The processing of staphylococcal biofilm images from Fluorescence and Confocal Microscopy to assess in vitro efficacy of antiseptic molecules

The in vitro efficacy of locally applied antiseptic molecules against staphylococcal biofilm is frequently assessed by a set of standard quantitative and semi-quantitative methods. The development of software for parametric image processing allowed to obtain parametric data also from microscopic images of biofilm dyed with a variety of dyes, especially with propidium iodine and SYTO-9, differentiating dead from live cells. In this work, using confocal/epifluorescent microscopy, we analyzed such major properties of staphylococcal biofilm in vitro as its thickness, cellular density and share of Live/Dead cells within its individual parts. We also scrutinized the impact of sample preparation and antiseptic introduction on the outcome obtained. As a result of our analyses we developed a revelatory method of assessment of the impact of antiseptic agents on staphylococcal biofilm in vitro, in which the microscopic images are processed with the use of ABE formula (Antiseptics Biofilm Eradication) which implements all the data and phenomena detected and revealed within the course of this study. We tested ABE with regard to polyhexanide, povidone-iodine and hypochlorous antiseptics and found a high correlation between this parameter and the results obtained by means of traditional techniques. Taking into account the fact that in vitro results of the efficacy of antiseptic agents against staphylococcal biofilm are frequently applied to back up their use in hospitals and ambulatory units, our work should be considered an important tool providing reliable, parametric data with this regard.

A fluorescence-based viability assay for Phytophthora agathidicida oospores

Viability staining is an essential tool in many fields of microbiology. In this study, we aimed to establish a dual fluorescence method for detecting the viability of Phytophthora oospores. Phytophthora is a genus of plant pathogens in the class Oomycete that cause disease in a wide range of agriculturally and ecologically important plants. During the Phytophthora disease cycle, thick-walled oospores can be produced via sexual reproduction. These oospores are essential for long-range dispersal and long-term survival of the pathogen, and therefore methods for the study of oospores are of great interest. In this study, we tested five fluorescent dyes for their ability to stain Phytophthora agathidicida oospores: SYTO 9, FUN-1, fluorescein diacetate, propidium iodide, and TOTO-3 iodide. These dyes represented a selection of total, viable and non-viable cell stains Each dye was assessed individually, and then the best dyes were combined for dual viability staining. In this study, we have identified three dyes — SYTO 9, fluorescein diacetate and TOTO-3 — that can be used to report total, viable and non-viable oospores respectively. We further demonstrate that fluorescein diacetate and TOTO-3 can be used together for dual viability staining. This new method is quantitative and compatible with automated image analysis, allowing oospores to be rapidly and accurately assessed for viability.

Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when Escherichia coli was treated with lytic antibiotics—ampicillin and polymyxin B—and stained with SYTO 9 and/or propidium iodide. Antibiotic concentration, bacterial growth phase, and treatment time used affected the efficacy of this detection method. Quantification methods of the lethal action and inhibitory action of the non-lytic antibiotics, ciprofloxacin and chloramphenicol, respectively, remain to be elucidated.

Rapid, Affordable and Scalable SARS-CoV-2 Detection from Saliva

Here we present an inexpensive, rapid, and robust RT-LAMP based SARS-CoV-2 detection method that is easily scalable, enabling point of care facilities and clinical labs to determine results from patients' saliva directly in 30 minutes for less than $2 a sample. The method utilizes a novel combination of widely available reagents that can be prepared in bulk, plated and frozen and remain stable until samples are received. This innovation dramatically reduces preparation time, enabling high-throughput automation and testing with time to results (including setup) in less than one hour for 96 patient samples simultaneously when using a 384 well format. By utilizing a dual-reporter (phenol red pH indicator for end-point detection and SYTO-9 fluorescent dye for real-time), the assay also provides internal validation of results and redundancy in the event of an instrument malfunction.

Clinical Biofilm Ring Test® Reveals the Potential Role of β-Lactams in the Induction of Biofilm Formation by P. aeruginosa in Cystic Fibrosis Patients

Biofilms are characterized by high tolerance to antimicrobials. However, conventional antibiograms are performed on planktonic microorganisms. Through the clinical Biofilm Ring Test® (cBRT), initially aimed to measure the adhesion propensity of bacteria, we discerned a variable distribution of biofilm-producer strains among P. aeruginosa samples isolated from expectorations of cystic fibrosis (CF) patients. Despite a majority of spontaneous adherent isolates, few strains remained planktonic after 5 h of incubation. Their analysis by an adapted protocol of the cBRT revealed an induction of the biofilm early formation by sub-inhibitory doses of β-lactams. Microscopic observations of bacterial cultures stained with Syto 9/Propidium Iodide (PI) confirmed the ability of antimicrobials to increase either the bacterial biomass or the biovolume occupied by induced sessile cells. Finally, the cBRT and its derivatives enabled to highlight in a few hours the potential inducer property of antibiotics on bacterial adhesion. This phenomenon should be considered carefully in the context of CF since patients are constantly under fluctuating antimicrobial treatments. To conclude, assays derived from the Biofilm Ring Test® (BRT) device, not only define efficient doses preventing biofilm formation, but could be useful for the antimicrobial selection in CF, to avoid inducer molecules of the early biofilm initiation.

Rapid identification of bacterial viability, culturable and non-culturable stages by using flow cytometry

The protocol details methods developed to optimize staining procedures and instrument settings for the rapid identification and unbiased enumeration of viable but non-culturable (VBNC) and viable-culturable (VC) cells as well as the membrane integrity of bacteria by flow cytometry (FCM). To detect viability, various Gram-negative bacteria were stained with numerous fluorescent membrane permeable (SYTO 9, SYTO 13, SYTO 17, SYTO 40) and impermeable (propidium iodide, PI) probes, and then quantified using the FCM. FCM data are then integrated with specific plate count results to calculate VC cells in different growth states. The cells were also exposed to heat (72° C) to monitor cellular membrane integrity. At late log phase (at 18 h incubation) of bacterial culture, the culturable, non-culturable and cells with damaged membranes varied from 40-98%, 1 to 64% and 0.7% to 4.5%, respectively. In this study, the cells with damaged cell membranes were considered dead (non-viable). This robust method preserves cell viability and takes a little more than an hour allowing the simultaneous quantification of phenotypes or cellular functions that is compatible with downstream cell-sorting and RNA-based assays.

Optimization of staining with SYTO 9/propidium iodide: interplay, kinetics and impact on Brevibacillus brevis

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Here we characterized the effects of these two dyes on Brevibacillus brevis under optimized staining. We found that SYTO 9 entered cells continuously while PI tended to adhere to the cell wall before entering the cell. In addition, results showed that a high amount of the dyes altered the physicochemical properties of membranes, improving their breakthrough. These results provide new perspectives and ideas for improving the characterization of bacterial viability using flow cytometry.

Effects of ultraviolet-C on the spores of Bacillus subtilis and Bacillus velezensis suspension in phosphate buffered saline with their structural and molecular analysis using Raman-AFM imaging

Bacterial spores are of concern in food processing due to their ubiquity and resistance. This study seeks to determine the effect of ultraviolet C (UV-C) in the inactivation of spores of Bacillus subtilis and Bacillus velezensis that can result in enzymatic spoilage in foods using PBS as the suspension medium. Purified spore samples were treated under 1 pass in a UV-C reactor using 10 mL of spore inoculum with one dose of the radiation (410 mJ/cm2) for 10secs at room temperature. Aliquots of the treated samples were plated on tryptone soy agar supplemented with 0.6% glucose and the colonies counted. Flow cytometry analysis was done using 500 μL of both treated and control samples with a cell concentration of a ≥106 CFU/ml with propidium iodide (15 μM) and SYTO 9 (500 nM) used as live/dead stains. Samples were processed for microscopy (SEM and Raman-AFM Imaging). The maximum lethality is 2.5 for B. velezensis and the minimum is 0.1 for B. subtilis. Microscopic imaging of treated spores shows significant morphological disruption of the spore structure. The Raman spectroscopy analysis reveals the B. subtilis isolates to have the highest concentrations of dipicolnic acid (Ca+2DPA) as well as other compounds belonging to other functional groups. Flow cytometric analysis of treated spores reveals sub-populations unaccounted for by plate count. UV-C shows a promising application in the inactivation of resistant spores during processing of liquid foods such as milk.

A quite sensitive fluorescent loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus

Introduction: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. Methodology: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. Results: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). Conclusions: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.