Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing

Abstract

Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed 'viable but nonculturable' (VNC) state. Initial difficulties in obtaining consistent reduction of ρ-iodonitrotetrazolium violet (INT) by H. pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions. The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied. Representation and interpretation of these enhancement profiles were facilitated by digital image processing. Nonculturable cells of H. pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37°C, while 99% of those produced at 4°C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, α-ketoglutarate, or aspartate. Activity was detected at similar levels in cells with coccoid and spiral shapes. In contrast, only 1% of nonculturable cells of V. vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population. Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms. Nonculturable cells of H. pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as 'bags of enzymes' remains to be established.