Rapid detection and characterization of mycobacteria using microchannel electrical impedance spectroscopy

The presence of virulent, pathogenic bacteria in our body, food, water and other consumables is harmful and causes enormous economic and personal losses. Here we will be looking at developing a platform technology that can rapidly detect pathogenic bacteria. The platform will be based on an electrical spectroscopic method called microchannel Electrical Impedance Spectroscopy (m-EIS). Initially, we will look at the growth of bacteria as our primary method to detect the presence of bacteria and find the time-to-detection. Following this, we will study the death of bacteria in a suspension using our technique of m-EIS to reduce the time-todetection further. We want to detect the pathogenic Mycobacteria Tuberculosis (that causes Tuberculosis in humans), which according to WHO, is the second leading cause of death due to infectious diseases. As proof of concept, surrogates like Mycobacterium bovis BCG and Mycobacterium smegmatis will be taken up for detection using our technique of m-EIS. However, our first focus is to prevent surgical site infections. Transmission of contagious infections like tuberculosis or infections occurring postsurgery like surgical site infections can be reduced and prevented, to some extent by proper treatment and correct use of antibiotics. For example, surgical site infection, occurring after insertion of implants can be reduced by use of coatings and other surface modifications. Many of these amendments ensure prevention of bacterial adhesion and kill surrounding bacteria near the implant. Further, in some cases, they promote the growth of desired cells that warrants proper integration of the implants with the bone. Surgical site infections occurring post-surgery can be reduced by following proper preoperative skin preparations. Several techniques are applied in the hospitals like 2-step scrubbing and painting, 2-step scrubbing and drying, and 1-step painting with a drying time. However, most of these techniques are time-consuming and labor-intensive. Here, we have demonstrated that the antimicrobial efficacy of a spray-on formulation containing Betadine is comparable to the existing techniques. The spray-on Betadine formulation is significantly less time-consuming and is not labor-intensive. Though prevention is necessary, often time, the pathogens have to be detected and identified as early as possible. In recent times, several molecular, serological and proteomic-based methods have been developed. However, these methods have several disadvantages such as they are expensive, labor-intensive, bulky, among others. The culture-based techniques are considered the gold standard. Though sensitive, they too suffer from inherent disadvantages of long time-to-detection. Hence, there is an urgent need for the development of rapid and cost-effective techniques for detection and identification of bacterial pathogens. Here, we present an approach that can detect the presence of viable microorganisms in suspensions, much faster than culture-based technique. The existing automated culture-based systems detect the metabolic changes in the growth media and the environment of the bottles containing the media (parameters monitored like pH, Oxygen, Carbon Dioxide among others) that change as the bacteria proliferates. As this changes in pH, Oxygen and others can be minuscule, a large number of bacteria is needed before the changes can be detected. This increases the time-to-detection significantly for culture-based techniques. Our technique, microchannel Electrical Impedance Spectroscopy (m-EIS), relies on the fact that on the application of an AC electric field to a bacterial suspension, the viable bacterial cells become polarized and store charges due to the presence of intact cell membranes. As a result, they behave as electrical capacitors. Any change in the number of bacterial cells in the suspension (like growth or death of cells) is reflected by a concomitant shift in the storage of bacterial charges and capacitance of the bulk. Due to the unique geometry of the measuring device (microfluidic cassettes) used by us, we can distinguish between the capacitance arising due to the bacterial cells and that of the parasitic double layer capacitance. A practical application of this technique has been found to be useful for detection of clinically significant slow-growing mycobacterial cultures. Most of the automated culture-based systems available in the market are based on the measurement of the growth dynamics of the microorganisms. However, as the generation time of the slow growing bacteria is long, these systems take a long time (6-8 weeks) to generate results. With the use of our m-EIS measurement technique, we can reduce the times-to-detection by [about]50%. Further, we observed that the time-to-detection is further reduced by monitoring cell death in real time using our technique. As only living entities can be killed, our technique can detect the presence of viable cells in a suspension by monitoring their death. It has been observed that using our technique following the death dynamics is much faster than growth dynamics. The death of microorganisms that have long generation times like mycobacteria can be achieved by use of antibiotics (depending on which antibiotics and its concentration) at a much faster rate than their growth in a nutrient media. Real-time monitoring of death shows a decrease in the bulk capacitance values which provides us the time-to-detection much more rapidly.Includes biblographical reference

Rapid culture-based detection of living mycobacteria using microchannel electrical impedance spectroscopy (m-EIS)

Abstract Background Multiple techniques exist for detecting Mycobacteria, each having its own advantages and drawbacks. Among them, automated culture-based systems like the BACTEC-MGIT™ are popular because they are inexpensive, reliable and highly accurate. However, they have a relatively long “time-to-detection” (TTD). Hence, a method that retains the reliability and low-cost of the MGIT system, while reducing TTD would be highly desirable. Methods Living bacterial cells possess a membrane potential, on account of which they store charge when subjected to an AC-field. This charge storage (bulk capacitance) can be estimated using impedance measurements at multiple frequencies. An increase in the number of living cells during culture is reflected in an increase in bulk capacitance, and this forms the basis of our detection. M. bovis BCG and M. smegmatis suspensions with differing initial loads are cultured in MGIT media supplemented with OADC and Middlebrook 7H9 media respectively, electrical “scans” taken at regular intervals and the bulk capacitance estimated from the scans. Bulk capacitance estimates at later time-points are statistically compared to the suspension’s baseline value. A statistically significant increase is assumed to indicate the presence of proliferating mycobacteria. Results Our TTDs were 60 and 36 h for M. bovis BCG and 20 and 9 h for M. smegmatis with initial loads of 1000 CFU/ml and 100,000 CFU/ml respectively. The corresponding TTDs for the commercial BACTEC MGIT 960 system were 131 and 84.6 h for M. bovis BCG and 41.7 and 12 h for M smegmatis, respectively. Conclusion Our culture-based detection method using multi-frequency impedance measurements is capable of detecting mycobacteria faster than current commercial systems

Direct-from-sputum rapid phenotypic drug susceptibility test for mycobacteria.

BackgroundThe spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof.Methods and findingsWe present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10μL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in ConclusionWe demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS

Pioneering Just-in-Time (JIT) Strategy for Accelerating Raman Method Development and Implementation for Biologic Continuous Manufacturing

Raman spectroscopy is a popular process analytical technology (PAT) tool that has been increasingly used to monitor and control the monoclonal antibody (mAb) manufacturing process. Although it allows the characterization of a variety of quality attributes by developing chemometric models, a large quantity of representative data is required, and hence, the model development process can be time-consuming. In recent years, the pharmaceutical industry has been expediting new drug development in order to achieve faster delivery of life-changing drugs to patients. The shortened development timelines have impacted the Raman application, as less time is allowed for data collection. To address this problem, an innovative Just-in-Time (JIT) strategy is proposed with the goal of reducing the time needed for Raman model development and ensuring its implementation. To demonstrate its capabilities, a proof-of-concept study was performed by applying the JIT strategy to a biologic continuous process for producing monoclonal antibody products. Raman spectroscopy and online two-dimensional liquid chromatography (2D-LC) were integrated as a PAT analyzer system. Raman models of antibody titer and aggregate percentage were calibrated by chemometric modeling in real-time. The models were also updated in real-time using new data collected during process monitoring. Initial Raman models with adequate performance were established using data collected from two lab-scale cell culture batches and subsequently updated using one scale-up batch. The JIT strategy is capable of accelerating Raman method development to monitor and guide the expedited biologics process development