Detection of induced β‐galactosidase activity in individual non‐culturable cells of pathogenic bacteria by quantitative cytological assay

Abstract

One Escherichia coli and two F' lac+ Salmonella strains were carbon and nitrogen stressed at 37 degrees C over 35 days in the presence or absence of chloramphenicol; the number, activity and culturability of cells in the resultant populations were studied. Active cells were enumerated by fluorescence microscopy after treatment with the lac inducer IPTG and cytological assay for beta-galactosidase. In all experiments, active and total cell counts remained within a three-fold range of each other and their initial values, while culturability fell by > 10(8)-fold and 10(3)-fold in chloramphenicol-treated and untreated preparations, respectively. Quantitative image analysis revealed different distributions of cell-specific fluorescence and indicated a progressive decline in the levels of induced enzyme activity in both E. coli and Salmonella enteritidis. It was concluded that the non-culturable cells studied retained inducible enzyme activity and that this activity did not result from a starvation-induced programme of gene expression. Whether or not such active but non-culturable cells are viable, they are clearly responsive and have the potential to influence their environment. The assay described can be applied to heterogeneous populations and environments and shows considerable potential for the study of gene expression at the single cell level.

Monitoring of active but non-culturable bacterial cells by flow cytometry

“…One of the survival strategies adopted by non-sporulating microorganisms when exposed to environmental stress, is the formation of the active but non-culturable (ABNC) state (Barer et al, 1993;Rozak and Colwell, 1987;McDougald et al, 1998), where the cell remains capable of undergoing metabolic activity but cannot be detected by culturable methods. This phenomenon was largely demonstrated based on the discrepancies observed between culturing methods and non-culture-based staining techniques (Baudart et al, 2002;McNamara et al, 2002;Rice et al, 2000), for example using E. coli strains (Davies et al, 1995;Grey and Steck, 2001;Nwoguh et al, 1995;Ohtomo and Saito, 2001;Pommepuy et al, 1996a;Roth et al, 1988). In general, it is difficult to establish the distribution of ABNC among the mixed population of live and dead cells and cells in the transition phase, as there is a difference between the culture-based measurements and non-culture-dependent techniques.…”

Fed‐batch production of recombinant β‐galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations

“…The images in the field of view were digitized at 8-bit resolution (0-255 gray scale) and recorded by using a cooled integrating charge-coupled device camera (Coolview, Photonic Science, East Sussex, UK). The signal intensity was quantified by constructing a binary image based on the phase-contrast image, laying the binary image over the corresponding fluorescence image and determining the integrated gray weight for each cells as described elsewhere (Nwoguh et al, 1995;Whiteley et al, 1998).…”

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

“…However, the reduction in enzyme activity was still less than the reduction in cfu, and an apparent increase in b-D-galactosidase activity calculated per cultivable FC cell was observed for increasing chlorine dose. The observed increase may be due to increased proportions of non-cultivable target bacteria with assayable activity (Davies et al 1995b ;Nwoguh et al 1995) and presence of b-D-galactosidase-positive, nontarget micro-organisms (Davies et al 1995a ;Fiksdal et al 1997 ;Van Poucke and Nelis 1997) that are more resistant to chlorine than FC bacteria, e.g. Bacillus (Ridgway and Olson 1982).…”