Research Accomplishment Reports 2007

Development of Peptides to Enhance Cheese Production and Bio-active Probes

C.L. Hicks
Department of Animal and Food Sciences

 

Project Description

Escherichia coli 15q and 15cc bacteria in TSB medium, at 32 deg. C for >5 h was monitored using contact acoustic sensors (20 to 50 khz and 50 to 200 khz) attached to the sides of the growth vessel. Acoustical emissions generated by the bacteria were picked up as a waveform and each waveform was referred to as a "hit". Hits were analyzed for rate of accumulation and periodic cycles. Fast Fourier transform analysis was used to calculate average peak frequency emissions.

Initial analysis showed that hit detection from the 20 to 50khz sensor began within 5 min after the medium was inoculated with E. coli 15cc. Hit detection from the 50 to 200 khz sensor became more apparent as the organism entered the log phase and displayed a linear natural log increase in hits during the log growth phase. Periodic cycles of 1.66 sec and 33.6 sec were observed during the early stages of growth suggesting that E. coli 15cc was involved in uniform sequenced activities, possibly quorum sensing. The average peak frequencies data showed shifting in frequencies as the bacteria moved from the lag, log, and stationary phases.

Differences between E. coli 15q and 15cc could be observed within the first 60 min of incubation (20 to 50 khz sensor) with 15q producing 7average peak domains (frequencies) and 15cc producing only 5. After 5 h of incubation 15q produced only one broad peak domain while 15cc generated one defined domain and two smaller domains. Average peak frequencies for E. coli 15cc and 15q were sufficiently different in frequency and intensity during the lag, log and stationary phases that specific strain identification might be possible.

Thus, acoustic emissions from bacteria may be unique enough to acoustically fingerprint bacteria and result in the most rapid of rapid assay methods.

Impact

Expected Significance. Rapid assay technologies are important to the medical, manufacturing, and regulatory sectors globally. Currently, rapid assays technologies are growing at a rapid rate and many startup companies are enjoying rapid growth due to financial opportunities that rapid assay technologies afford. Rapid assays are a multi-billion dollar industry globally. If the AE fingerprint technology is successful for a non-pathogenic organism (model system) then the technology should work as well for pathogenic organisms. If the assay can measure 102 bacteria /ml or less and each assay costs less than $5.00 (including operator time), this rapid assay system should replace most other assays that require greater cost, greater preparation time, and higher skilled technicians. The value of an AE fingerprint assay that is specific for a pathogen and that could measure a pathogen at a minimum concentration within a mixture of bacteria would have a retail value of $30/ assay when compared to other assays that are currently on the market. Currently, companies pay identification laboratories from $19.00 (negative test) per assay for a standard plate assays that requires 7 days. ELISA tests cost about $25.00 per test and require 24 to 48 hours. PCR tests cost approximately $30.00 per test and take at least 12 hours.

The authors believe that AE fingerprint assays would replace all PCR and ELISA tests currently being used by testing laboratories if assay times could be cut to 30 min. Thus, several million assays/year would be performed for the food and regulatory industry per year at a cost of greater than 60 million dollars per year. It is anticipated that the medical industry would use even more assays than the food industry. For example, every person admitted to a hospital for any kind of infection or fever gets blood cultures drawn and these samples are submitted for antibiotic susceptibility tests. On the average, 0.3% of these admissions turn out to be severe sepsis, i.e. a run-away bacterial attack in the blood, and the interval between hospital admission and treatment with the correct antibiotic is a life-or-death countdown of between 24-96 hours (7). On the contrary, AE data obtained to date show the detection of bacterial growth, no growth or death within one hour of inoculation and a sensitivity at approximately 105 cfu/ml (current state of art). Sepsis occurs when the bacterial count is approximately 105cfu/g.

Publications

Hicks, C. L., J.M. Stencel, H. Song, and F. A. Payne. 2007. Acoustical emissions generated by E. coli. J. Dairy Sci. 90 (Suppl. 1):426. Abst. 576.

Hicks, C. L., J.M. Stencel, and H. Song. 2007. Acoustical emissions generated by Lactococcus lactis ssp lactis C2. J. Dairy Sci. 90 (Suppl. 1):488. Abst. W82.