DETECTION OF BACILLUS ANTHRACIS SPORES IN SOURCE AND FINISHED WATER

Authors: PEREZ, ABRIL - PHOENIX CTY WATER SERVICE; HOHN, CHRISTINA; HIGGINS, JAMES

Submitted to: Water Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/29/2005
Publication Date: 11/1/2005
Citation: Perez, A., Hohn, C.A., Higgins, J.A. 2006. Detection of Bacillus anthracis spores in source and finished water. Water Research. (39):5199-5211.

Interpretive Summary: Bacillus anthracis, the spore-forming bacterium which is responsible for the disease anthrax, was used in a bioterror attack in the Washington DC area in the Fall of 2001. This event heightened concerns about the ability to detect this organism in a variety of environmental sample types. In this project, the researchers developed a method to detect anthrax spores in water. The method relies on the use of filtration to recover spores from spiked 100 ml, 1 L, and 10 L volumes of tap water, followed by overnight culture of the recovered spores, which allows them to form distinctive colonies of bacterial cells on agar plates. The polymerase chain reaction (PCR) was used to provide molecular confirmation of the identity of the suspect colonies. Results indicate that as few as 10 spores can be detected in 10 L of tap water. Attempting to detect spores spiked into source water was less successful, mainly because of the increased concentration of particulate matter in source water, and the increased prevalence of other spore-forming, non-anthracis bacteria, which can overgrow the agar plates. In addition to the use of overnight culture to detect the anthrax spores, the researchers also developed a method based on the use of PCR to directly detect spores washed from the filters. This protocol is much faster than the culture step but has reduced sensitivity, being capable of detecting 534 spores spiked into 1 L of tap water. Overall, both the culture-based and direct detection-based methods are affordable and readily adaptable for use by water quality testing laboratories.

Technical Abstract: Spores of Bacillus anthracis Sterne strain were recovered from 100 ml and 1 L volumes of tap and source waters using filtration through a 0.45 um filter, followed by overnight culture on sheep red blood cell agar plates. With a spiking dose of 100 spores in 100 ml drinking water, the average recovery was 48 spores. Spiking doses of 35 and 10 spores in 1 L tap water were successful, but recovery efforts from spiked 1 L volumes of source water were problematic due to the concomitant growth of normal spore-forming flora. Recoveries were also attempted on 10 L volumes of tap water, using a combination of 0.2 um capsule and 0.45 um filtrations. For a spiking dose of 100 spores, mean recovery from six replicates was 11 spores (± 6.8, range 2 - 20), and for a spiking dose of 10 spores, mean recovery from six replicates was 2.3 spores (± 3.5, range 0 - 9). PCR for the lethal factor gene (lef) was used to confirm the identity of suspect colonies. Efforts were also made to "direct detect" spores via PCR on washes from filters, without recourse to overnight culture; a bead-beating step was used to lyse the spores and release DNA for extraction. When spiking 534 spores in 100 ml, 9/9 replicates of spiked tap water, 6/6 source water replicates, and 0/3 unspiked controls were positive by lef PCR. When 534 spores were spiked into 1 L tap water, the lef PCR was unsuccessful; however, using the nested vrrA PCR resulted in 4/9 spiked samples, and 0/3 unspiked controls, testing positive. Attempting to detect only 100 spores in 100 ml was unsuccessful. Our results indicate that an inexpensive and user-friendly method, utilizing filtration apparatus commonly present in many water quality testing labs, can readily be adapted for use in detecting this potential threat agent.