Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux.

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.

Visible-Light-Activated Molecular Machines Kill Fungi by Necrosis Following Mitochondrial Dysfunction and Calcium Overload.

Invasive fungal infections are a growing public health threat. As fungi become increasingly resistant to existing drugs, new antifungals are urgently needed. Here, it is reported that 405-nm-visible-light-activated synthetic molecular machines (MMs) eliminate planktonic and biofilm fungal populations more effectively than conventional antifungals without resistance development. Mechanism-of-action studies show that MMs bind to fungal mitochondrial phospholipids. Upon visible light activation, rapid unidirectional drilling of MMs at ≈3 million cycles per second (MHz) results in mitochondrial dysfunction, calcium overload, and ultimately necrosis. Besides their direct antifungal effect, MMs synergize with conventional antifungals by impairing the activity of energy-dependent efflux pumps. Finally, MMs potentiate standard antifungals both in vivo and in an ex vivo porcine model of onychomycosis, reducing the fungal burden associated with infection.

Probiotics: insights and new opportunities for Clostridioides difficile intervention.

Clostridioides difficile infection (CDI) is a life-threatening disease caused by the Gram-positive, opportunistic intestinal pathogen C. difficile. Despite the availability of antimicrobial drugs to treat CDI, such as vancomycin, metronidazole, and fidaxomicin, recurrence of infection remains a significant clinical challenge. The use of live commensal microorganisms, or probiotics, is one of the most investigated non-antibiotic therapeutic options to balance gastrointestinal (GI) microbiota and subsequently tackle dysbiosis. In this review, we will discuss major commensal probiotic strains that have the potential to prevent and/or treat CDI and its recurrence, reassess the efficacy of probiotics supplementation as a CDI intervention, delve into lessons learned from probiotic modulation of the immune system, explore avenues like genome-scale metabolic network reconstructions, genome sequencing, and multi-omics to identify novel strains and understand their functionality, and discuss the current regulatory framework, challenges, and future directions.

Hemithioindigo-Based Visible Light-Activated Molecular Machines Kill Bacteria by Oxidative Damage.

Antibiotic resistance is a growing health threat. There is an urgent and critical need to develop new antimicrobial modalities and therapies. Here, a set of hemithioindigo (HTI)-based molecular machines capable of specifically killing Gram-positive bacteria within minutes of activation with visible light (455 nm at 65 mW cm-2 ) that are safe for mammalian cells is described. Importantly, repeated exposure of bacteria to HTI does not result in detectable development of resistance. Visible light-activated HTI kill both exponentially growing bacterial cells and antibiotic-tolerant persister cells of various Gram-positive strains, including methicillin-resistant S. aureus (MRSA). Visible light-activated HTI also eliminate biofilms of S. aureus and B. subtilis in as little as 1 h after light activation. Quantification of reactive oxygen species (ROS) formation and protein carbonyls, as well as assays with various ROS scavengers, identifies oxidative damage as the underlying mechanism for the antibacterial activity of HTI. In addition to their direct antibacterial properties, HTI synergize with conventional antibiotics in vitro and in vivo, reducing the bacterial load and mortality associated with MRSA infection in an invertebrate burn wound model. To the best of the authors' knowledge, this is the first report on the antimicrobial activity of HTI-based molecular machines.

Light-activated molecular machines are fast-acting broad-spectrum antibacterials that target the membrane.

The increasing occurrence of antibiotic-resistant bacteria and the dwindling antibiotic research and development pipeline have created a pressing global health crisis. Here, we report the discovery of a distinctive antibacterial therapy that uses visible (405 nanometers) light-activated synthetic molecular machines (MMs) to kill Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, in minutes, vastly outpacing conventional antibiotics. MMs also rapidly eliminate persister cells and established bacterial biofilms. The antibacterial mode of action of MMs involves physical disruption of the membrane. In addition, by permeabilizing the membrane, MMs at sublethal doses potentiate the action of conventional antibiotics. Repeated exposure to antibacterial MMs is not accompanied by resistance development. Finally, therapeutic doses of MMs mitigate mortality associated with bacterial infection in an in vivo model of burn wound infection. Visible light-activated MMs represent an unconventional antibacterial mode of action by mechanical disruption at the molecular scale, not existent in nature and to which resistance development is unlikely.

Light-based technologies for management of COVID-19 pandemic crisis.

The global dissemination of the novel coronavirus disease (COVID-19) has accelerated the need for the implementation of effective antimicrobial strategies to target the causative agent SARS-CoV-2. Light-based technologies have a demonstrable broad range of activity over standard chemotherapeutic antimicrobials and conventional disinfectants, negligible emergence of resistance, and the capability to modulate the host immune response. This perspective article identifies the benefits, challenges, and pitfalls of repurposing light-based strategies to combat the emergence of COVID-19 pandemic.

The value of antimicrobial peptides in the age of resistance.

Accelerating growth and global expansion of antimicrobial resistance has deepened the need for discovery of novel antimicrobial agents. Antimicrobial peptides have clear advantages over conventional antibiotics which include slower emergence of resistance, broad-spectrum antibiofilm activity, and the ability to favourably modulate the host immune response. Broad bacterial susceptibility to antimicrobial peptides offers an additional tool to expand knowledge about the evolution of antimicrobial resistance. Structural and functional limitations, combined with a stricter regulatory environment, have hampered the clinical translation of antimicrobial peptides as potential therapeutic agents. Existing computational and experimental tools attempt to ease the preclinical and clinical development of antimicrobial peptides as novel therapeutics. This Review identifies the benefits, challenges, and opportunities of using antimicrobial peptides against multidrug-resistant pathogens, highlights advances in the deployment of novel promising antimicrobial peptides, and underlines the needs and priorities in designing focused development strategies taking into account the most advanced tools available.

Early network properties of the COVID-19 pandemic - The Chinese scenario.

OBJECTIVES: To control epidemics, sites more affected by mortality should be identified. METHODS: Defining epidemic nodes as areas that included both most fatalities per time unit and connections, such as highways, geo-temporal Chinese data on the COVID-19 epidemic were investigated with linear, logarithmic, power, growth, exponential, and logistic regression models. A z-test compared the slopes observed. RESULTS: Twenty provinces suspected to act as epidemic nodes were empirically investigated. Five provinces displayed synchronicity, long-distance connections, directionality and assortativity - network properties that helped discriminate epidemic nodes. The rank I node included most fatalities and was activated first. Fewer deaths were reported, later, by rank II and III nodes, while the data from rank I-III nodes exhibited slopes, the data from the remaining provinces did not. The power curve was the best fitting model for all slopes. Because all pairs (rank I vs. rank II, rank I vs. rank III, and rank II vs. rank III) of epidemic nodes differed statistically, rank I-III epidemic nodes were geo-temporally and statistically distinguishable. CONCLUSIONS: The geo-temporal progression of epidemics seems to be highly structured. Epidemic network properties can distinguish regions that differ in mortality. This real-time geo-referenced analysis can inform both decision-makers and clinicians.

Assessing the Dynamics and Complexity of Disease Pathogenicity Using 4-Dimensional Immunological Data.

Investigating disease pathogenesis and personalized prognostics are major biomedical needs. Because patients sharing the same diagnosis can experience different outcomes, such as survival or death, physicians need new personalized tools, including those that rapidly differentiate several inflammatory phases. To address these topics, a pattern recognition-based method (PRM) that follows an inverse problem approach was designed to assess, in <10 min, eight concepts: synergy, pleiotropy, complexity, dynamics, ambiguity, circularity, personalized outcomes, and explanatory prognostics (pathogenesis). By creating thousands of secondary combinations derived from blood leukocyte data, the PRM measures synergic, pleiotropic, complex and dynamic data interactions, which provide personalized prognostics while some undesirable features-such as false results and the ambiguity associated with data circularity-are prevented. Here, this method is compared to Principal Component Analysis (PCA) and evaluated with data collected from hantavirus-infected humans and birds that appeared to be healthy. When human data were examined, the PRM predicted 96.9 % of all surviving patients while PCA did not distinguish outcomes. Demonstrating applications in personalized prognosis, eight PRM data structures sufficed to identify all but one of the survivors. Dynamic data patterns also distinguished survivors from non-survivors, as well as one subset of non-survivors, which exhibited chronic inflammation. When the PRM explored avian data, it differentiated immune profiles consistent with no, early, or late inflammation. Yet, PCA did not recognize patterns in avian data. Findings support the notion that immune responses, while variable, are rather deterministic: a low number of complex and dynamic data combinations may be enough to, rapidly, unmask conditions that are neither directly observable nor reliably forecasted.

Options and Limitations in Clinical Investigation of Bacterial Biofilms.

Bacteria can form single- and multispecies biofilms exhibiting diverse features based upon the microbial composition of their community and microenvironment. The study of bacterial biofilm development has received great interest in the past 20 years and is motivated by the elegant complexity characteristic of these multicellular communities and their role in infectious diseases. Biofilms can thrive on virtually any surface and can be beneficial or detrimental based upon the community's interplay and the surface. Advances in the understanding of structural and functional variations and the roles that biofilms play in disease and host-pathogen interactions have been addressed through comprehensive literature searches. In this review article, a synopsis of the methodological landscape of biofilm analysis is provided, including an evaluation of the current trends in methodological research. We deem this worthwhile because a keyword-oriented bibliographical search reveals that less than 5% of the biofilm literature is devoted to methodology. In this report, we (i) summarize current methodologies for biofilm characterization, monitoring, and quantification; (ii) discuss advances in the discovery of effective imaging and sensing tools and modalities; (iii) provide an overview of tailored animal models that assess features of biofilm infections; and (iv) make recommendations defining the most appropriate methodological tools for clinical settings.

Attaching NorA efflux pump inhibitors to methylene blue enhances antimicrobial photodynamic inactivation of Escherichia coli and Acinetobacter baumannii in vitro and in vivo.

Resistance of bacteria to antibiotics is a public health concern worldwide due to the increasing failure of standard antibiotic therapies. Antimicrobial photodynamic inactivation (aPDI) is a promising non-antibiotic alternative for treating localized bacterial infections that uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species and kill microbes. Phenothiazinium photosensitizers like methylene blue (MB) and toluidine blue O are hydrophobic cations that are naturally expelled from bacterial cells by multidrug efflux pumps, which reduces their effectiveness. We recently reported the discovery of a NorA efflux pump inhibitor-methylene blue (EPI-MB) hybrid compound INF55-(Ac)en-MB that shows enhanced photodynamic inactivation of the Gram-positive bacterium methicillin-resistant Staphylococcus aureus (MRSA) relative to MB, both in vitro and in vivo. Here, we report the surprising observation that INF55-(Ac)en-MB and two related hybrids bearing the NorA efflux pump inhibitors INF55 and INF271 also show enhanced aPDI activity in vitro (relative to MB) against the Gram-negative bacteria Escherichia coli and Acinetobacter baumannii, despite neither species expressing the NorA pump. Two of the hybrids showed superior effects to MB in murine aPDI infection models. The findings motivate wider exploration of aPDI with EPI-MB hybrids against Gram-negative pathogens and more detailed studies into the molecular mechanisms underpinning their activity.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

Attaching the NorA Efflux Pump Inhibitor INF55 to Methylene Blue Enhances Antimicrobial Photodynamic Inactivation of Methicillin-Resistant Staphylococcus aureus in Vitro and in Vivo.

Antimicrobial photodynamic inactivation (aPDI) uses photosensitizers (PSs) and harmless visible light to generate reactive oxygen species (ROS) and kill microbes. Multidrug efflux systems can moderate the phototoxic effects of PSs by expelling the compounds from cells. We hypothesized that increasing intracellular concentrations of PSs by inhibiting efflux with a covalently attached efflux pump inhibitor (EPI) would enhance bacterial cell phototoxicity and reduce exposure of neighboring host cells to damaging ROS. In this study, we tested the hypothesis by linking NorA EPIs to methylene blue (MB) and examining the photoantimicrobial activity of the EPI-MB hybrids against the human pathogen methicillin-resistant Staphylococcus aureus (MRSA). Photochemical/photophysical and in vitro microbiological evaluation of 16 hybrids carrying four different NorA EPIs attached to MB via four linker types identified INF55-(Ac)en-MB 12 as a lead. Compound 12 showed increased uptake into S. aureus cells and enhanced aPDI activity and wound healing effects (relative to MB) in a murine model of an abrasion wound infected by MRSA. The study supports a new approach for treating localized multidrug-resistant MRSA infections and paves the way for wider exploration of the EPI-PS hybrid strategy in aPDI.

Photoantimicrobials-are we afraid of the light?

Although conventional antimicrobial drugs have been viewed as miraculous cure-alls for the past 80 years, increasing antimicrobial drug resistance requires a major and rapid intervention. However, the development of novel but still conventional systemic antimicrobial agents, having only a single mode or site of action, will not alleviate the situation because it is probably only a matter of time until any such agents will also become ineffective. To continue to produce new agents based on this notion is unacceptable, and there is an increasing need for alternative approaches to the problem. By contrast, light-activated molecules called photoantimicrobials act locally via the in-situ production of highly reactive oxygen species, which simultaneously attack various biomolecular sites in the pathogenic target and therefore offer both multiple and variable sites of action. This non-specificity at the target circumvents conventional mechanisms of resistance and inhibits the development of resistance to the agents themselves. Photoantimicrobial therapy is safe and easy to implement and, unlike conventional agents, the activity spectrum of photoantimicrobials covers bacteria, fungi, viruses, and protozoa. However, clinical trials of these new, truly broad-spectrum, and minimally toxic agents have been few, and the funding for research and development is almost non-existent. Photoantimicrobials constitute one of the few ways forward through the morass of drug-resistant infectious disease and should be fully explored. In this Personal View, we raise awareness of the novel photoantimicrobial technologies that offer a viable alternative to conventional drugs in many relevant application fields, and could thus slow the pace of resistance development.

Pneumonia and Pleural Empyema due to a Mixed Lactobacillus spp. Infection as a Possible Early Esophageal Carcinoma Signature.

Lactobacilli are human commensals found in the gastrointestinal and genitourinary tract. Although generally conceived as non-pathogenic microorganisms, the existence of several reports implicating them in certain severe pathological entities renders this species as opportunistic pathogens. The case of a 58-year-old woman with mixed Lactobacillus infection is described. The patient was admitted in an outpatient clinic with community acquired pneumonia, and on the third day of hospitalization she presented rapid pneumonia deterioration. Subsequent imaging techniques revealed increased pleural empyema in alignment with the general deterioration of her clinical condition. Pleural fluid culture revealed the presence of Lactobacillus delbrueckii and Lactobacillus gasseri and the infection was successfully treated with clindamycin. Five months after hospital discharge and an overall good condition, the patient developed signs of dysphagia and upon re-admission an inoperable esophageal carcinoma was diagnosed. The patient succumbed to the cancer 11 months later. Herein, we report for the first time a mixed respiratory infection due to lactobacilli, possibly associated with a formerly unveiled esophageal malignancy.

Preventing Data Ambiguity in Infectious Diseases with Four-Dimensional and Personalized Evaluations.

BACKGROUND: Diagnostic errors can occur, in infectious diseases, when anti-microbial immune responses involve several temporal scales. When responses span from nanosecond to week and larger temporal scales, any pre-selected temporal scale is likely to miss some (faster or slower) responses. Hoping to prevent diagnostic errors, a pilot study was conducted to evaluate a four-dimensional (4D) method that captures the complexity and dynamics of infectious diseases. METHODS: Leukocyte-microbial-temporal data were explored in canine and human (bacterial and/or viral) infections, with: (i) a non-structured approach, which measures leukocytes or microbes in isolation; and (ii) a structured method that assesses numerous combinations of interacting variables. Four alternatives of the structured method were tested: (i) a noise-reduction oriented version, which generates a single (one data point-wide) line of observations; (ii) a version that measures complex, three-dimensional (3D) data interactions; (iii) a non-numerical version that displays temporal data directionality (arrows that connect pairs of consecutive observations); and (iv) a full 4D (single line-, complexity-, directionality-based) version. RESULTS: In all studies, the non-structured approach revealed non-interpretable (ambiguous) data: observations numerically similar expressed different biological conditions, such as recovery and lack of recovery from infections. Ambiguity was also found when the data were structured as single lines. In contrast, two or more data subsets were distinguished and ambiguity was avoided when the data were structured as complex, 3D, single lines and, in addition, temporal data directionality was determined. The 4D method detected, even within one day, changes in immune profiles that occurred after antibiotics were prescribed. CONCLUSIONS: Infectious disease data may be ambiguous. Four-dimensional methods may prevent ambiguity, providing earlier, in vivo, dynamic, complex, and personalized information that facilitates both diagnostics and selection or evaluation of anti-microbial therapies.

Terms of endearment: Bacteria meet graphene nanosurfaces.

Microbial multidrug resistance poses serious risks in returning the human species into the pre-antibiotic era if it remains unsolved. While conventional research approaches to combat infectious diseases have been inadequate, nanomaterials are a promising alternative for the development of sound antimicrobial countermeasures. Graphene, a two-dimensional ultra-thin nanomaterial, possesses excellent electronic and biocompatibility properties, which position it in the biotechnology forefront for diverse applications in biosensing, therapeutics, diagnostics, drug delivery and device development. Yet, several questions remain unanswered. For instance, the way these nanosurfaces interact with the microbial entities is poorly understood. The mechanistic elucidation of this interface seems critical to determine the feasibility of applications under development. Are graphene derivatives appropriate materials to design potent antimicrobial agents, vehicles or effective diagnostic microsensors? Has the partition of major microbial resistance phenotypic determinants been sufficiently investigated? Can toxicity become a limiting factor? Are we getting closer to clinical implementation? To facilitate research conducive to answer such questions, this review describes the features of the graphene-bacterial interaction. An overview on paradigms of graphene-microbial interactions is expected to shed light on the range of materials available, and identify possible applications, serving the ultimate goal to develop deeper understanding and collective conscience for the true capabilities of this nanomaterial platform.

Harnessing the power of light to treat staphylococcal infections focusing on MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become the most important drug-resistant microbial pathogen in countries throughout the world. Morbidity and mortality due to MRSA infections continue to increase despite efforts to improve infection control measures and to develop new antibiotics. Therefore alternative antimicrobial strategies that do not give rise to development of resistance are urgently required. A group of therapeutic interventions has been developed in the field of photomedicine with the common theme that they rely on electromagnetic radiation with wavelengths between 200 and 1000 nm broadly called "light". These techniques all use simple absorption of photons by specific chromophores to deliver the killing blow to microbial cells while leaving the surrounding host mammalian cells relatively unharmed. Photodynamic inactivation uses dyes called photosensitizers (PS) that bind specifically to MRSA cells and not host cells, and generate reactive oxygen species including singlet oxygen and singlet oxygen upon illumination. Sophisticated molecular strategies to target the PS to MRSA cells have been designed. Ultraviolet C radiation can damage microbial DNA without unduly harming host DNA. Blue light can excite endogenous porphyrins and flavins in MRSA cells that are not present in host cells. Near-infrared lasers can interfere with microbial membrane potentials without raising the temperature of the tissue. Taken together these innovative approaches towards harnessing the power of light suggest that the ongoing threat of MRSA may eventually be defeated.