An Optical Fiber-Based Nanomotion Sensor for Rapid Antibiotic and Antifungal Susceptibility Tests.

The emergence of antibiotic and antifungal resistant microorganisms represents nowadays a major public health issue that might push humanity into a post-antibiotic/antifungal era. One of the approaches to avoid such a catastrophe is to advance rapid antibiotic and antifungal susceptibility tests. In this study, we present a compact, optical fiber-based nanomotion sensor to achieve this goal by monitoring the dynamic nanoscale oscillation of a cantilever related to microorganism viability. High detection sensitivity was achieved that was attributed to the flexible two-photon polymerized cantilever with a spring constant of 0.3 N/m. This nanomotion device showed an excellent performance in the susceptibility tests of Escherichia coli and Candida albicans with a fast response in a time frame of minutes. As a proof-of-concept, with the simplicity of use and the potential of parallelization, our innovative sensor is anticipated to be an interesting candidate for future rapid antibiotic and antifungal susceptibility tests and other biomedical applications.

Mitochondrial nanomotion measured by optical microscopy.

Nanometric scale size oscillations seem to be a fundamental feature of all living organisms on Earth. Their detection usually requires complex and very sensitive devices. However, some recent studies demonstrated that very simple optical microscopes and dedicated image processing software can also fulfill this task. This novel technique, termed as optical nanomotion detection (ONMD), was recently successfully used on yeast cells to conduct rapid antifungal sensitivity tests. In this study, we demonstrate that the ONMD method can monitor motile sub-cellular organelles, such as mitochondria. Here, mitochondrial isolates (from HEK 293 T and Jurkat cells) undergo predictable motility when viewed by ONMD and triggered by mitochondrial toxins, citric acid intermediates, and dietary and bacterial fermentation products (short-chain fatty acids) at various doses and durations. The technique has superior advantages compared to classical methods since it is rapid, possesses a single organelle sensitivity, and is label- and attachment-free.

Modulation of the nanoscale motion rate of Candida albicans by X-rays.

Introduction: Patients undergoing cancer treatment by radiation therapy commonly develop Candida albicans infections (candidiasis). Such infections are generally treated by antifungals that unfortunately also induce numerous secondary effects in the patient. Additional to the effect on the immune system, ionizing radiation influences the vital activity of C. albicans cells themselves; however, the reaction of C. albicans to ionizing radiation acting simultaneously with antifungals is much less well documented. In this study, we explored the effects of ionizing radiation and an antifungal drug and their combined effect on C. albicans. Methods: The study essentially relied on a novel technique, referred to as optical nanomotion detection (ONMD) that monitors the viability and metabolic activity of the yeast cells in a label and attachment-free manner. Results and discussion: Our findings demonstrate that after exposure to X-ray radiation alone or in combination with fluconazole, low-frequency nanoscale oscillations of whole cells are suppressed and the nanomotion rate depends on the phase of the cell cycle, absorbed dose, fluconazole concentration, and post-irradiation period. In a further development, the ONMD method can help in rapidly determining the sensitivity of C. albicans to antifungals and the individual concentration of antifungals in cancer patients undergoing radiation therapy.

Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics.

Background and Objectives: Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method's applicability.

Nanomotion Detection-Based Rapid Antibiotic Susceptibility Testing.

Rapid antibiotic susceptibility testing (AST) could play a major role in fighting multidrug-resistant bacteria. Recently, it was discovered that all living organisms oscillate in the range of nanometers and that these oscillations, referred to as nanomotion, stop as soon the organism dies. This finding led to the development of rapid AST techniques based on the monitoring of these oscillations upon exposure to antibiotics. In this review, we explain the working principle of this novel technique, compare the method with current ASTs, explore its application and give some advice about its implementation. As an illustrative example, we present the application of the technique to the slowly growing and pathogenic Bordetella pertussis bacteria.

Single yeast cell nanomotions correlate with cellular activity.

Living single yeast cells show a specific cellular motion at the nanometer scale with a magnitude that is proportional to the cellular activity of the cell. We characterized this cellular nanomotion pattern of nonattached single yeast cells using classical optical microscopy. The distribution of the cellular displacements over a short time period is distinct from random motion. The range and shape of such nanomotion displacement distributions change substantially according to the metabolic state of the cell. The analysis of the nanomotion frequency pattern demonstrated that single living yeast cells oscillate at relatively low frequencies of around 2 hertz. The simplicity of the technique should open the way to numerous applications among which antifungal susceptibility tests seem the most straightforward.

Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria.

Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed.

Outer membrane protein OmpQ of Bordetella bronchiseptica is required for mature biofilm formation.

Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48 h. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.