CICEET Progress Report for the period 3/16/04 through 9/15/04

Project Title: Assay and Sensor Development to Identify, Detect, and Quantify Microbial Contaminants
Principal Investigator(s): Jack Fell, Kelly Goodwin, Peter Ortner

Accomplishments
This project aims to apply advances in biotechnology to water quality monitoring. Specifically, the near term goal is a high throughput species-specific assay to detect sewage indicating bacteria and toxic algae.

Scheduled Tasks:
a) Test Luminex assay for detection of fecal bacteria.
b) Sequence bacteria from environmental samples to compare Luminex against traditional culture-based methods.
c) Use Luminex for harmful algae detection.

Progress on Tasks
We have made substantial progress on the tasks outlined above. Progress is described in the “Preliminary Data” section.

• Basic Luminex probe design and testing was completed for both fecal bacteria and a variety of harmful algae.
  
• Environmental samples were assayed by culture-based methods and Luminex. Sequencing was used to verify the culture-based results.
  
• A new postdoc was successfully hired to work on the project.

Difficulties Encountered
The postdoc working on this project moved to a new position. Although replacing personnel was costly for time, it expanded the educational opportunities provided by this project.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
We anticipate being able to meet project objectives.

Preliminary Data
a) Detection of Sewage-Indicating Bacteria.
Species-specific probes and group-specific probes were designed for the Luminex system from sequence analysis of the 16s rRNA gene for several species and groups of bacteria associated with fecal contamination. Each probe was tested to validate the specificity and to determine the stringency of the hybridization conditions necessary for discrimination among other species or groups. The probe designed to identify the total coliform group was called ENTI2. ENTI2 is a modification of ENT1, originally designed by Loge et al. (1999). We found that the original sequence had strong secondary structure so ENT1 was shifted 3 basepairs 3’and lengthened by 3 basepairs on the 3’ end. ENTI2 binds from basepairs 1251-1270 in the 16s rRNA gene (based on E. coli sequence X80723). The probe to identify E. coli (COLINSITU2), originally called Ec637 was designed by Regnault et al. (2000). COLINSITU2 binds from basepairs 623-646 in the 16s rRNA gene (based on E. coli sequence X80723).

Efaec2 was designed to specifically identify the species Enterococcus faecalis. Efaec2 binds to basepairs 471-494 in the 16s rRNA gene (based on E. faecalis sequence AF515223). Ehir was designed to identify Enterococcus faecium, Enterococcus durans and Enterococcus hirae but will also hybridize with Enterococcus ratti and Enterococcus villorum. Ehir binds to basepairs 476-499 in the 16s rRNA gene (based on E. faecium sequence AY172570). Eflav was designed to identify Enterococcus casseliflavus and Enterococcus flavescens but will also hybridize with Enterococcus dispar, Enterococcus pseudoavium, Enterococcus sulfureus and Enterococcus gilvus. Eflav binds to basepairs 446-470 in the 16s rRNA gene (based on E. flavescens sequence Y18295).

Bfra3, a modification of Bfra602, originally designed by Franks et al. (1998), specifically identifies the Bacteroides fragilis group. We modified Bfra602 by adding 3 basepairs onto the 5’ end. Bfra3 binds at basepairs 586-607 in the 16s rRNA gene (based on B. fragilis sequence AB050106). Bdis3 was designed to specifically identify Bacteroides distasonis and it binds at basepairs 711-732 in the 16s rRNA gene (based on B. distasonis sequence M86695).

The probes described above have been successfully utilized with cultures and on environmental samples. Figure 1 shows Luminex probes used to detect a variety of fecal indicating bacteria. The primers Unifor/Unirev822 were used to amplify a short piece of 16s rDNA. Results were as expected: Enti 2, Bdis 3, and Bfra 3 gave positive signals. Other probes did not because they were designed outside the region of this amplicon (Enti2) or they were labeled on an alternative strand (Efaec, Ehir and Eflav). To increase speed and utility of the assay, multiplex PCR is used (Figure 2). Seven primers and 6 DNA types were successfully multiplexed in this Luminex assay. Results were as expected. Ehir and Eflav were not expected to produce a signal since they should not have bound to any of the DNA types present; therefore, no cross-reactivity was observed.

REFERENCES
Loge et al. Water Env. Res. 1999, V71, pp. 76-83.
Franks et al. Appl. Environ. Microbiol. 1998, V 64, pp. 3336-3345.
Regnault et al. Res. Microbiol. 2000, V151, pp. 521-533

b) Sequence bacteria from environmental samples to compare Luminex against traditional culture-based methods.

In addition to extracting DNA from environmental samples and testing on the Luminex system, samples were plated using traditional culture-based methods to enumerate fecal-indicating bacteria. Selective media were used to grow Bacteroides species, E. coli, Enterococci species, fecal coliform, and total coliform. The culture methods were used to test the sensitivity of the Luminex assay. However, it was important to verify that the media were indeed producing the expected bacteial taxa.. Therefore, colonies growing on the plates were picked and sequenced. Water from the Miami River sampled during three separate collections was plated and a subset of the resulting bacteria sequenced. Approximately 700 colonies were picked for sequencing and readable results were obtained for 432. Sequences were obtained for 115 colonies picked from plates selective for Bacteroides, 62 from E. coli, 82 from Enterococci: 140 from total coliform, and 33 from fecal coliform plates. Results are summarized below.

• Bacteroides medium was not specific for Bacteroides, in fact only 1 out of 115 colonies was Bacteroides. Most colonies were actually Cetobacterium somerae. Such information is critical if analysis of the sensitivity of Luminex is based, in part, on traditional culture methods
• The E. coli specific medium was not entirely specific for E. coli. Only 45/62 colonies sequenced were E. coli colonies. Other colonies were members of or closely related to Acinetobacter, Enterobacter, Shigella, Klebsiella, and Aeromonas. Again, these results are significant if molecular methods are to be evaluated on and culture-based methods.
• Medium specific for Enterococci appeared to be fairly specific for Enterococci. Colonies whose sequence matched that of Streptococcus macedonicus/S. bovis AF459431/AF104114 were obtained (25 out of 82 colonies). However, Enterococcus species were once included in the genus Streptococcus, thus this could reflect a issue with nomenclature.
• Colonies picked from total and fecal coliform plates produced a variety of sequences. Most were either total or fecal coliforms, except for Aeromonas species, which were highly represented.

c) Use Luminex for harmful algae detection.
Luminex probes were designed for the following 6 species of harmful algae and successfully tested with DNA from algal cultures. A Luminex probe was also designed for Karenia mikimotoi, but DNA was not available for testing. The Kbrev probe was used on environmental samples from the Rookery Bay National Estuarine Research Reserve. The Luminex assay successful detected K. brevis in those samples.

probe name	harmful algal species
Kbrev	Karenia brevis
AC2	Amphidinium carterae
Plim	Prorocentrum lima
Pmic	Prorocentrum micans
Phoff	Prorocentrum hoffmanium
Pparv2	Prymnesium parvum

Tasks and activities for the next reporting period
Continue optimization of the Luminex assay for application to coastal water quality monitoring.

Tasks for the next reporting period
a) Explore DNA extraction methods to increase sensitivity and throughput of the assay.
b) Continue to sequence bacteria from environmental samples to compare culture based and molecular based methods.
c) Test Luminex assay using samples collected from beach surf zone.
d) Expand Luminex assay to include analysis of beach sand, as well as water column samples.

Work plan to accomplish tasks
We will continue to optimize the Luminex assay by exploring DNA extractions methods in an effort to increase sensitivity up stream of the assay itself. We will apply the Luminex assay to recreational waters. In addition, we will expand assay application to beach sand because of the growing evidence that sand may be a source of fecal bacteria to overlying waters. This will necessitate development and optimization of extraction techniques for this matrix. Recreational water sampling will be performed in conjunction with traditional microbiological assay to “ground-truth” the molecular approaches. In turn, sequencing will be used to verify the types of colonies grown using the traditional assays. A local beach is being used because Rookery Bay NERR samples were not found to be contaminated with fecal bacteria and thus did not offer appropriate samples with which to develop the assay.

Overall Project Timeline Update
Presently, a new postdoc on the project is completing basic testing of the probes mentioned above. In October, she will travel to Luminex to receive formalized training. Afterward, the focus will be to improve sensitivity with environmental samples by understanding and increasing the efficiencies of DNA extraction and PCR.

Expenditures
Expenditures were in the expected range.

Dissemination
Publications:
A DNA hybridization assay to identify toxic dinoflagellates in coastal waters: detection of Karenia Brevis in the Rookery Bay National Estuarine Research Reserve. K.D. Goodwin, S.A. Cotton, G. Scorzetti, and J.W. Fell, Harmful Algae, in press (2004).

Detection of Karenia brevis by a Microtiter Plate Assay. K.D. Goodwin, G. Scorzetti, S.A. Cotton, T.L. Kiesling, P.B. Ortner, and J.W. Fell. IN: Harmful Algae 2002. Proceedings of the Xth International Conference on Harmful Algae. Steidinger, K.A., Landsberg, J.H. Tomas, C.R., and Vargo, G.A. (Eds.). Florida Fish and Wildlife Conservation Commission and Intergovernmental Oceanographic Commission of UNESCO, in press (2004).

Workshops:
K.D. Goodwin. “Molecular Biological Water Quality Monitoring to Protect Human Health.” NOAA Workshop on Oceans and Human Health: Research Programs, and Related Activities. Silver Spring, MD, May 3-4, 2004.

Conferences:
K.D. Goodwin, S.A. Cotton, G. Scorzetti, T.L. Kiesling, and J.W. Fell. 2004. Use of Immobilized DNA Probes to Rapidly Detect Sewage-Indicating Bacteria and Toxic Dinoflagellates, NIEHS-MFBS Center/ARCH Program Science Symposium, March 18, Miami, FL.

J. Fell, C. Sinigalliano, M. Diaz, T. Kiesling, G. Scorzetti, K.D. Goodwin, S. Cotton, and M. LaGier. Molecular Detection of HAB’s and Other Little Critters, NIEHS-MFBS Center/ARCH Program Science Symposium, March 18, Miami, FL.

T.L. Kiesling, M. Diaz, K.D. Goodwin, S.A. Cotton, J.W. Fell. 2004. Hybridization Based Detection of Fecal Bacteria Using the Luminex 100 System, ASLO, February 15-20, Honolulu, HI and NIEHS-MFBS Center/ARCH Program Science Symposium, March 18, Miami, FL.

K.D. Goodwin. G. Scorzetti, S.A. Cotton, and J.W. Fell. 2003. Microplate Detection of Microbial Contaminants. EPA Workshop, January 29-31, Orlando, FL.

Training:
A postdoc hired in July is receiving training on this project.

Contact with End Users:
The assays being developed under this project are undergoing market analysis by a technology transfer company.