Skin repair and infection control in diabetic, obese mice using bioactive laser-activated sealants.

Conventional wound approximation devices, including sutures, staples, and glues, are widely used but risk of wound dehiscence, local infection, and scarring can be exacerbated in these approaches, including in diabetic and obese individuals. This study reports the efficacy and quality of tissue repair upon photothermal sealing of full-thickness incisional skin wounds using silk fibroin-based laser-activated sealants (LASEs) containing copper chloride salt (Cu-LASE) or silver nanoprisms (AgNPr-LASE), which absorb and convert near-infrared (NIR) laser energy to heat. LASE application results in rapid and effective skin sealing in healthy, immunodeficient, as well as diabetic and obese mice. Although lower recovery of epidermal structure and function was seen with AgNPr-LASE sealing, likely because of the hyperthermia induced by laser and presence of this material in the wound space, this approach resulted in higher enhancement in recovery of skin biomechanical strength compared to sutures and Cu-LASEs in diabetic, obese mice. Histological and immunohistochemical analyses revealed that AgNPr-LASEs resulted in significantly lower neutrophil migration to the wound compared to Cu-LASEs and sutures, indicating a more muted inflammatory response. Cu-LASEs resulted in local tissue toxicity likely because of effects of copper ions as manifested in the form of a significant epidermal gap and a 'depletion zone', which was a region devoid of viable cells proximal to the wound. Compared to sutures, LASE-mediated sealing, in later stages of healing, resulted in increased angiogenesis and diminished myofibroblast activation, which can be indicative of lower scarring. AgNPr-LASE loaded with vancomycin, an antibiotic drug, significantly lowered methicillin-resistant Staphylococcus aureus (MRSA) load in a pathogen challenge model in diabetic and obese mice and also reduced post-infection inflammation of tissue compared to antibacterial sutures. Taken together, these attributes indicate that AgNPr-LASE demonstrated a more balanced quality of tissue sealing and repair in diabetic and obese mice and can be used for combating local infections, that can result in poor healing in these individuals.

M. tuberculosis PrrA binds the dosR promoter and regulates mycobacterial adaptation to hypoxia.

The PrrAB two-component system (TCS) is essential for Mycobacterium tuberculosis viability. Previously, it was demonstrated that PrrA binds DNA in the absence of PrrB-mediated transphosphorylation and that non-cognate serine/threonine-kinases phosphorylate PrrA threonine-6 (T6). Therefore, we investigated the differential binding affinity and regulatory properties of the M. tuberculosis-derived wild-type PrrA, PrrA phosphomimetic (D58E, T6E), and PrrA phosphoablative (D58A, T6A) proteins with the prrAMtb, dosRMtb, and cydAMtb genes. While we hypothesized greater DNA binding affinity and more pronounced regulation by PrrA phosphomimetic variants, recombinant, wild-type PrrAMtb bound DNA with greatest affinity. Collectively, wild-type PrrAMtb recombinant protein displayed the highest binding affinity to the dosRMtb promoter (KD 3.46 ± 2.09 nM), followed by the prrAMtb promoter (KD 9.00 ± 2.66 nM). To establish PrrAMtb regulatory activity, we constructed M. smegmatis ΔprrABMsmeg::prrAMtb strains with each of the PrrAMtb variants and extrachromosomal prrAMtb, dosRMtb, and cydAMtb promoter-mCherry reporter fusions. Our findings showed that PrrAMtb is autoregulatory and induces dosRMtb expression only during in vitro, hypoxic growth. Combined expression of prrABMtb in M. smegmatis ΔprrAB significantly induced cydAMtb promoter-mCherry expression. Our studies advanced the understanding of PrrA function and PrrAB phosphorylation-mediated regulatory mechanisms and control of mycobacterial dosR and cydA hypoxic and low-oxygen responsive genes.

Deep Learning-Based Culture-Free Bacteria Detection in Urine Using Large-Volume Microscopy.

Bacterial infections, increasingly resistant to common antibiotics, pose a global health challenge. Traditional diagnostics often depend on slow cell culturing, leading to empirical treatments that accelerate antibiotic resistance. We present a novel large-volume microscopy (LVM) system for rapid, point-of-care bacterial detection. This system, using low magnification (1-2×), visualizes sufficient sample volumes, eliminating the need for culture-based enrichment. Employing deep neural networks, our model demonstrates superior accuracy in detecting uropathogenic Escherichia coli compared to traditional machine learning methods. Future endeavors will focus on enriching our datasets with mixed samples and a broader spectrum of uropathogens, aiming to extend the applicability of our model to clinical samples.

Rapid Detection of Urinary Tract Infection in 10 min by Tracking Multiple Phenotypic Features in a 30 s Large-Volume Scattering Video of Urine Microscopy.

Rapid point-of-care (POC) diagnosis of bacterial infection diseases provides clinical benefits of prompt initiation of antimicrobial therapy and reduction of the overuse/misuse of unnecessary antibiotics for nonbacterial infections. We present here a POC compatible method for rapid bacterial infection detection in 10 min. We use a large-volume solution scattering imaging (LVSi) system with low magnifications (1-2×) to visualize bacteria in clinical samples, thus eliminating the need for culture-based isolation and enrichment. We tracked multiple intrinsic phenotypic features of individual cells in a short video. By clustering these features with a simple machine learning algorithm, we can differentiate Escherichia coli from similar-sized polystyrene beads, distinguish bacteria with different shapes, and distinguish E. coli from urine particles. We applied the method to detect urinary tract infections in 104 patient urine samples with a 30 s LVSi video, and the results showed 92.3% accuracy compared with the clinical culture results. This technology provides opportunities for rapid bacterial infection diagnosis at POC settings.

High-Throughput Screening Identifies Synthetic Peptides with Antibacterial Activity against Mycobacterium abscessus and Serum Stability.

The rise in antibiotic resistance in bacteria has spawned new technological approaches for identifying novel antimicrobials with narrow specificity. Current antibiotic treatment regimens and antituberculosis drugs are not effective in treating Mycobacterium abscessus. Meanwhile, antimicrobial peptides are gaining prominence as alternative antimicrobials due to their specificity toward anionic bacterial membranes, rapid action, and limited development of resistance. To rapidly identify antimicrobial peptide candidates, our group has developed a high-density peptide microarray consisting of 125,000 random synthetic peptides screened for interaction with the mycobacterial cell surface of M. abscessus morphotypes. From the array screening, peptides positive for interaction were synthesized and their antimicrobial activity was validated. Overall, six peptides inhibited the M. abscessus smooth morphotype (IC50 = 1.7 µM for all peptides) and had reduced activity against the M. abscessus rough morphotype (IC50 range: 13-82 µM). Peptides ASU2056 and ASU2060 had minimum inhibitory concentration values of 32 and 8 µM, respectively, against the M. abscessus smooth morphotype. Additionally, ASU2060 (8 µM) was active against Escherichia coli, including multidrug-resistant E. coli clinical isolates, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. ASU2056 and ASU2060 exhibited no significant hemolytic activity at biologically relevant concentrations, further supporting these peptides as promising therapeutic candidates. Moreover, ASU2060 retained antibacterial activity after preincubation in human serum for 24 h. With antimicrobial resistance on the rise, methods such as those presented here will streamline the peptide discovery process for targeted antimicrobial peptides.

Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.

Complete Genome Sequence of the Methicillin-Resistant Staphylococcus aureus Strain SQL1/USA300, Used for Testing the Antimicrobial Properties of Clay Phyllosilicates and Customized Aluminosilicates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium that causes community-acquired and health care-acquired infections. We previously demonstrated that clay phyllosilicates and customized aluminosilicates display antimicrobial activity against the MRSA strain SQL1. The SQL1 annotated genome reveals a USA300 lineage and contributes critical knowledge of the MRSA virulence factors associated with tissue infection.

Fibrinolytic Serine Proteases, Therapeutic Serpins and Inflammation: Fire Dancers and Firestorms.

The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, "off-the-shelf" system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the serine protease inhibitors, termed serpins. Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a "respiratory" coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.

Rapid Antimicrobial Susceptibility Testing on Clinical Urine Samples by Video-Based Object Scattering Intensity Detection.

To combat the ongoing public health threat of antibiotic-resistant infections, a technology that can quickly identify infecting bacterial pathogens and concurrently perform antimicrobial susceptibility testing (AST) in point-of-care settings is needed. Here, we develop a technology for point-of-care AST with a low-magnification solution scattering imaging system and a real-time video-based object scattering intensity detection method. The low magnification (1-2×) optics provides sufficient volume for direct imaging of bacteria in urine samples, avoiding the time-consuming process of culture-based bacterial isolation and enrichment. Scattering intensity from moving bacteria and particles in the sample is obtained by subtracting both spatial and temporal background from a short video. The time profile of scattering intensity is correlated with the bacterial growth rate and bacterial response to antibiotic exposure. Compared to the image-based bacterial tracking and counting method we previously developed, this simple image processing algorithm accommodates a wider range of bacterial concentrations, simplifies sample preparation, and greatly reduces the computational cost of signal processing. Furthermore, development of this simplified processing algorithm eases implementation of multiplexed detection and allows real-time signal readout, which are essential for point-of-care AST applications. To establish the method, 130 clinical urine samples were tested, and the results demonstrated an accuracy of ∼92% within 60-90 min for UTI diagnosis. Rapid AST of 55 positive clinical samples revealed 98% categorical agreement with both the clinical culture results and the on-site parallel AST validation results. This technology provides opportunities for prompt infection diagnosis and accurate antibiotic prescriptions in point-of-care settings.

Antimicrobial laser-activated sealants for combating surgical site infections.

Surgical-site infections (SSIs) occur in 2-5% of patients undergoing surgery in the US alone, impacting 300 000-500 000 lives each year, and presenting up to 11 times greater risk of death compared to patients without SSIs. The most common cause of SSI is Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA) is the most common pathogen in community hospitals. Current clinical devices used for approximating incisions and traumatic lacerations include sutures, adhesives, tapes, or staples with or without antimicrobial incorporation. However, current closure technologies may not provide adequate protection against infection, are susceptible to wound dehiscence, and can result in delayed biomechanical recoveries. Laser-activated tissue repair is a sutureless technique in which chromophore-loaded sealants convert laser light energy to heat in order to induce rapid tissue sealing. Here, we describe the generation and evaluation of laser-activated sealant (LASE) biomaterials, in which, indocyanine green (ICG), an FDA-approved dye, was embedded in a silk fibroin matrix and cast into films as wound sealants. Silk-ICG films were subjected to different near-infrared (NIR) laser powers to identify temperatures optimal for laser sealing of soft tissues. A mathematical model was developed in order to determine the photothermal conversion efficiency of LASEs following laser irradiation. NIR laser activation of silk-ICG LASEs increased the recovery of skin biomechanical strength compared to sutured skin in full-thickness incisional wounds in immunocompetent mice, and live animal imaging indicated persistence of silk-ICG LASEs over several days. LASEs loaded with the antibiotic vancomycin demonstrated higher efficacies for combating MRSA infections in a mouse model of surgical site infection compared to antibacterial sutures. Our results demonstrate that LASEs can be loaded with antimicrobial drugs and may serve as new multifunctional biomaterials for rapid tissue sealing, repair and surgical site protection following surgery.

Direct Antimicrobial Susceptibility Testing on Clinical Urine Samples by Optical Tracking of Single Cell Division Events.

With the increasing prevalence of antibiotic resistance, the need to develop antimicrobial susceptibility testing (AST) technologies is urgent. The current challenge has been to perform the antibiotic susceptibility testing in short time, directly with clinical samples, and with antibiotics over a broad dynamic range of clinically relevant concentrations. Here, a technology for point-of-care diagnosis of antimicrobial-resistant bacteria in urinary tract infections, by imaging the clinical urine samples directly with an innovative large volume solution scattering imaging (LVSi) system and analyzing the image sequences with a single-cell division tracking method is developed. The high sensitivity of single-cell division tracking associated with large volume imaging enables rapid antibiotic susceptibility testing directly on the clinical urine samples. The results demonstrate direct detection of bacterial infections in 60 clinical urine samples with a 60 min LVSi video, and digital AST of 30 positive clinical samples with 100% categorical agreement with both the clinical culture results and the on-site agar plating validation results. This technology provides opportunities for precise antibiotic prescription and proper treatment of the patient within a single clinic visit.

Synthesized Geopolymers Adsorb Bacterial Proteins, Toxins, and Cells.

Pore-forming and hemolytic toxins are bacterial cytotoxic proteins required for virulence in many pathogens, including staphylococci and streptococci, and are notably associated with clinical manifestations of disease. Inspired by adsorption properties of naturally occurring aluminosilicates, we engineered inexpensive, laboratory-synthesized, aluminosilicate geopolymers with controllable pore and surface characteristics to remove pathogenic or cytotoxic material from the surrounding environment. In this study, macroporous and mesoporous geopolymers were produced with and without stearic acid surface modifications. Geopolymer binding efficacies were assessed by measuring adsorption of methicillin-resistant Staphylococcus aureus (MRSA) culture filtrate proteins, α-hemolysin and streptolysin-O toxins, MRSA whole cells, and antibiotics. Macroporous and mesoporous geopolymers were strong non-selective adsorbents for bacterial protein, protein toxins, and bacteria. Although some geopolymers adsorbed antibiotics, these synthesized geopolymers could potentially be used in non-selective adsorptive applications and optimized for adsorption of specific biomolecules.

Rapid antibiotic susceptibility testing based on bacterial motion patterns with long short-term memory neural networks.

Antibiotic resistance is an increasing public health threat. To combat it, a fast method to determine the antibiotic susceptibility of infecting pathogens is required. Here we present an optical imaging-based method to track the motion of single bacterial cells and generate a model to classify active and inactive cells based on the motion patterns of the individual cells. The model includes an image-processing algorithm to segment individual bacterial cells and track the motion of the cells over time, and a deep learning algorithm (Long Short-Term Memory network) to learn and determine if a bacterial cell is active or inactive. By applying the model to human urine specimens spiked with an Escherichia coli lab strain, we show that the method can accurately perform antibiotic susceptibility testing as fast as 30 minutes for five commonly used antibiotics.

Comparative transcriptomics reveals PrrAB-mediated control of metabolic, respiration, energy-generating, and dormancy pathways in Mycobacterium smegmatis.

BACKGROUND: Mycobacterium smegmatis is a saprophytic bacterium frequently used as a genetic surrogate to study pathogenic Mycobacterium tuberculosis. The PrrAB two-component genetic regulatory system is essential in M. tuberculosis and represents an attractive therapeutic target. In this study, transcriptomic analysis (RNA-seq) of an M. smegmatis ΔprrAB mutant was used to define the PrrAB regulon and provide insights into the essential nature of PrrAB in M. tuberculosis. RESULTS: RNA-seq differential expression analysis of M. smegmatis wild-type (WT), ΔprrAB mutant, and complementation strains revealed that during in vitro exponential growth, PrrAB regulates 167 genes (q < 0.05), 57% of which are induced in the WT background. Gene ontology and cluster of orthologous groups analyses showed that PrrAB regulates genes participating in ion homeostasis, redox balance, metabolism, and energy production. PrrAB induced transcription of dosR (devR), a response regulator gene that promotes latent infection in M. tuberculosis and 21 of the 25 M. smegmatis DosRS regulon homologues. Compared to the WT and complementation strains, the ΔprrAB mutant exhibited an exaggerated delayed growth phenotype upon exposure to potassium cyanide and respiratory inhibition. Gene expression profiling correlated with these growth deficiency results, revealing that PrrAB induces transcription of the high-affinity cytochrome bd oxidase genes under both aerobic and hypoxic conditions. ATP synthesis was ~ 64% lower in the ΔprrAB mutant relative to the WT strain, further demonstrating that PrrAB regulates energy production. CONCLUSIONS: The M. smegmatis PrrAB two-component system regulates respiratory and oxidative phosphorylation pathways, potentially to provide tolerance against the dynamic environmental conditions experienced in its natural ecological niche. PrrAB positively regulates ATP levels during exponential growth, presumably through transcriptional activation of both terminal respiratory branches (cytochrome c bc1-aa3 and cytochrome bd oxidases), despite transcriptional repression of ATP synthase genes. Additionally, PrrAB positively regulates expression of the dormancy-associated dosR response regulator genes in an oxygen-independent manner, which may serve to fine-tune sensory perception of environmental stimuli associated with metabolic repression.

Rapid Antimicrobial Susceptibility Testing of Patient Urine Samples Using Large Volume Free-Solution Light Scattering Microscopy.

The emergence of antibiotic resistance has prompted the development of rapid antimicrobial susceptibility testing (AST) technologies that will enable evidence-based treatment and promote antimicrobial stewardship. To date, many rapid AST methods have been developed, but few are able to be performed on clinical samples directly. Here we developed a large volume light scattering microscopy technique that tracks phenotypic features of single bacterial cells directly in clinical urine samples without sample enrichment or culturing. The technique demonstrated rapid (90 min) detection of Escherichia coli in 24 clinical urine samples with 100% sensitivity and 83% specificity and rapid (90 min) AST in 12 urine samples with 87.5% categorical agreement with two antibiotics, ampicillin and ciprofloxacin.

Correction to: Comparative transcriptomics reveals PrrABmediated control of metabolic, respiration, energy-generating, and dormancy pathways in Mycobacterium smegmatis.

Following the publication of the original article [1], the authors reported an error in Fig. 2 of the PDF version of their article.

Superior ion release properties and antibacterial efficacy of nanostructured zeolites ion-exchanged with zinc, copper, and iron.

Antimicrobial zeolites ion-exchanged with inexpensive transition metal ions (such as zinc, copper, and iron) are critically important for a broader adoption of the materials for public health applications. Due to the high surface area and small particle sizes, nanozeolites are particularly promising in enhancing the efficacy of the zeolite-based antimicrobial materials. By using highly-crystalline nanostructured zeolites (FAU) with textural mesoporosity, we report a comprehensive study on the materials characteristics of zinc-, copper-, and iron-ion exchanged nanozeolites, the ion release properties, and antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA), as well as a comparison of the properties to those obtained for the corresponding microsized zeolites. Superior ion release properties were observed for both zinc and copper ion-exchanged nanostructured zeolite X, with ion release up to 73% for zinc and 36% for copper of their initial loadings, as compared to 50% and 12%, respectively, for the corresponding microsized zeolites, validating the importance of nanostructuring for enhanced ion diffusion through zeolite pore channels. The 2 hours minimum bactericidal concentration (MBC) in saline for the copper ion-exchanged nanostructured zeolite X was 32 µg mL-1, half the corresponding microsized zeolite X MBC of 64 µg mL-1. Our results established nanostructured zeolite X as a superior host material for metal ion-based antimicrobials, with the aforementioned improvements for copper-exchanged nanozeolites compared to previous studies.

Mycobacterium smegmatis PrrAB two-component system influences triacylglycerol accumulation during ammonium stress.

The PrrAB two-component system is conserved across all sequenced mycobacterial species and is essential for viability in Mycobacterium tuberculosis, thus making it a promising drug target. The prrAB operon was successfully deleted in nonpathogenic Mycobacterium smegmatis, and the ∆prrAB mutant strain exhibited clumping in ammonium-limited medium and significantly reduced growth during ammonium and hypoxic stress. To assess the influence of M. tuberculosis PrrA overexpression, we constructed a recombinant M. smegmatis ∆prrAB mutant strain which overexpresses M. tuberculosis prrA. M. smegmatis prrAB and M. tuberculosis prrA complemented the M. smegmatis ∆prrAB deletion mutant in Middlebrook M7H9 and ammonium-limited media and during hypoxic and ammonium stress. Based on quantitative untargeted mass spectrometry-based lipidomics, triacylglycerol lipid species were significantly upregulated in the ∆prrAB mutant strain compared to the wild-type when cultured in ammonium-limited medium, revealing that M. smegmatis PrrAB influences triacylglycerol levels during ammonium stress. These results were qualitatively corroborated by thin-layer chromatography. Furthermore, the ∆prrAB mutant significantly upregulated expression of several genes (glpK, GPAT, WS/DGAT, accA3, accD4, accD6 and Ag85C) that participate in triacylglycerol and lipid biosynthetic pathways, thus corroborating the lipidomics analyses.

Building and Breaking the Cell Wall in Four Acts: A Kinesthetic and Tactile Role-Playing Exercise for Teaching Beta-Lactam Antibiotic Mechanism of Action and Resistance.

"Building and breaking the cell wall" is designed to review the bacterial cell envelope, previously learned in lower-division biology classes, while introducing new topics such as antibiotics and bacterial antibiotic resistance mechanisms. We developed a kinesthetic and tactile modeling activity where students act as cellular components and construct the cell wall. In the first two acts, students model a portion of the gram-positive bacterial cell envelope and then demonstrate in detail how the peptidoglycan is formed. Act III involves student demonstration of the addition of ß-lactam antibiotics to the environment and how they inhibit the formation of peptidoglycan, thereby preventing bacterial replication. Using Staphylococcus aureus as a model for gram-positive bacteria, students finish the activity (Act IV) by acting out how S. aureus often becomes resistant to ß-lactam antibiotics. A high level of student engagement was observed, and the activity received positive feedback. In an assessment administered prior to and two months after the activity, significant improvements in scores were observed (p < 0.0001), demonstrating increased understanding and retention. This activity allows students to (i) visualize, role play, and kinesthetically "build" the cell envelope and form the peptidoglycan layer, (ii) understand the mechanism of action for ß-lactam antibiotics, as well as how gene acquisition and protein changes result in resistance, and (iii) work cooperatively and actively to promote long-term retention of the subject material.