Accurate Prediction of Antimicrobial Susceptibility for Point-of-Care Testing of Urine in Less than 90 Minutes via iPRISM Cassettes.

The extensive and improper use of antibiotics has led to a dramatic increase in the frequency of antibiotic resistance among human pathogens, complicating infectious disease treatments. In this work, a method for rapid antimicrobial susceptibility testing (AST) is presented using microstructured silicon diffraction gratings integrated into prototype devices, which enhance bacteria-surface interactions and promote bacterial colonization. The silicon microstructures act also as optical sensors for monitoring bacterial growth upon exposure to antibiotics in a real-time and label-free manner via intensity-based phase-shift reflectometric interference spectroscopic measurements (iPRISM). Rapid AST using clinical isolates of Escherichia coli (E. coli) from urine is established and the assay is applied directly on unprocessed urine samples from urinary tract infection patients. When coupled with a machine learning algorithm trained on clinical samples, the iPRISM AST is able to predict the resistance or susceptibility of a new clinical sample with an Area Under the Receiver Operating Characteristic curve (AUC) of ∼ 0.85 in 1 h, and AUC > 0.9 in 90 min, when compared to state-of-the-art automated AST methods used in the clinic while being an order of magnitude faster.

Shining light in blind alleys: deciphering bacterial attachment in silicon microstructures.

With new advances in infectious disease, antifouling surfaces, and environmental microbiology research comes the need to understand and control the accumulation and attachment of bacterial cells on a surface. Thus, we employ intrinsic phase-shift reflectometric interference spectroscopic measurements of silicon diffraction gratings to non-destructively observe the interactions between bacterial cells and abiotic, microstructured surfaces in a label-free and real-time manner. We conclude that the combination of specific material characteristics (i.e., substrate surface charge and topology) and characteristics of the bacterial cells (i.e., motility, cell charge, biofilm formation, and physiology) drive bacteria to adhere to a particular surface, often leading to a biofilm formation. Such knowledge can be exploited to predict antibiotic efficacy and biofilm formation, and enhance surface-based biosensor development, as well as the design of anti-biofouling strategies.

Hearing Aid Use in Children With Unilateral Hearing Loss: A Randomized Crossover Clinical Trial.

OBJECTIVES/HYPOTHESIS: In children with mild to moderately severe unilateral hearing loss (UHL), assess whether subject-reported quality of life (QOL) and teacher- and parent-reported perception of listening difficulty are affected by use of a hearing aid (HA) with baseline accommodations, compared to children receiving only baseline accommodations. STUDY DESIGN: Randomized crossover clinical trial. METHODS: Thirty-seven children 6-12 years of age with mild to moderately severe UHL and ≥80% word recognition scores in the poorer hearing ear were randomized into arm 1, using baseline accommodations (frequency-modulated system and strategic seating) for 12 weeks, followed by addition of a HA for 12 weeks. The other participants were randomized into the reverse methodology: arm 2, using a HA in addition to baseline accommodations for 12 weeks, followed by baseline accommodations alone. Surveys of QOL (Hearing Environments and Reflection on Quality of Life) and listening difficulties or challenges with hearing amplification (CHILD and LIFE-R questionnaires) were administered at 6-week intervals. Differences in mean survey scores, percent change, and improvement over time were computed between the two arms and inter-arm intervals. Per-protocol analysis was used. RESULTS: Of the 37 children enrolled, 34 children underwent the study interventions and were included in the analysis, (arm 1 = 20, arm 2 = 14) (mean [standard deviation] age = 8 [1.5] years; 21 boys [61.8%]). Survey scores averaged across both arms during the HA interval (77.79 [15.13]) were significantly higher than during the baseline-only interval (69.67 [14.69], P = .036). There was no significant difference between trial arms in mean scores between the two HA intervals (P = .450) and two baseline-only intervals (P = .539). CONCLUSIONS: Hearing-related QOL and listening ability improved in children who met eligibility criteria with mild to moderately severe UHL with HA use compared with baseline accommodations alone. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02269124. LEVEL OF EVIDENCE: 1 Laryngoscope, 132:881-888, 2022.

Antifungal Susceptibility Testing of Aspergillus niger on Silicon Microwells by Intensity-Based Reflectometric Interference Spectroscopy.

There is a demonstrated and paramount need for rapid, reliable infectious disease diagnostics, particularly those for invasive fungal infections. Current clinical determinations for an appropriate antifungal therapy can take up to 3 days using current antifungal susceptibility testing methods, a time-to-readout that can prove detrimental for immunocompromised patients and promote the spread of antifungal resistant pathogens. Herein, we demonstrate the application of intensity-based reflectometric interference spectroscopic measurements (termed iPRISM) on microstructured silicon sensors for use as a rapid, phenotypic antifungal susceptibility test. This diagnostic platform optically tracks morphological changes of fungi corresponding to conidia growth and hyphal colonization at a solid-liquid interface in real time. Using Aspergillus niger as a model fungal pathogen, we can determine the minimal inhibitory concentration of clinically relevant antifungals within 12 h. This assay allows for expedited detection of fungal growth and provides a label-free alternative to broth microdilution and agar diffusion methods, with the potential to be used for point-of-care diagnostics.

Designing Bacterial Chemotactic Receptors Guided by Photonic Femtoliter Well Arrays for Quantifiable, Label-Free Measurement of Bacterial Chemotaxis.

Whole cell bioreporters, such as bacterial cells, can be used for environmental and clinical sensing of specific analytes. However, the current methods implemented to observe such bioreporters in the form of chemotactic responses heavily rely on microscope analysis, fluorescent labels, and hard-to-scale microfluidic devices. Herein, we demonstrate that chemotaxis can be detected within minutes using intrinsic optical measurements of silicon femtoliter well arrays (FMAs). This is done via phase-shift reflectometric interference spectroscopic measurements (PRISM) of the wells, which act as silicon diffraction gratings, enabling label-free, real-time quantification of the number of trapped bacteria cells in the optical readout. By generating unsteady chemical gradients over the wells, we first demonstrate that chemotaxis toward attractants and away from repellents can be easily differentiated based on the signal response of PRISM. The lowest concentration of chemorepellent to elicit an observed bacterial response was 50 mM, whereas the lowest concentration of chemoattractant to elicit a response was 10 mM. Second, we employed PRISM, in combination with a computational approach, to rapidly scan for and identify a novel synthetic histamine chemoreceptor strain. Consequently, we show that by using a combined computational design approach, together with a quantitative, real-time, and label-free detection method, it is possible to manufacture and characterize novel synthetic chemoreceptors in Escherichia coli (E. coli).

Recent Advances in the Race to Design a Rapid Diagnostic Test for Antimicrobial Resistance.

Even with advances in antibiotic therapies, bacterial infections persistently plague society and have amounted to one of the most prevalent issues in healthcare today. Moreover, the improper and excessive administration of antibiotics has led to resistance of many pathogens to prescribed therapies, rendering such antibiotics ineffective against infections. While the identification and detection of bacteria in a patient's sample is critical for point-of-care diagnostics and in a clinical setting, the consequent determination of the correct antibiotic for a patient-tailored therapy is equally crucial. As a result, many recent research efforts have been focused on the development of sensors and systems that correctly guide a physician to the best antibiotic to prescribe for an infection, which can in turn, significantly reduce the instances of antibiotic resistance and the evolution of bacteria "superbugs." This review details the advantages and shortcomings of the recent advances (focusing from 2016 and onward) made in the developments of antimicrobial susceptibility testing (AST) measurements. Detection of antibiotic resistance by genomic AST techniques relies on the prediction of antibiotic resistance via extracted bacterial DNA content, while phenotypic determinations typically track physiological changes in cells and/or populations exposed to antibiotics. Regardless of the method used for AST, factors such as cost, scalability, and assay time need to be weighed into their design. With all of the expansive innovation in the field, which technology and sensing systems demonstrate the potential to detect antimicrobial resistance in a clinical setting?

Unraveling Antimicrobial Susceptibility of Bacterial Networks on Micropillar Architectures Using Intrinsic Phase-Shift Spectroscopy.

With global antimicrobial resistance becoming increasingly detrimental to society, improving current clinical antimicrobial susceptibility testing (AST) is crucial to allow physicians to initiate appropriate antibiotic treatment as early as possible, reducing not only mortality rates but also the emergence of resistant pathogens. In this work, we tackle the main bottlenecks in clinical AST by designing biofunctionalized silicon micropillar arrays to provide both a preferable solid-liquid interface for bacteria networking and a simultaneous transducing element that monitors the response of bacteria when exposed to chosen antibiotics in real time. We harness the intrinsic ability of the micropillar architectures to relay optical phase-shift reflectometric interference spectroscopic measurements (referred to as PRISM) and employ it as a platform for culture-free, label-free phenotypic AST. The responses of E. coli to various concentrations of five clinically relevant antibiotics are optically tracked by PRISM, allowing for the minimum inhibitory concentration (MIC) values to be determined and compared to both standard broth microdilution testing and clinic-based automated AST system readouts. Capture of bacteria within these microtopologies, followed by incubation of the cells with the appropriate antibiotic solution, yields rapid determinations of antibiotic susceptibility. This platform not only provides accurate MIC determinations in a rapid manner (total assay time of 2-3 h versus 8 h with automated AST systems) but can also be employed as an advantageous method to differentiate bacteriostatic and bactericidal antibiotics.

Biogenesis of phycobiliproteins. III. CpcM is the asparagine methyltransferase for phycobiliprotein beta-subunits in cyanobacteria.

All phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their beta-subunits. Methylation of this Asn to produce gamma-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-3H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the beta-subunits of phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding alpha-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo.

CpcM posttranslationally methylates asparagine-71/72 of phycobiliprotein beta subunits in Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803.

Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved gamma-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of phycobiliproteins are discussed.