Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Background: Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Methods: Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. Results: We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. Conclusions: We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining.

Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.

Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy.

Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage.

Antimicrobial susceptibility testing for Gram positive cocci towards vancomycin using scanning electron microscopy.

The rapid detection of resistant bacteria has become a challenge for microbiologists worldwide. Numerous pathogens that cause nosocomial infections are still being treated empirically and have developed resistance mechanisms against key antibiotics. Thus, one of the challenges for researchers has been to develop rapid antimicrobial susceptibility testing (AST) to detect resistant isolates, ensuring better antibiotic stewardship. In this study, we established a proof-of-concept for a new strategy of phenotypic AST on Gram-positive cocci towards vancomycin using scanning electron microscopy (SEM). Our study evaluated the profiling of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus after brief incubation with vancomycin. Sixteen isolates were analysed aiming to detect ultrastructural modifications at set timepoints, comparing bacteria with and without vancomycin. After optimising slides preparation and micrographs acquisition, two analytical strategies were used. The high magnification micrographs served to analyse the division of cocci based on the ratio of septa, along with the bacterial size. Susceptible strains with vancomycin showed a reduced septa percentage and the average surface area was consequently double that of the controls. The resistant bacteria revealed multiple septa occurring at advanced timepoints. Low magnification micrographs made it possible to quantify the pixels at different timepoints, confirming the profiling of cocci towards vancomycin. This new phenotypic AST strategy proved to be a promising tool to discriminate between resistant and susceptible cocci within an hour of contact with vancomycin. The analysis strategies applied here would potentially allow the creation of artificial intelligence algorithms for septa detection and bacterial quantification, subsequently creating a rapid automated SEM-AST assay.

Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation.

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results. Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains. Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 µm] and with [2.50 ± 0.68 µm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 µm] and with [1.28 ± 0.19 µm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem. Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity.

We present here a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification.

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research.

Noninvasively measuring respiratory activity of rat primary hepatocyte spheroids by scanning electrochemical microscopy.

Construction of an in vitro drug screening method for evaluating drug metabolism and toxicity by using cells is required instead of the conventional in vivo one that uses animals. In order to realize the in vitro study, analyzing the cellular activity or viability noninvasively in advance of the screening is essential. The aim of the current study is to establish a method that can evaluate the cellular activity depending on spheroid sizes by means of oxygen consumption and to determine the valid diameter of hepatocyte spheroids. To measure the respiratory activity of the spheroids, which were formed on a nanopillar sheet, we applied scanning electrochemical microscopy (SECM). From the viewpoint of high respiratory activity and its small variation, we determined that spheroids with 70 µm in diameter were adequate. We then performed a gene expression analysis by using a real-time PCR to evaluate the correlation with respiratory activity. As a result, a higher expression level of Hnf4α, which is essential for hepatocytes to fulfill many liver functions and is the indicator of well-differentiated hepatocytes, showed relatively higher respiratory activity. We concluded that the noninvasive SECM technique could evaluate the cellular activity of a single spheroid. Noninvasively measuring cellular activity by SECM makes it possible to evaluate the cellular activity prior to a nonclinical test and enables the continued monitoring of the drug response by using single spheroid. SECM becomes a powerful tool to satisfying the increasing demand for an in vitro system in the course of new drug development.

A Pt layer/Pt disk electrode configuration to evaluate respiration and alkaline phosphatase activities of mouse embryoid bodies.

A Pt layer/Pt disk electrode configuration was used as a scanning electrochemical microscopy (SECM) probe. The glass seal part of the insulator was covered with a Pt layer to form an exposed pseudo reference electrode. In a HEPES-based medium at pH 7.5, the half-wave potential (E(1/2)) for [Fe(CN)(6)](4-) oxidation and O(2) reduction measured versus the internal Pt pseudo reference was shifted by about -0.2V, compared with the E(1/2) measured versus the external Ag/AgCl reference electrode. The shape and the current of the cyclic voltammograms (CVs) did not change notably over time, indicating that the Pt layer is sufficiently stable to be used as an integrated pseudo reference for voltammetric measurements. To demonstrate the suitability for SECM applications, the Pt/Pt probe configuration was used for measuring the oxygen consumption and the alkaline phosphatase (ALP) activity of a single mouse embryoid body (mEB). Ten individual mEB samples were characterized to monitor the oxygen concentration profile. Oxygen reduction currents were monitored at -0.7 V versus the Pt pseudo reference and compared with those monitored at -0.5 V versus Ag/AgCl. The respiration rate of mEBs becomes greater with increasing cultivation dates. We have plotted the oxygen consumption rate (F(O(2))) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. The linearity of the plot was excellent (coefficient of determination R(2)=0.90). The slope of the least squares method was 1. In a 1.0mM p-aminophenylphospate (PAPP) HEPES buffer (pH 9.5) solution, APL activity of mEBs can be characterized, to monitor the p-aminophenol (PAP) oxidation current. ALP catalyzes the hydrolysis of PAPP to PAP. The E(1/2) for PAP oxidation measured versus the Pt layer was not shifted, compared with the E(1/2) versus Ag/AgCl. The mEB samples were characterized to monitor the PAP concentration profile. PAP oxidation currents were monitored at +0.3 V versus the Pt layer and compared with those monitored at +0.3 V versus Ag/AgCl. We have plotted the PAP production rate (F(PAP)) of each mEB sample, measured versus the Pt layer and versus Ag/AgCl. In this case, the linearity of the plot became slightly scattered, but it was found to be possible to evaluate ALP activities of mEB samples utilizing the Pt/Pt probe configuration. This type of probe is very useful because it is not necessary to insert a reference electrode into the measuring solution to obtain an electrical connection, and thus electrochemical measurement in a small volume becomes much easier.

Differential effect of scaffold shape on dentin regeneration.

The effect of scaffold shape on dentin regeneration is not well understood. In this study, porous hydroxyapatite/beta-tricalcium phosphate (HAp/beta-TCP), powdered HAp/beta-TCP, and polyglycolic acid (PGA) fiber mesh were used as scaffolds and transplanted with cultured porcine dental pulp-derived cells into the backs of nude mice. Samples were harvested after 6 weeks. Newly-formed hard tissue was observed in all transplants. When porous HAp/beta-TCP was used, dentin-like hard tissue was observed on the inner wall with minimum cell inclusions and odontoblast-like cells were aligned adjacent to the hard tissue. When HAp/beta-TCP powders or PGA were used, bone-like hard tissues showed cell inclusions and cell alignment was not observed. Hard tissue from the HAp/beta-TCP block group was positive for type I collagen, osteonectin, bone sialoprotein and dentin sialoprotein (DSP), which are markers for dentin. This result was confirmed by in situ hybridization with a dsp probe. Only the aligned cells were positive with an antisense probe. On the other hand, hard tissue from other scaffolds showed incomplete expression of both bone and dentin markers and they were negative for osteonectin and DSP. These results suggest that scaffold shape affects the type of tissue regenerated by dental pulp-derived cells.

Formation of hepatocyte spheroids with structural polarity and functional bile canaliculi using nanopillar sheets.

We developed a method for controlling the spheroid formation of adult rat primary hepatocytes simply by optimizing the pillar diameters and patterns of nanopillar sheets. To investigate the effects of the pillar parameters on the spheroid formation, rat primary hepatocytes were cultured on nanopillar sheets with pillars that had one of five different diameters and that had been precoated with a solution containing one of two different concentrations of type I collagen. Spheroids with a compact morphology that were adhesive to the substratum and had an optimal size (50 to 100 microm) were obtained using a sheet with a pillar diameter of 2.0 microm that was precoated with 100 ng/mL of type I collagen solution. Immunohistochemistry revealed that the spheroids had a structure similar to that of native liver tissue. We then assessed the effect of overlaying reconstituted spheroids with Matrigel with the aim of achieving a simulated in vivo environment. The mRNA expression levels of MRP2, albumin, and P450-3A3 for spheroids determined by semiquantitative real-time PCR were significantly higher than those for spheroids cultured without the Matrigel overlay or for hepatocytes cultured using a conventional two-dimensional method. The spheroids obtained exhibited higher structural polarity and functional bile canaliculi compared with hepatocytes cultured using a conventional two-dimensional method.

Phytochrome A requires jasmonate for photodestruction.

The plant photoreceptor phytochrome is organised in a small gene family with phytochrome A (phyA) being unique, because it is specifically degraded upon activation by light. This so called photodestruction is thought to be important for dynamic aspects of sensing such as measuring day length or shading by competitors. Signal-triggered proteolytic degradation has emerged as central element of signal crosstalk in plants during recent years, but many of the molecular players are still unknown. We therefore analyzed a jasmonate (JA)-deficient rice mutant, hebiba, that in several aspects resembles a mutant affected in photomorphogenesis. In this mutant, the photodestruction of phyA is delayed as shown by in vivo spectroscopy and Western blot analysis. Application of methyl-JA (MeJA) can rescue the delayed phyA photodestruction in the mutant in a time- and dose-dependent manner. Light regulation of phyA transcripts thought to be under control of stable phytochrome B (phyB) is still functional. The delayed photodestruction is accompanied by an elevated sensitivity of phytochrome-dependent growth responses to red and far-red light.

A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis.

The plant vacuole is a multifunctional organelle which is essential for growth and development. To visualize the dynamics of plant vacuolar membranes, gamma-TIP (tonoplast intrinsic protein) was fused to GFP and expressed in Arabidopsis thaliana. The marker molecule was targeted to the vacuolar membranes in most tissues, as expected. In rapidly expanding cells, some additional spherical structures were often observed within the lumen of vacuoles, which emitted strong fluorescence. To confirm their normal presence, we examined wild-type Arabidopsis cotyledons by transmission electron microscopy. The metal-contact rapid-freezing method revealed that the vacuolar lumen of epidermal cells contained many cytoplasmic projections, which often formed spherical structures (1-3 microm diameter) consisting of double membranes. Thus we concluded that these structures are authentic and named them 'bulbs'. Three-dimensional reconstruction from serial electron microscopic images demonstrates that bulbs are very intricately folded, but are continuous with the limiting vacuolar membrane. The fluorescence intensity of bulbs is about threefold higher than that of vacuolar membrane. GFP-AtRab75c, another marker of the vacuole, did not give fluorescent signals of bulbs in transgenic plants, but the existence of bulbs was still confirmed by electron microscopy. These results suggest that bulbs define a subregion in the continuous vacuolar membrane, where some proteins are concentrated and others segregated.