Rapid Detection of Staphylococcus aureus Using Paper-Derived Electrochemical Biosensors.

Several groups have recently explored the idea of developing electrochemical paper-based wearable devices, specifically targeting metabolites in sweat. While these sensors have the potential to provide a breadth of analytical information, there are several key challenges to address before these sensors can be widely adopted for clinical interventions. Toward this goal, we describe the development of a paper-based electrochemical sensor for the detection of Staphylococcus aureus. Enabling the application, this report describes the use of paper-derived carbon electrodes, which were modified with a thin layer of sputtered gold (that minimizes lateral resistivity and significantly improves the electron transfer process) and with chitosan (used as a binder, to offer flexibility). The resulting material was laser-patterned and applied for the development of an electrochemical biosensor controlled (via a wireless connection) by a custom-built, portable potentiostat. As no interference was observed when exposed to other bacteria or common metabolites, this wearable system (paper-derived electrodes + potentiostat) has the potential to detect the presence of S. aureus in the skin, a commonly misdiagnosed and mistreated infection.

Does Nesting Material Affect Wood Duck (Aix sponsa) Nest Box Selection, Reproduction, and Eggshell Bacteria?

Wood Ducks (Aix sponsa) are secondary cavity nesters that use natural cavities and artificial nest boxes, the latter of which has been attributed to the recovery of populations across the southeastern US. Continual use of these boxes results in a buildup of bacteria, parasites, and other pathogens. To avoid the accumulation of these deleterious organisms, best management practices include the occasional removal of old nesting material (i.e., wood shavings) and replacement with fresh wood shavings. No studies have been performed on the effects of shaving material on nest box selection, nest success, and bacterial growth. We monitored 142 and 111 nest boxes in Florida and Georgia, USA, respectively, and filled a random sample with aspen or cedar shavings. We then swabbed the surface of 144 and 150 eggs during 2020 and 2021, respectively, to screen for culturable bacteria. We detected no effect of shaving type on nest box selection, nest success, or egg surface bacterial growth. We found 3-8 bacterial colony types (1-123 colony-forming units [CFU]/box) and 1-8 bacterial colony types (3-382 CFU/box) among the Georgia and Florida samples, respectively. We detected no effect from shaving type on Wood Duck reproduction or bacterial growth in the sampled nest boxes. We concluded that both shaving types are suitable nesting materials for box-nesting Wood Duck populations and the continued use of either would be a reasonable decision for managers.

Ultradense Electrochemical Chips with Arrays of Nanostructured Microelectrodes to Enable Sensitive Diffusion-Limited Bioassays.

Nanostructured microelectrodes (NMEs) are an attractive alternative to yield sensitive bioassays in unprocessed samples. However, although valuable for different applications, nanoporous NMEs usually cannot boost the sensitivity of diffusion-limited analyses because of the enlarged Debye length within the nanopores, which reduces their accessibility. To circumvent this limitation, nanopore-free gold NMEs were electrodeposited from 45 µm SU-8 apertures, featuring nanoridged microspikes on a recessed surface of gold thin film while carrying interconnected crown-like and spiky structures along the edge of a SU-8 passivation layer. These structures were grown onto ultradense, vertical array chips that offer a promising strategy for translating reproducible, high-resolution, and cost-effective sensors into real-world applications. The NMEs yielded reproducible analyses, while machine learning allowed us to predict the analytical responses from NME electrodeposition data. By taking advantage of the high surface area and accessible structure of the NMEs, these structures provided a sensitivity for [Fe(CN)6]3-/4- that was 5.5× higher than that of bare WEs while also delivering a moderate antibiofouling property in undiluted human plasma. As a proof of concept, these electrodes were applied toward the fast (22 min) and simple determination of Staphylococcus aureus by monitoring the oxidation of [Fe(CN)6]4-, which acted as a cellular respiration rate redox reporter. The sensors also showed a wide dynamic range, spanning 5 orders of magnitude, and a calculated limit of detection of 0.2 CFU mL-1.

Exploring the interactive mechanism of acarbose with the amylase SusG in the starch utilization system of the human gut symbiont Bacteroides thetaiotaomicron through molecular modeling.

The α-amylase, SusG, is a principal component of the Bacteroides thetaiotaomicron (Bt) starch utilization system (Sus) used to metabolize complex starch molecules in the human gastrointestinal (GI) tract. We previously reported the non-microbicidal growth inhibition of Bt by the acarbose-mediated arrest of the Sus as a potential therapeutic strategy. Herein, we report a computational approach using density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation to explore the interactive mechanism between acarbose and SusG at the atomic level in an effort to understand how acarbose shuts down the Bt Sus. The docking analysis reveals that acarbose binds orthosterically to SusG with a binding affinity of -8.3 kcal/mol. The MD simulation provides evidence of conformational variability of acarbose at the active site of SusG and also suggests that acarbose interacts with the main catalytic residues via a general acid-base double-displacement catalytic mechanism. These results suggest that small molecule competitive inhibition against the SusG protein could impact the entire Bt Sus and eliminate or reduce the system's ability to metabolize starch. This computational strategy could serve as a potential avenue for structure-based drug design to discover other small molecules capable of inhibiting the Sus of Bt with high potency, thus providing a holistic approach for selective modulation of the GI microbiota.

Non-lethal growth inhibition by arresting the starch utilization system of clinically relevant human isolates of Bacteroides dorei.

We describe the inhibition of the starch utilization system (Sus) belonging to various strains of Bacteroides dorei in a non-lethal manner using the small molecule probe, acarbose. Concentrations of acarbose as low as 5 µM significantly impede the growth of B. dorei and increase the doubling time of cultures. The successful inhibition of this species of Bacteroides is relevant to several disease states including type I diabetes mellitus. This method continues to explore a new, potential route to intervene in illnesses associated with aberrant changes in the composition of the human gut microbiota through the strategic manipulation of its constituents.

Murine Adherent and Invasive E. coli Induces Chronic Inflammation and Immune Responses in the Small and Large Intestines of Monoassociated IL-10-/- Mice Independent of Long Polar Fimbriae Adhesin A.

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) is preferentially associated with ileal Crohn's disease (CD). The role of AIEC in the development of inflammation and its regional tropism is unresolved. The presence of long polar fimbriae (LPF) in 71% of ileal CD AIEC suggests a role for LPF in the tropism and virulence of AIEC. The aim of our study is to determine if AIEC, with or without LpfA, induces intestinal inflammation in monoassociated IL-10-/- mice. METHODS: We compared murine AIEC strains NC101 (phylogroup B2, LpfA-) and CUMT8 (phylogroup B1, LpfA+), and isogenic mutant CUMT8 lacking lpfA154, with a non-AIEC (E. coli K12), evaluating histologic inflammation, bacterial colonization, mucosal adherence and invasion, and immune activation. RESULTS: IL-10-/- mice monoassociated with AIEC (either CUMT8, CUMT8:ΔlpfA, or NC101) but not K12 developed diffuse small intestinal and colonic inflammation. There was no difference in the magnitude and distribution of inflammation in mice colonized with CUMT8:ΔlpfA compared with wild-type CUMT8. Bacterial colonization was similar for all E. coli strains. Fluorescence in situ hybridization revealed mucosal adherence and tissue invasion by AIEC but not K12. Production of the cytokines IL-12/23 p40 by the intestinal tissue and IFN-γ and IL-17 by CD4 T cells correlated with inflammation. CONCLUSIONS: IL-10-/- mice monoassociated with murine AIEC irrespective of LpfA expression developed chronic inflammation accompanied by IL-12/23 p40 production in the small and large intestines and IFN-γ/IL-17 production by CD4 T cells that model the interplay between enteric pathosymbionts, host susceptibility, and enhanced immune responses in people with IBD.

Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730.

Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to the low pH in the stomach and bile in the small intestine. The probiotic microorganism Lactobacillus reuteri ATCC 55730 has previously been shown to survive in the human small intestine. To address how L. reuteri can resist bile stress, we performed microarray experiments to determine gene expression changes that occur when the organism is exposed to physiological concentrations of bile. A wide variety of genes that displayed differential expression in the presence of bile indicated that the cells were dealing with several types of stress, including cell envelope stress, protein denaturation, and DNA damage. Mutations in three genes were found to decrease the strain's ability to survive bile exposure: lr1864, a Clp chaperone; lr0085, a gene of unknown function; and lr1516, a putative esterase. Mutations in two genes that form an operon, lr1584 (a multidrug resistance transporter in the major facilitator superfamily) and lr1582 (unknown function), were found to impair the strain's ability to restart growth in the presence of bile. This study provides insight into the possible mechanisms that L. reuteri ATCC 55730 may use to survive and grow in the presence of bile in the small intestine.

Nonmicrobicidal Small Molecule Inhibition of Polysaccharide Metabolism in Human Gut Microbes: A Potential Therapeutic Avenue.

A new approach for the nonmicrobicidal phenotypic manipulation of prominent gastrointestinal microbes is presented. Low micromolar concentrations of a chemical probe, acarbose, can selectively inhibit the Starch Utilization System and ablate the ability of Bacteroides thetaiotaomicron and B. fragilis strains to metabolize potato starch and pullulan. This strategy has potential therapeutic relevance for the selective modulation of the GI microbiota in a nonmicrobicidal manner.

Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.