title: Filtration methods for recovery of Bacillus anthracis spores spiked into source and finished water.
abstract: Spores of Bacillus anthracis Sterne strain were recovered from 100ml and 1L volumes of tap and source waters using filtration through a 0.45um filter, followed by overnight culture on agar plates. In a set of experiments comparing sheep red blood cell (SRBC) plates with a chromogenic agar formulation designed by R & F Laboratories, with a spiking dose of 47 plate-enumerated spores in 100 ml tap water, the mean spore recoveries were 34.0 and 30.8 spores, respectively. When a spiking dose of 100 fluorescence activated cell sorter(FACS)-enumerated spores was used in 100 ml potable water, the average recovery with SRBC plates was 48 spores. Detection efforts with 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. 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). Efforts were also made to "direct detect" spores via polymerase chain reaction (PCR) on washes from filters. 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. 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.
authors.affiliation: City of Phoenix Water Services Laboratory, 2474 South 22nd Avenue, Phoenix, AZ 85009, USA.
authors.forename: Abril
authors.initials: A
authors.lastname: Perez
authors.forename: Christina
authors.initials: C
authors.lastname: Hohn
authors.forename: James
authors.initials: J
authors.lastname: Higgins
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics; D005374:Filtration / Q000379:methods; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000302:isolation & purification; D014871:Water Microbiology; D014873:Water Pollutants / Q000302:isolation & purification; D018508:Water Purification / Q000379:methods*; D014881:Water Supply
chemical_list: D004269:DNA, Bacterial; D014873:Water Pollutants