Accurate and rapid antibiotic susceptibility testing using a machine learning-assisted nanomotion technology platform.

Antimicrobial resistance (AMR) is a major public health threat, reducing treatment options for infected patients. AMR is promoted by a lack of access to rapid antibiotic susceptibility tests (ASTs). Accelerated ASTs can identify effective antibiotics for treatment in a timely and informed manner. We describe a rapid growth-independent phenotypic AST that uses a nanomotion technology platform to measure bacterial vibrations. Machine learning techniques are applied to analyze a large dataset encompassing 2762 individual nanomotion recordings from 1180 spiked positive blood culture samples covering 364 Escherichia coli and Klebsiella pneumoniae isolates exposed to cephalosporins and fluoroquinolones. The training performances of the different classification models achieve between 90.5 and 100% accuracy. Independent testing of the AST on 223 strains, including in clinical setting, correctly predict susceptibility and resistance with accuracies between 89.5% and 98.9%. The study shows the potential of this nanomotion platform for future bacterial phenotype delineation.

Nanomotion technology in combination with machine learning: a new approach for a rapid antibiotic susceptibility test for Mycobacterium tuberculosis.

Nanomotion technology is a growth-independent approach that can be used to detect and record the vibrations of bacteria attached to cantilevers. We have developed a nanomotion-based antibiotic susceptibility test (AST) protocol for Mycobacterium tuberculosis (MTB). The protocol was used to predict strain phenotype towards isoniazid (INH) and rifampicin (RIF) using a leave-one-out cross-validation (LOOCV) and machine learning techniques. This MTB-nanomotion protocol takes 21 h, including cell suspension preparation, optimized bacterial attachment to functionalized cantilever, and nanomotion recording before and after antibiotic exposure. We applied this protocol to MTB isolates (n = 40) and were able to discriminate between susceptible and resistant strains for INH and RIF with a maximum sensitivity of 97.4% and 100%, respectively, and a maximum specificity of 100% for both antibiotics when considering each nanomotion recording to be a distinct experiment. Grouping recordings as triplicates based on source isolate improved sensitivity and specificity to 100% for both antibiotics. Nanomotion technology can potentially reduce time-to-result significantly compared to the days and weeks currently needed for current phenotypic ASTs for MTB. It can further be extended to other anti-TB drugs to help guide more effective TB treatment.

Investigating nanomotion-based technology (Resistell AST) for rapid antibiotic susceptibility testing among adult patients admitted to a tertiary-care hospital with Gram-negative bacteraemia: protocol for a prospective, observational, cross-sectional, single-arm study.

INTRODUCTION: Effective treatment of bloodstream infections (BSIs) is relying on rapid identification of the causing pathogen and its antibiotic susceptibility. Still, most commercially available antibiotic susceptibility testing (AST) methods are based on monitoring bacterial growth, thus impacting the time to results. The Resistell AST is based on a new technology measuring the nanomotion caused by physiologically active bacterial cells and detecting the changes in nanomotion caused by the exposure to a drug. METHODS AND ANALYSIS: This is a single-centre, prospective, cross-sectional, single-arm diagnostic accuracy study to determine the agreement of the Resistell AST on Gram-negative bacteria isolated from blood cultures among patients admitted to a tertiary-care hospital with the reference method. Up to 300 patients will be recruited. Starting with a pilot phase, enrolling 10%-20% of the subjects and limited to Escherichia coli BSI tested for ceftriaxone susceptibility, the main phase will follow, extending the study to Klebsiella pneumoniae and ciprofloxacin. ETHICS AND DISSEMINATION: This study has received ethical approval from the Swiss Ethics Committees (swissethics, project 2020-01622). All the case report forms and clinical samples will be assigned a study code by the local investigators and stored anonymously at the reference centre (Lausanne University Hospital). The results will be broadly distributed through conference presentations and peer-reviewed publications. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05002413).

In Vitro Activity of Ebselen and Diphenyl Diselenide Alone and in Combination with Drugs against Trichophyton mentagrophytes Strains.

BACKGROUND: Dermatophytoses are one of the most prevalent infectious diseases in the world for which the pace of developing new drugs has not kept pace with the observed therapeutic problems. Thus, searching for new antifungals with an alternative and novel mechanism of action is necessary. OBJECTIVE: This study aimed to evaluate the antifungal activity of ebselen and diphenyl diselenide against Trichophyton mentagrophytes clinical isolates. METHODS: In vitro antifungal susceptibility was assessed for organoselenium compounds used alone or in combination with allylamines and azoles according to the 3rd edition of the CLSI M38 protocol. RESULTS: Ebselen demonstrated high antifungal activity with MICGM equal to 0.442 µg/mL and 0.518 µg/mL in the case of human and animal origin strains, respectively. The values of MICGM of diphenyl diselenide were higher: 17.36 µg/mL and 13.45 µg/mL for the human and animal isolates, respectively. Synergistic or additive effects between terbinafine and ebselen or diphenyl diselenide were observed in the case of 12% and 20% strains, respectively. In turn, the combination of itraconazole with diphenyl diselenide showed a synergistic effect only in the case of 6% of the tested strains, whereas no synergism was shown in the combination with ebselen. CONCLUSIONS: The results highlight the promising activity of organoselenium compounds against Trichophyton mentagrophytes. However, their use in combinational therapy with antifungal drugs seems to be unjustified due to the weak synergistic effect observed.

Quartz tuning fork mass change sensing for FIB/SEM technology.

In this paper we present a metrological method for determination of mass density of focused ion beam induced deposition (FIBID) materials using quartz tuning fork (QTF) mass change sensors. Dimension and density determination of FIBID deposited nanostructures is necessary to develop and reliable and repeatable microfabribrication technology of the highest versatility. The proposed metrological methodology allows to determine mass change with 5 pg resolution and accuracy below 5 % if density is considered. The described method is suitable for precise FIBID precursor parameters determination conducted during the deposition as actuation and signal read-out of the applied QTF can be performed electrically. High accuracy, resolution and stability are ensured due to excellent properties of quartz forming the sensor structure.

Multifrequency Kelvin probe force microscopy on self assembled molecular layers and quantitative assessment of images' quality.

The application of single-pass multifrequency Kelvin probe force microscopy (KPFM) for topography and contact potential difference (CPD) measurements of organic self-assembled monolayers (SAM) is demonstrated. Four modes of mechanical and electrical cantilever excitation were tested in order to obtain the best possible resolution in the CPD measurements. The algorithm using maximum capacity of information channel for quantitative image quality assessment was proposed to compare and assess the quality of the recorded images and imaging modes. The improvement of the quality of CPD imaging in multiresonance operation was confirmed.

Carrier density distribution in silicon nanowires investigated by scanning thermal microscopy and Kelvin probe force microscopy.

The use of scanning thermal microscopy (SThM) and Kelvin probe force microscopy (KPFM) to investigate silicon nanowires (SiNWs) is presented. SThM allows imaging of temperature distribution at the nanoscale, while KPFM images the potential distribution with AFM-related ultra-high spatial resolution. Both techniques are therefore suitable for imaging the resistance distribution. We show results of experimental examination of dual channel n-type SiNWs with channel width of 100 nm, while the channel was open and current was flowing through the SiNW. To investigate the carrier distribution in the SiNWs we performed SThM and KPFM scans. The SThM results showed non-symmetrical temperature distribution along the SiNWs with temperature maximum shifted towards the contact of higher potential. These results corresponded to those expressed by the distribution of potential gradient along the SiNWs, obtained using the KPFM method. Consequently, non-uniform distribution of resistance was shown, being a result of non-uniform carrier density distribution in the structure and showing the pinch-off effect. Last but not least, the results were also compared with results of finite-element method modeling.

Investigation of thermal effects in through-silicon vias using scanning thermal microscopy.

Results of quantitative investigations of copper through-silicon vias (TSVs) are presented. The experiments were performed using scanning thermal microscopy (SThM), enabling highly localized imaging of thermal contrast between the copper TSVs and the surrounding material. Both dc and ac active-mode SThM was used and differences between these variants are shown. SThM investigations of TSVs may provide information on copper quality in TSV, as well as may lead to quantitative investigation of thermal boundaries in micro- and nanoelectronic structures. A proposal for heat flow analysis in a TSV, which includes the influence of the boundary region between the TSV and the silicon substrate, is presented; estimation of contact resistance and boundary thermal conductance is also given.

Thermal mapping of a scanning thermal microscopy tip.

Scanning thermal microscopy (SThM) is a very promising technique for local investigation of temperature and thermal properties of nanostructures with great application potential in contemporary nanoelectronics and nanotechnology. In order to increase the localization of SThM measurements, the size of probes has recently substantially decreased, which results in novel types of SThM probes manufactured with the use of modern silicon microfabrication technology. Quantitative SThM measurements with these probes need methods, which enable to assess the quality of thermal contact between the probe and the investigated surface. In this paper we propose a tip thermal mapping (TThM) procedure, which is used to estimate experimentally the distribution of power dissipated by the tip of an SThM probe. We also show that the proposed power dissipation model explains the results of active-mode SThM measurements and that the TThM procedure is reversible for a given probe and sample.

Tunneling/shear force microscopy using piezoelectric tuning forks for characterization of topography and local electric surface properties.

Characterization of novel nanoelectronic structures and materials requires advanced and high-resolution diagnostic methods. In this article new approach for high sensitivity measurements of electric surface properties using scanning probe microscopy is presented. In this procedure topography and tunneling current flowing between the metallic tip and the surface are observed simultaneously. In our design piezoelectric tuning fork equipped with metallic tip in shear force microscope is used. In our experiments we also applied an additional feedback loop to maintain constant tunneling current while scanning over electrical inhomogeneous surfaces. In this way crosstalk between topography and tunneling current measurements is reduced. The described method was tested on nanocrystalline diamond and gold thin films deposited on silicon substrates.

TLC of alkaloids on cyanopropyl bonded stationary phases. Part II. Connection with RP18 and silica plates.

Some standards of the alkaloids and synthetic or natural mixtures are separated by two-dimensional thin-layer chromatography (TLC) on different adsorbent layers. Normal- and reversed-phase systems are used to obtain significant differences in the separation selectivity. Optimization of the one-dimensional TLC separation of the alkaloids' standards is performed on cyanopropyl-silica, RP18W, and silica layers in various eluents containing (besides diluent and modifier) silanol blockers, such as diethyl amine or ammonia. The most selective systems are used for the separation of the alkaloids' mixtures by two-dimensional TLC with an adsorbent gradient method. The mixtures of alkaloids or plant extracts (Chelidonium majus, Fumaria officinalis, or Glaucium flavum) are chromatographed in system I; the plates are connected with the plate pre-coated with various adsorbent, and partly separated fractions are transferred to the second layer and developed in system II. CN-silica-RP18W and CN-silica-silica are used as the connected layers. The alkaloids are identified by R(F) values of standards, and the components of plant extracts are identified in both systems, and by the comparison of UV spectra obtained in diode array detector densitometry.

Influence of sample application mode on performance of pressurized planar electrochromatography in completely closed system.

Three modes of sample application on the chromatographic plate are applied at present investigations of pressurized planar electrochromatography (PPEC) systems taking into special attention their influence on performance of the separating system. These modes are as follows: application of the sample solution directly on the chromatographic plate with microsyringe, deposition of sample solution on scrap of adsorbent layer followed by location oft this scrap on the chromatographic plate, application of the sample solution with commercially available aerosol applicator. These modes were combined with prewetting procedures of the chromatographic plates which lead to an accomplishment of equilibration of the stationary phase-mobile phase system. The plots of plate height versus linear flow rate of the mobile phase are presented for PPEC systems for the first time. The best separation performance has been obtained in PPEC system when prewetting of the chromatographic plate followed the sample application with commercially available aerosol applicator. The higher repeatability of migration distance of the solute bands has been obtained in PPEC experiments when the sample application was followed by prewetting the chromatographic plate in comparison to the experiments when these operations were performed in reversed order.

Thin-layer chromatography of alkaloids on cyanopropyl bonded stationary phases. Part I.

Selected alkaloids are chromatographed on cyanopropyl-silica thin layers using various nonaqueous and aqueous eluents. Because of the strong retention of these basic compounds, nonaqueous eluents containing medium polar diluents, strongly polar modifiers, and silanol blockers (ammonia or diethylamine) are required for separation. Likewise, aqueous eluents containing modifiers (acetonitrile, methanol, and tetrahydrofuran), buffered aqueous solutions at pH 2-8, ion-pair reagents [octane sulfonic acid sodium salt, sodium dodecyl sulphate, and bis-(2-ethylhexyl)phosphoric acid], or silanol blockers (ammonia, tetrabutyl ammonium chloride, and diethyl amine) are investigated. The separation selectivity as well as spot symmetry and efficiency system in the applied eluent systems are analyzed. The most selective and efficient systems are used in two-dimensional separations of isoquinoline alkaloids' mixture and the plant extracts Chelidonium majus, Fumaria officinalis, and Glaucium flavum. Two-dimensional thin-layer chromatography on cyanopropyl layer with diode array detection densitometry enables the separation and identification of some alkaloids in plant extracts.