Recipient Organization
IMPEDX DIAGNOSTICS INC.
8318 W 102ND ST
OVERLAND PARK,KS 662123420
Performing Department
(N/A)
Non Technical Summary
We aim to develop an electrical-sensor that can detect low-levels of Lactic Acid Bacteria (LAB) in the yeast inocula used to start large-scale ethanol cultures, and thus reduce the resulting loss of productivity of the culture and the incidence of "stuck" fermentations [Topic-Area 8.8: Biofuels and Bio-based products, wherein we will be "developing new and improved technologies that will lead to improved competitiveness"] Currently contamination by LAB is detected indirectly: by measuring products (lactic-acid) in the fermentation-broth using HPLC or LC-MS. These methods have a high limit-of-detection (corresponding to > 1 Million CFU/mL). The proposed sensing-approach relies on two phenomena. First: LAB can be killed rapidly using antibiotics that have minimal effect on yeast-cells. Second: this death can be monitored in real-time using microchannel Electrical Impedance Spectroscopy (m-EIS). Briefly, m-EIS relies on the fact that because living cells have a non-zero membrane-potential, exposing them to a high-frequency AC field results in charge accumulation at the cell-membranes (electrical-capacitance). Loss of membrane-potential accompanying cell death results in a drop in this capacitance. Hence, if an aliquot from a yeast inoculum is exposed to said antibiotics and monitored using m-EIS, one should see cell-death (drop in capacitance) only if LAB are present. In Phase I, we will demonstrate the ability to detect LAB at concentrations as low as 100 -1000 CFU/ml (3 orders-of-magnitude lower than incumbent methods). It is expected that screening inocula using this technology will result in fewer incidences of "stuck fermentations" and also increase the productivity of other cultures.
Animal Health Component
65%
Research Effort Categories
Basic
10%
Applied
65%
Developmental
25%
Goals / Objectives
We aim to develop an electrical-sensor to detect low-levels of Lactic Acid Bacteria (LAB) in yeast inocula used to start large-scale ethanol cultures, reducing the loss of productivity of the culture and the incidence of "stuck" fermentations (Topic-Area 8.8: Biofuels and Bio-based products), wherein we will be "developing new and improved technologies that will lead to improved competitiveness."In order for the R&D effort to be successful, we need to verify the following: (1) Low levels of lactic acid bacteria may be present at the end of aerobic culture without there being detectable levels of lactic present, (2) most (> 75%) of these bacteria present at low concentrations can be collected using MNPs, and re-suspended in smaller volumes,effectively concentration them by 100X, and (3) Upon re-suspension, these bacteria can be detected in < 2 hours using the "detection by death" approach. In consideration of the above, our Specific Aims will be the following:1. Verify that low levels of Lactic Acid Bacteria can be present in fermentations without there being detectable levels of acetic acid present.2. Quantify our ability of the MNPs to capture "contaminant" Lactic Acid Bacteria (LAB) (in the presence of yeast) from matrices of interest/ relevance3. Quantify (and improve upon) the ability of our sensing methodology ("detection by death" using m-EIS) to detect LAB in the presence of levels of MNPs and yeast that one would expect in real world situationsIn Phase I, we will demonstrate the ability to detect LAB at concentrations as low as 100 -1000 CFU/ml (3 orders-of-magnitude lower than incumbent methods). It is expected that screening inocula using this technology will result in fewer incidences of "stuck fermentations" and also increase the productivity of other cultures.
Project Methods
Tools:A. m-EIS has been applied to a number of problems in Biomedical Diagnostics such as rapid blood culture, rapid detection of live bacteria, phenotypic antibiotic susceptibility testing (AST). B. Magnetic Nano-Particles (MNPs) used to successfully capture live target microbes (lactic acid bacteria) from culture broths with an efficiency of >50%, resuspended captured microbes to concentrate. C. Beta-lactam antibiotics such as penicillin, ampicillin etc. at relatively high concentrations to kill a significant number of the target lactic acid bacteria (LAB) in 30min-1hr, without affecting yeast present.D. m-EIS to monitor bacterial death in real time (via decrease measured "bulk capacitance" of the suspension). Also, should bacteria not be present, the signal (decrease in bulk capacitance) will not be observed.Approach:Based on initial tests conducted, commercially available MNPs from Microsens Biotechnologies, London (UK) will be used. ImpeDx has modified the extraction protocol recommended by the manufacturer to maximize the capture efficiency of live cells. Modified protocols developed by ImpeDx Diagnostics were used to capture bacteria from numerous clinically relevant biological matrices.Capture efficiency is obtained using methods described in literature. Briefly, volumes of the original sample taken for analysis, the discarded supernatants, and the re-suspended pellet containing MNPs and microorganisms were recorded. Microbial concentrations in suspensions were then estimated using standard agar-plate counts. Product of estimated concentration and recorded volume yield estimated microbial load in samples. The ratio of the estimated load (in log) in the isolate (re-suspension) to that in the starting sample yields the capture efficiency of the extraction process. We plan to apply similar protocols to 5-10mL aliquots of fermentation broth containing 100 CFU/mL of lactic acid bacteria (LAB). 10mL of the suspension containing yeast and (LAB) (PBS or DME) is mixed with equal volume of commercially available MNPs and sits for 10min. The MNPs (and bound microbes) are collected using a magnet. The supernatant is discarded, and the MNPs-microbe complexes are washed and resuspended in 100 µL of PBS. Efficiencies of 80% for LAB in dry malt extract is achieved.The effect of beta lactam antibiotics on LAB and yeast Beta lactam antibiotics work by inhibiting the synthesis of peptidoglycan, thereby specifically killing the bacteria and not yeasts.Examples of beta-lactam drugs include penicillin, ampicillin, cephalosporin etc. LAB remain almost universally susceptible, with >90% of wild strains having Minimum Inhibitory Concentration (MIC) values at or below 4 µg/mL for ampicillin. Hence, beat lactams at 4X the cutoff values can serve as effective agents to selectively kill LAB.We have developed (and patented) a novel method to detect the presence of proliferating microorganisms in suspensions. Aqueous solutions containing species cells can be modeled electrically by our circuit. The charge stored at the electrode-solution interfaces is accounted for by the two interfacial Capacitances (Ce), and the charge stored by elements dispersed in the interior by the "bulk capacitance" (aka "medium" or "geometric" capacitance) (Cb). When cells proliferate, we observe an increase in bulk capacitance (Cb). Also, such large amount of charge accumulate only at intact membranes over which a potential difference exists (live cells with a non-zero membrane potential). Cell death is accompanied by loss of membrane potential and electrical polarization, death of cells is manifested as a decrease in bulk capacitance (Cb).Detection by Death: Our ability to monitor cell death in real time forms the basis of our ability to use this method to assay for antibiotic susceptibility / resistance in bacteria isolated from clinical samples. In the previously published study, turbidometrically standard bacterial solutions were prepared from bacterial colonies according to established protocols. These protocols were developed to ensure that extraneous factors do not interfere with either the mechanism of action of the antibiotic on the microorganism, or with the sensing method that is used to determine growth, or lack thereof.While each sample electrically assayed may have a different value of Cb due to differences in protein concentration, number of cells present, and concentration of MNPs, the Cb value will change over time only when cells grow or die.To ensure unbiased analysis of the data, the following statistical approach is adopted to determine whether one is observing growth, death or stasis in a given experiment: The slope of the Cb vs. time for the first 3 hours is then calculated, along with the 90% confidence interval of its value. We plot the slopes recorded (our estimates of the rates of increase/decrease) along with the confidence intervals (error bars).We call the above approach "Detection by Death", and we have previously shown that mycobacteria that have doubling times of ~ 20 hours (days to detect by culture) is detected in 5 hours using Detection by Death.We use betalactam antibiotics as our killing agents, and since yeasts are resistant to beta-lactams, "cells that can be killed" equates to just the contaminating LAB. The effect of the antibiotic on the cells is monitored both electrically, and by taking small aliquots at pre-determined time intervals (every ½ hour) and introducing them into our designed and fabricated microfluidic cassettes, and plating them. Cells are re-suspended in an inert medium (PBS), and number of live yeast cells remains constant. When placed in growth media instead, they proliferate, and in this case, the signal from the growth of yeast cells overwhelms the signal from the death of bacteria.To advance our rationale that this limit of detection represents a case where the concentration of LAB in the sample is fairly high, we note that literature indicates that, for a typical LAB under static conditions, the (non-growth) specific rates of lactic acid production are ~10 mmol of lactic acid per gram of bacteria per hour. Given that a bacterial cell has a dry weight of 12 grams, the cell mass of 1 gram represents 1012 cells. So, 1012 cells produce 1 g of lactic acid per hour. In order to bring about a lactic acid concentration of 0.5 mg/L (0.5 x 10-6 g/mL), one needed 2e6 CFU/mL of lactic acid bacteria to have been "working" for an hour, and thus the limit of detection in terms of CFU/mL is certainly at or above 106 CFU/mL. Interestingly, this "threshold of detection" (e7 to e8 CFU/mL) is very similar to that of medical instruments such as the BACTEC which use bacterial metabolism to detect the presence of live bacteria in blood culture broth.In contrast, if there are ~100 CFU/mL of bacteria in the suspension, one can take a 10mL aliquot, use MNPs to isolate the ~1000 CFU of bacteria present into 100µL in minutes, thereby achieving local concentrations of e4 CFU/mL. We can then check for death of collected bacteria in antibiotic (ampicillin). Thus, we detect lactic acid bacteria at levels ~ 5 logs lower than currently done routinely, and 3 logs lower than what can be achieved using significantly more expensive equipment. The detection of these low levels of pathogens in the inoculum potentially prevents LAB from reaching levels that significantly reduce productivity and/or lead to stuck fermentations.