Mimicking kidney flow shear efficiently induces aggregation of LECT2, a protein involved in renal amyloidosis.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Persister control by leveraging dormancy associated reduction of antibiotic efflux.

Persistent bacterial infections do not respond to current antibiotic treatments and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 µg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 µg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles' heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.

Cadiolide analogues and their precursors as new inhibitors of bacterial quorum sensing and biofilm formation.

Bacterial quorum sensing (QS) and biofilm formation are promising targets for developing new therapies to treat chronic infections. Herein, we report the stereoselective synthesis of 18 new analogs of natural cadiolides. Among the new compounds, substances 8b, 8f, 8i, 9a, 9b and 9e completely inhibited the biofilm formation of Escherichia coli RP347 in vitro. In addition, compound 8b interfered acyl-homoserine lactone (AHL) mediated QS, while 9e interrupted the QS via autoinducer-2 (AI-2). Biological assays also revealed that synthetic intermediates alkynones are potent inhibitors of AI-2 and AHL-mediated QS. These results indicate that cadiolides and alkynones are good candidates for further structural modification for a new generation of more potent antimicrobial agents.

Development of the computational antibiotic screening platform (CLASP) to aid in the discovery of new antibiotics.

Bacterial colonization of biotic and abiotic surfaces and antibiotic resistance are grand challenges with paramount societal impacts. However, in the face of increasing bacterial resistance to all known antibiotics, efforts to discover new classes of antibiotics have languished, creating an urgent need to accelerate the antibiotic discovery pipeline. A major deterrent in the discovering of new antibiotics is the limited permeability of molecules across the bacterial envelope. Notably, the Gram-negative bacteria have nutrient specific protein channels (or porins) that restrict the permeability of non-essential molecules, including antibiotics. Here, we have developed the Computational Antibiotic Screening Platform (CLASP) for screening of potential drug molecules through the porins. The CLASP takes advantage of coarse grain (CG) resolution, advanced sampling techniques, and a parallel computing environment to maximize its performance. The CLASP yields comprehensive thermodynamic and kinetic output data of a potential drug molecule within a few hours of wall-clock time. Its output includes the potential of mean force profile, energy barrier, the rate constant, and contact analysis of the molecule with the pore-lining residues, and the orientational analysis of the molecule in the porin channel. In our first CLASP application, we report the transport properties of six carbapenem antibiotics-biapenem, doripenem, ertapenem, imipenem, meropenem, and panipenem-through OccD3, a major channel for carbapenem uptake in Pseudomonas aeruginosa. The CLASP is designed to screen small molecule libraries with a fast turnaround time to yield structure-property relationships to discover antibiotics with high permeability. The CLASP will be freely distributed to enable accelerated antibiotic drug discovery.

High-Level Antibiotic Tolerance of a Clinically Isolated Enterococcus faecalis Strain.

Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 µg · ml-1) were 25.4% ± 4.3% and 51.9% ± 4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections.IMPORTANCEEnterococcus faecalis represents a major group of pathogens causing nosocomial infections that are resistant to multiple classes of antibiotics. An important challenge associated with E. faecalis infection is the emergence of multidrug-tolerant strains, which have normal MICs but do not respond to antibiotic treatment. Here, we report a strain of E. faecalis that was isolated from a cystic fibrosis patient and demonstrated high-level tolerance to ampicillin, vancomycin, and tetracycline. Whole-genome sequencing revealed critical substitutions in one of the tetracycline efflux pump repressors that are consistent with the increased tolerance of E. faecalis UM001B to tetracycline. These findings provide new information about bacterial antibiotic tolerance and may help develop more effective therapeutics.

Antibiotic Susceptibility of Escherichia coli Cells during Early-Stage Biofilm Formation.

Bacteria form complex multicellular structures on solid surfaces known as biofilms, which allow them to survive in harsh environments. A hallmark characteristic of mature biofilms is the high-level antibiotic tolerance (up to 1,000 times) compared with that of planktonic cells. Here, we report our new findings that biofilm cells are not always more tolerant to antibiotics than planktonic cells in the same culture. Specifically, Escherichia coli RP437 exhibited a dynamic change in antibiotic susceptibility during its early-stage biofilm formation. This phenomenon was not strain specific. Upon initial attachment, surface-associated cells became more sensitive to antibiotics than planktonic cells. By controlling the cell adhesion and cluster size using patterned E. coli biofilms, cells involved in the interaction between cell clusters during microcolony formation were found to be more susceptible to ampicillin than cells within clusters, suggesting a role of cell-cell interactions in biofilm-associated antibiotic tolerance. After this stage, biofilm cells became less susceptible to ampicillin and ofloxacin than planktonic cells. However, when the cells were detached by sonication, both antibiotics were more effective in killing the detached biofilm cells than the planktonic cells. Collectively, these results indicate that biofilm formation involves active cellular activities in adaption to the attached life form and interactions between cell clusters to build the complex structure of a biofilm, which can render these cells more susceptible to antibiotics. These findings shed new light on bacterial antibiotic susceptibility during biofilm formation and can guide the design of better antifouling surfaces, e.g., those with micron-scale topographic structures to interrupt cell-cell interactions.IMPORTANCE Mature biofilms are known for their high-level tolerance to antibiotics; however, antibiotic susceptibility of sessile cells during early-stage biofilm formation is not well understood. In this study, we aim to fill this knowledge gap by following bacterial antibiotic susceptibility during early-stage biofilm formation. We found that the attached cells have a dynamic change in antibiotic susceptibility, and during certain phases, they can be more sensitive to antibiotics than planktonic counterparts in the same culture. Using surface chemistry-controlled patterned biofilm formation, cell-surface and cell-cell interactions were found to affect the antibiotic susceptibility of attached cells. Collectively, these findings provide new insights into biofilm physiology and reveal how adaptation to the attached life form may influence antibiotic susceptibility of bacterial cells.

Conjugation in Escherichia coli Biofilms on Poly(dimethylsiloxane) Surfaces with Microtopographic Patterns.

Bacterial biofilms are highly tolerant to antimicrobials and play an important role in the development and spread of antibiotic resistance based on horizontal gene transfer due to close cell-to-cell contact. As an important surface property, topography has been shown to affect bacterial adhesion and biofilm formation. Here, we demonstrate that micrometer-scale surface topographies also affect horizontal gene transfer through conjugation in bacterial biofilms. Specifically, biofilm formation and associated conjugation on poly(dimethylsiloxane) (PDMS) surfaces with 10 µm tall protruding patterns were studied using fluorescently labeled donor and recipient strains of Escherichia coli. The results demonstrate that square-shaped topographic patterns with side length of 20, 50, and 100 µm and interpattern distance equal to or larger than 10 µm promote biofilm formation and conjugation compared to the smooth control. The vertical sides of these topographic features were found to be the "hot spots" for bacterial conjugation compared to the top of patterns and grooves between topographic features. The increase in conjugation frequency on the sides of topographic patterns was attributed to the high cell density of recipient cells at these locations. A motility (motB) mutant of the recipient strain exhibited defects in biofilm formation at the "hot spots" and conjugation, which were recovered by complementing the motB gene on a plasmid. These results also provided guidance for designing surface topographies that can reduce conjugation. Specifically, 10 µm tall hexagon-shaped topographic patterns with side length of 15 µm and interpattern distance of 2 µm were prepared to reduce biofilm formation on the side of protruding patterns and interrupt cell-cell interaction in the grooves. This topography exhibited 85% and 46% reduction of biofilm formation and associated conjugation, respectively, compared to the smooth control.

Phagocytosis of Escherichia coli biofilm cells with different aspect ratios: a role of substratum material stiffness.

Bacterial biofilms play an important role in chronic infections due to high-level tolerance to antibiotics. Thus, it is important to eradicate bacterial cells that are attached to implanted medical devices of different materials. Phagocytosis is a key process of the innate immunity to eliminate invading pathogens. Previous research demonstrated that the efficiency of phagocytosis is affected by the aspect ratio of polymer beads. Recently, we reported that the stiffness of polydimethylsiloxane (PDMS) influences Escherichia coli biofilm formation and the biofilm cells on stiff (5:1) PDMS are 46.2% shorter than those on soft (40:1) PDMS. Based on these findings, we hypothesized that E. coli cells attached on stiff PDMS can be more effectively removed via phagocytosis. This hypothesis was tested in the present study using viability assays, flow cytometry, and cell tracking. The results revealed that shorter E. coli cells detached from stiff PDMS were easier to be phagocytized than the longer cells from soft PDMS surfaces. Furthermore, macrophage cells were found to be more motile on stiff PDMS surfaces and more effective at phagocytosis of E. coli cells attached on these surfaces. These results may help the design of better biomaterials to reduce fouling and associated infections.

Signaling factor interactions with polysaccharide aggregates of bacterial biofilms.

Biofilms are surface-attached colonies of bacteria embedded in an extracellular polymeric substance (EPS). Inside the eukaryotic hosts, bacterial biofilms interact with the host cells through signaling factors (SFs). These signaling processes play important roles in the interaction between bacteria and host cells and the outcome of infections and symbiosis. However, how host immune factors diffuse through biofilms is not well understood. Here, we describe synergistic molecular dynamics and experimental approaches for studying the translocation of signaling factors through polysaccharide chain aggregates present in the extracellular matrix of bacterial biofilms. The effect of polysaccharide chain degradation on the energetics of SF-EPS interactions was examined by simulating an EPS consisting of various polysaccharide chain lengths. It is shown that the SF stabilization energy, defined as the average potential of mean force difference between the environments outside and within the matrix, increases linearly with decreasing chain length. This effect has been explained based on the changes in the polysaccharide configurations around the SF. Specifically, shorter chains are packed tightly around the SF, promoting favorable SF-EPS interactions, while longer chains are packed loosely resulting in screening of interactions with neighboring chains. We further investigated the translocation of SFs through the host cell membrane using molecular dynamics simulations. Further, simulations predict the existence of energy barriers greater than 1000 kJ mol(-1) associated with the translocation of the signaling factors necrosis factor-alpha (TNF-α) and granulocyte macrophage colony stimulating factor (GM-CSF) across the lipid bilayer. The agreement of computational and experimental findings motivates future computational studies using a more detailed description of the EPS aimed at understanding the role of the extracellular matrix on biofilm drug resistance.

Synthetic dendrimeric peptide active against biofilm and persister cells of Pseudomonas aeruginosa.

Antimicrobial dendrimeric peptides (AMDP) are a relatively new class of agents displaying repetitive functional groups on a branched core. Previously, we have investigated the length requirement for antimicrobial activity of peptides consisting of repeated arginine (R) and tryptophan (W) side chains and found that even short linear RW repeats are active, providing a starting point for a de novo design of multivalent structures. In this study, we synthesized and tested a new synthetic dendrimer, 2D-24, for its antimicrobial activity against Pseudomonas aeruginosa, including the wild-type PAO1 and its mucoid mutant PDO300. This synthetic AMDP was found to kill planktonic cells of both PAO1 and PDO300 in a dose-dependent manner, with nearly complete killing of both strains observed when treated with 50 µM of this agent. In addition to planktonic cells, 2D-24 was also found to kill biofilm cells of both strains in a dose-dependent manner. For example, treatment with 30 µM 2D-24 led to 94.4 ± 1.4 and 93.9 ± 4.2 % killing of PAO1 and PDO300 biofilm cells, respectively. Furthermore, 2D-24 was effective in killing multidrug-tolerant persister cells of PAO1 and PDO300. While higher concentrations of 2D-24 were required to kill persister cells, combinations of 2D-24 with ciprofloxacin, tobramycin, or carbenicillin showed synergistic effects on killing persister cells of both strains. Based on hemolysis assays using sheep erythrocytes and a coculture model of PAO1 and human epithelial cells, 2D-24 was found to kill P. aeruginosa cells at concentrations that are not toxic to mammalian cells.

Stiffness of cross-linked poly(dimethylsiloxane) affects bacterial adhesion and antibiotic susceptibility of attached cells.

In this study, Escherichia coli RP437 and Pseudomonas aeruginosa PAO1 were used as model strains to investigate the early stage biofilm formation on poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness, which were prepared by controlling the degree of cross-linking (base:curing agent ratios of 5:1, 10:1, 20:1, and 40:1 were tested). An inverse correlation between cell adhesion and substrate stiffness was observed for both species. Interestingly, it was found that the cells attached on relatively stiff substrates (5:1 PDMS) were significantly smaller than those on relatively soft substrates (40:1 PDMS). In addition to the difference in size, the cells on 5:1 PDMS substrates were also found to be less susceptible to antibiotics, such as ofloxacin, ampicillin, and tobramycin, than the cells attached on 40:1 PDMS substrates. These results reveal that surface stiffness is an important material property that influences the attachment, growth, and stress tolerance of biofilm cells.

Micron-scale topographies affect phagocytosis of bacterial cells on polydimethylsiloxane surfaces.

Many medical devices implanted in patients to mitigate diseases and medical conditions have different types of topographic features. While appropriate textures can promote the integration of host cells and reduce scar tissue formation, some textured implants with inappropriate topographies have been associated with inflammation, bacterial colonization, or even malignant complications. To better understand how surface topography affects host immune response to colonizing bacteria, a protocol was developed to investigate phagocytosis of bacterial cells attached on polydimethylsiloxane (PDMS) surfaces with different square-shaped recessive patterns. The interaction between activated RAW 264.7 macrophages and Escherichia coli in recessive wells was visualized in 3D using multiple fluorescent markers. The results revealed that there is a threshold dimension of topography, below which phagocytosis of attached bacterial cells is significantly impeded. Specifically, under our experimental condition, up to 100-fold reduction in phagocytosis was observed in square-shaped patterns with 5 µm side length and 10 µm depth compared to the flat control and patterns with 10 µm or longer side length. The spacing between wells also showed significant effects; e.g., phagocytosis in the wells further decreased when spacing increased to 50 µm. These results are helpful for understanding how undesired topographies may contribute to bacterial colonization and thus infection and other associated complications. STATEMENT OF SIGNIFICANCE: Surface topography plays an important role in bacteria-material infections and thus the safety of implantable medical devices. Undesired topographic features can cause biofilm formation and related complications. However, how surface topography affects the capability of host immune cells to clear colonizing bacteria is not well understood. In this study, the interaction between macrophage RAW264.7 and colonizing E. coli cells on polydimethylsiloxane (PDMS) with recessive features is investigated. It was discovered that the size of recessive features and the spacing between these features have significant effects on phagocytosis of bacteria by macrophages. These new results are helpful for understanding the complex interaction among host cells, bacteria, and implanted biomaterials, which will help guide the rational design of safer medical devices.

Persistence and Culturability of Escherichia coli under Induced Toxin Expression.

BACKGROUND/OBJECTIVES: Bacteria are well known to enter dormancy under stress conditions. However, the mechanisms of different dormancy-related phenotypes are still under debate and many questions remain unanswered. This study aims to better understand the effects of toxin gene expression on the dormancy of Escherichia coli. METHODS: The effects of toxin gene expression on growth, persistence, and culturability were characterized. Specifically, we detailed dose- and time-dependent dormancy of E. coli and its susceptibility to ofloxacin via arabinose-induced hipA toxin gene expression under the PBAD promoter. A new plot was developed to better describe the dynamic changes in culturability and persistence. The expression level of hipA was determined using qPCR and cellular activities were monitored using fluorescence imaging and flow cytometry. RESULTS: High-level persister formation and strong tolerance to ofloxacin were observed after high-level hipA induction. The new plot reveals more information than the changes in persistence alone, e.g., reduced culturability of E. coli and thus deeper dormancy under high-level hipA induction. Consistently, controlled hipA induction led to decreased cellular activities at promoter PrrnBP1 and an increase in the non-culturable subpopulation. CONCLUSIONS: Overall, this study provides new insights into dormancy induced by toxin gene expression and a more comprehensive view of persistence and culturability. The findings may help develop better control agents against dormant bacterial cells.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

Structural effects on persister control by brominated furanones.

Bacterial persister cells are a small population of dormant cells that are tolerant to essentially all antibiotics. Recently, we reported that a quorum sensing (QS) inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can revert antibiotic tolerance of Pseudomonas aeruginosa persister cells. To better understand this phenomenon, several synthetic brominated furanones with similar structures were compared for their activities in persister control and inhibition of acyl-homoserine lactone (AHL) mediated QS. The results show that some other furanones in addition to BF8 are also AHL QS inhibitors and can revert antibiotic tolerance of P. aeruginosa PAO1 persister cells. However, not all QS inhibiting BFs can revert persistence at growth non-inhibitory concentrations, suggesting that QS inhibition itself is not sufficient for persister control.

Controlling persister cells of Pseudomonas aeruginosa PDO300 by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one.

Pseudomonas aeruginosa is a major pathogen causing chronic pulmonary infections; for example, 80% of cystic fibrosis patients get infected by this bacterium as the disease progresses. Such chronic infections are challenging because P. aeruginosa exhibits high-level tolerance to antibiotics by forming biofilms (multicellular structures attached to surfaces), by entering dormancy and forming antibiotic tolerant persister cells, and by conversion to the mucoid phenotype. Recently, we reported that a synthetic quorum sensing inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can sensitize both planktonic and biofilm-associated persister cells of P. aeruginosa PAO1 to antibiotics at the concentrations non-inhibitory to its growth. In this study, we further characterized the effects of this compound on the mucoid strain P. aeruginosa PDO300. BF8 was found to reduce persistence during the growth of PDO300 and effectively kill the persister cells isolated from PDO300 cultures. In addition to planktonic cells, BF8 was also found to inhibit biofilm formation of PDO300 and reduce associated persistence. These findings broaden the activities of this class of compounds and indicate that BF8 also has other targets in P. aeruginosa in addition to quorum sensing.

(Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one sensitizes Escherichia coli persister cells to antibiotics.

Persisters are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics. The intrinsic antibiotic tolerance of persisters also facilitates the development of multidrug resistance through acquired mechanisms based on drug resistance genes. In this study, we demonstrate that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can reduce persistence during Escherichia coli growth and revert the antibiotic tolerance of its persister cells. The effects of BF8 were more profound when the pH was increased from 6 to 8.5. Although BF8 is a quorum sensing (QS) inhibitor, similar effects were observed for the wild-type E. coli RP437 and its ΔluxS mutant, suggesting that these effects did not occur solely through inhibition of AI-2-mediated QS. In addition to its effects on planktonic persisters, BF8 was also found to disperse RP437 biofilms and to render associated cells more sensitive to ofloxacin. At the doses that are effective against E. coli persister cells, BF8 appeared to be safe to the tested normal mammalian cells in vitro and exhibited no long-term cytotoxicity to normal mouse tissues in vivo. These findings broadened the activities of brominated furanones and shed new light on persister control.

A novel inductively coupled capacitor wireless sensor system for rapid antibiotic susceptibility testing.

BACKGROUND: The increasing prevalence and severity of antimicrobial resistance (AMR) present a major challenge to our healthcare system. Rapid detection of AMR is essential for lifesaving under emergent conditions such as sepsis. The current gold standard phenotypic antibiotic susceptibility testing (AST) takes more than a day to obtain results. Genotypic ASTs are faster (hours) in detecting the presence of resistance genes but require specific probes/knowledge of each AMR gene and do not provide specific information at the phenotype level. To address this unmet challenge, we developed a new rapid phenotypic AST. RESULT: We designed a new electrochemical biosensor based on the concept of magnetically coupled LC sensors. The engineered LC sensors can be placed in 96-well plates and communicate the reading remotely with a receiver coil for signal analysis. The sensors were validated by monitoring the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in the presence and absence of different antibiotics. Drug-resistant strains were used as controls. Bacterial growth was detected within 30 min after inoculation, allowing rapid determination of antibiotic susceptibility at the phenotype level. The sensor also functions in the presence of host proteins when tested with 2% FBS in growth media. CONCLUSIONS: With the compatibility with 96-well plates, this label-free rapid 30-min AST has the potential for low-cost applications with simple integration into the existing workflow in clinical settings.

Tailored Fabrication of Plasmonic Film Light Filters for Enhanced Microalgal Growth and Biomass Composition.

Through plasmon resonance, silver and gold nanoparticles can selectively backscatter light within different regions of the visible electromagnetic spectrum. We engineered a plasmonic film technology that utilizes gold and silver nanoparticles to enhance light at the necessary wavelengths for microalgal photosynthetic activities. Nanoparticles were embedded in a polymeric matrix to fabricate millimeter-thin plasmonic films that can be used as light filters in microalgal photobioreactors. Experiments conducted with microalga Chlamydomonas reinhardtii proved that microalgal growth and photosynthetic pigment production can be increased by up to 50% and 78%, respectively, by using these plasmonic film light filters. This work provides a scalable strategy for the efficient production of specialty chemicals and biofuels from microalgae through irradiation control with plasmonic nanoparticles.