Semi-automated water sampling module for repeated sampling and concentration of Bacillus cereus group spores

Abstract

Monitoring the presence of pathogenic Bacillus spores is important for industrial applications, as well as necessary for ensuring human health. Bacillus thuringiensis is used as a biopesticide against several insect pests. Bacillus cereus spores are a significant cause of food poisoning, and Bacillus anthracis is a recognized biosecurity threat. Laboratory-based methods, such as polymerase chain reaction, enzyme-linked immunosorbent assay, or matrix-assisted laser desorption ionization spectroscopy provide sensitive detection of bacteria and spores, but the application of those methods for quasi-continuous environmental monitoring presents a significant challenge requiring frequent human intervention. To address this challenge, we developed a workstation for quasi-autonomous monitoring of water reservoirs for the presence of bacteria and spores, and designed and validated the functionality of a microprocessor-controlled module capable of repetitive collection and pre-concentration of spores in liquid samples tested with fiberglass (FG), polyether sulfone and polyvinylidene fluoride filters. The best results were obtained with FG filters delivering a 20× concentration of B. thuringiensis and B. cereus spores from saline suspensions. The successful 20× pre-concentration of Bacillus spores demonstrated with FG filters could be repeated up to 3 times when bleach decontamination is applied between filtrations. Taken together, our results demonstrate an attractive instrument suitable for semi-automated, quasi-continuous sampling and pre-processing of water samples for biosensing of bacterial spores originating from a complex environment.

Figure 1

Schematic diagram of the water sampling module (WSM-S) used to filter and concentrate spores into a 5 mL backwash solution from a 250 mL water reservoir. The filtration (blue arrows) and backwash (red arrows) runs are indicated by respective flow directions controlled by the peristaltic pump and a set of electromagnetic pinch valves.

Figure 2

Concentration factors of B. thuringiensis kurstaki and B. cereus spores obtained with FG, PES, and PVDF filters for suspensions at initial concentration of 3.14.2 × 10² spores/mL. The data bars represent standard errors of the mean from three separate experiments.

Figure 3

Images of stamp-grown B. cereus spores from backwashed FG (a), PVDF (b) and PES (c) filters used to retain spores from 250 mL suspensions at 1 × 10² spores/mL.

Figure 4

Confocal microscopy images of (a) a fresh FG filter, and (b) a FG filter after 3 filtration runs with backwashing and bleach decontamination. The scale bar corresponds to 10 μm.