Rapid Enumeration of Active Bacteria in Water After Disinfection 981761

Drinking water aboard spacecraft and on earth must be monitored to ensure that harmful bacteria are absent. NASA needs rapid methods for this purpose, to avoid possible launch delays and limit potential water-related health risks aboard spacecraft on orbit. Determination of bacterial viability after exposure to disinfection has significant health importance since oxidatively injured pathogenic bacteria have been shown to retain their virulence. This problem is compounded by the observation that injured bacteria are recovered at significantly lower frequencies using standard agar plate assays, leading to an underestimation of infection risks. Escherichia coli O157:H7 was exposed to 0.5 ppm free chlorine, retained on membrane filters and tested for physiological activity using a variety of assays. Physiological activity was assessed by detection of membrane potential [rhodamine 123 (Rh123)], substrate responsiveness [direct viable counts (DVC)], respiratory activity [5-cyano-2,3-di-4-tolyltetrazolium chloride (CTC)] and the LIVE/DEAD BacLight Viability kit. Loss of physiological activity was determined by detection of the absence of membrane potential {bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]} and the LIVE/DEAD BacLight Viability kit. Injury and total culturable counts were determined using TLY, TLYD and R2A agars. Total cell counts were determined using DAPI. CTC detected 20.6 to 42.7% more active bacteria than the other physiological assays and 19.0% more than R2A agar prior to disinfection. Following disinfection, CTC detected 16 to 35.4% and 42% more active bacteria than the other assays and R2A agar, respectively. When DiBAC4(3) was used in conjunction with CTC, 102% and 103% of the total cell count was accounted for prior to and following disinfection, respectively. The LIVE/DEAD BacLight Viability kit accounted for 109 and 107% of the total cell count under identical conditions. A method is described where CTC, DiBAC4(3) and a tetramethyl rhodamine isothiocyanate (TRITC) labeled fluorescent antibody (FAb) are used concurrently permitting rapid evaluation of levels of activity (estimates of viability) and total cell counts on a single membrane filter. Immunomagnetic separation (IMS) has been used to enhance sensitivity. Superparamagnetic beads coated with an antibody which reacts with O157 cells were added to water samples, and a magnet was used to separate the beads with bacteria attached to them. Following CTC incubation, filtration and staining with an O157-specific fluorescein isothiocyanate (FITC) labeled antibody, the cells were enumerated by fluorescence microscopy or a rapid solid-phase laser scanner (Scan RDI, Chemunex). Resultant enumeration was closely related to plate counts, and detection of as few as one E. coli O157:H7 cell was possible with the laser scanning method. Methods such as those described are candidates for future spacecraft use by NASA, and will also be useful in the microbiological examination of water and food on earth. A military application is the detection of pathogenic bacteria in combat zones. The prototype CTC/FAb technique for E. coli O157:H7 detection can be applied to the enumeration of any bacterium for which a suitable antibody can be produced. The procedure can be completed with 6-8 hours, and is much more sensitive than existing procedures. Ultimately, techniques like these which do not require cultivation of bacteria are likely to replace existing culture methods because of their rapidity, sensitivity, and specificity.


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