ID, and PepID (unique to B-AMP). Further, AMPs are annotated to existing biofilm literature, consisting of a vast library of over 10,000 articles, enhancing the functional capabilities of B-AMP. To provide an example of the usability of B-AMP, we use the sortase C biofilm target of the emerging pathogen Corynebacterium striatum as a case study. For this, 100 structural AMP models from B-AMP were subject to in silico protein-peptide molecular docking against the catalytic site residues of the C. striatum sortase C protein. Based on docking scores and interacting residues, we suggest a preference scale using which candidate AMPs could be taken up for further in silico, in vitro and in vivo testing. The 3D protein-peptide interaction models and preference scale are available in B-AMP. B-AMP is a comprehensive structural and functional repository of AMPs, and will serve as a starting point for future studies exploring AMPs for biofilm studies. B-AMP is freely available to the community at https://b-amp.karishmakaushiklab.com and will be regularly updated with AMP structures, interaction models with potential biofilm targets, and annotations to biofilm literature.

Reduced serum methods for contact-based coculture of human dermal fibroblasts and epidermal keratinocytes.

Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems and a range of supplemental factors. These approaches are technically demanding and labor intensive, and pose scientific and ethical limitations associated with the high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the proliferation of one or both cell types when they are cultured together. We have developed two reduced serum approaches (1-2% serum) that support the contact-based coculture of human dermal fibroblasts and immortalized keratinocytes and enable the study of cell migration and wound closure.

From Treatise to Test: Evaluating Traditional Remedies for Anti-Biofilm Potential.

Traditional plant-based remedies hold vast potential as novel antimicrobial agents, particularly for recalcitrant infection states such as biofilms. To explore their potential, it is important to bring these remedies out of historical treatises, and into present-day scientific evaluation. Using an example of Indian traditional medicine (Ayurveda), we present a perspective toward evaluating historical remedies for anti-biofilm potential. Across compendia, we identified three plant-based remedies (of Kalanchoe pinnata, Cynodon dactylon, and Ocimum tenuiflorum) recommended for wounds. The remedies were reconstituted in accordance with historical practices, and tested for their effects on biofilm formation and eradication assays of wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Based on our approach and the results obtained, we provide insights into the considerations and challenges related to identifying potential remedies in historical texts, and testing them in the laboratory with standard biofilm assays. We believe this will be relevant for future studies exploring anti-biofilm approaches at the interface of historical medicine and present-day scientific practices.

Recent Advances in Non-Conventional Antimicrobial Approaches for Chronic Wound Biofilms: Have We Found the 'Chink in the Armor'?

Chronic wounds are a major healthcare burden, with huge public health and economic impact. Microbial infections are the single most important cause of chronic, non-healing wounds. Chronic wound infections typically form biofilms, which are notoriously recalcitrant to conventional antibiotics. This prompts the need for alternative or adjunct 'anti-biofilm' approaches, notably those that account for the unique chronic wound biofilm microenvironment. In this review, we discuss the recent advances in non-conventional antimicrobial approaches for chronic wound biofilms, looking beyond standard antibiotic therapies. These non-conventional strategies are discussed under three groups. The first group focuses on treatment approaches that directly kill or inhibit microbes in chronic wound biofilms, using mechanisms or delivery strategies distinct from antibiotics. The second group discusses antimicrobial approaches that modify the biological, chemical or biophysical parameters in the chronic wound microenvironment, which in turn enables the disruption and removal of biofilms. Finally, therapeutic approaches that affect both, biofilm bacteria and microenvironment factors, are discussed. Understanding the advantages and limitations of these recent approaches, their stage of development and role in biofilm management, could lead to new treatment paradigms for chronic wound infections. Towards this end, we discuss the possibility that non-conventional antimicrobial therapeutics and targets could expose the 'chink in the armor' of chronic wound biofilms, thereby providing much-needed alternative or adjunct strategies for wound infection management.

Advances in Chemical and Biological Methods to Identify Microorganisms-From Past to Present.

Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations.

Molecular characterization of clinical varicella-zoster strains from India and differentiation from the oka vaccine strain.

With the introduction of varicella vaccination in India, surveillance of circulating varicella-zoster strains has gained significance. The aim of the present study was to achieve molecular characterization of circulating varicella-zoster virus (VZV) strains and differentiation from the Oka vaccine strain. In this study, the genotype of 100 clinical VZV strains was analyzed. Vesicle fluid was collected from patients with VZV infections (92 cases of varicella and 8 cases of herpes zoster). The PCR-RFLP analysis of two polymorphic loci--a PstI restriction site in ORF 38 and a BglI restriction site in ORF 54 was used to characterize and differentiate them from the vaccine strain. All the wild-type strains were positive for the PstI restriction site in ORF 38. This differentiated them from the Oka vaccine strain, which is PstI negative. The wild-type strains as well as the Oka vaccine strain were positive for the BglI restriction site in ORF 54. Thus, the genotype of all the VZV strains examined had the wild-type pattern represented as PstI(+) BglI(+). None of the strains had the PstI(-) BglI(+) genotype characteristic of the Oka strain or the PstI(+) BglI(-) wild-type pattern. To conclude, PstI and BglI serve as good reference markers in the genotyping of circulating varicella strains in India and serve to differentiate them from the vaccine strain as well as other wild-type strains.

Tobramycin and bicarbonate synergise to kill planktonic Pseudomonas aeruginosa, but antagonise to promote biofilm survival.

Increasing antibiotic resistance and the declining rate at which new antibiotics come into use create a need to increase the efficacy of existing antibiotics. The aminoglycoside tobramycin is standard-of-care for many types of Pseudomonas aeruginosa infections, including those in the lungs of cystic fibrosis (CF) patients. P. aeruginosa is a nosocomial and opportunistic pathogen that, in planktonic form, causes acute infections and, in biofilm form, causes chronic infections. Inhaled bicarbonate has recently been proposed as a therapy to improve antimicrobial properties of the CF airway surface liquid and viscosity of CF mucus. Here we measure the effect of combining tobramycin and bicarbonate against P. aeruginosa, both lab strains and CF clinical isolates. Bicarbonate synergises with tobramycin to enhance killing of planktonic bacteria. In contrast, bicarbonate antagonises with tobramycin to promote better biofilm growth. This suggests caution when evaluating bicarbonate as a therapy for CF lungs infected with P. aeruginosa biofilms. We analyse tobramycin and bicarbonate interactions using an interpolated surface methodology to measure the dose-response function. These surfaces allow more accurate estimation of combinations yielding synergy and antagonism than do standard isobolograms. By incorporating predictions based on Loewe additivity theory, we can consolidate information on a wide range of combinations that produce a complex dose-response surface, into a single number that measures the net effect. This tool thus allows rapid initial estimation of the potential benefit or harm of a therapeutic combination. Software code is freely made available as a resource for the community.

The spatial profiles and metabolic capabilities of microbial populations impact the growth of antibiotic-resistant mutants.

Antibiotic resistance adversely affects clinical and public health on a global scale. Using the opportunistic human pathogen Pseudomonas aeruginosa, we show that increasing the number density of bacteria, on agar containing aminoglycoside antibiotics, can non-monotonically impact the survival of antibiotic-resistant mutants. Notably, at high cell densities, mutant survival is inhibited. A wide range of bacterial species can inhibit antibiotic-resistant mutants. Inhibition results from the metabolic breakdown of amino acids, which results in alkaline by-products. The consequent increase in pH acts in conjunction with aminoglycosides to mediate inhibition. Our work raises the possibility that the manipulation of microbial population structure and nutrient environment in conjunction with existing antibiotics could provide therapeutic approaches to combat antibiotic resistance.