VBNC hypothesis- the foundation on which the 'autochthonous V. cholerae theory' is built

Traditionally, the viability of bacteria is related to its ability to form colonies on agar plates. When they lose the ability to form colonies, bacteria are considered dead. However, in 1982, the laboratory of Dr. Rita Colwell reported that bacteria grown in salt water microcosms for two weeks remained viable even though they lost the ability to form colonies. This state is known as viable but non-culturable (VBNC) state. Later it was reported that a number of bacterial organisms enter VBNC state when grown under different stress conditions like lack of nutrients, low temperature, presence of stressors etc. Even though bacteria under VBNC state lose their ability to form colonies, the removal of stress and/or the addition of nutrients can result in the resuscitation of their culturability. Thus, V. cholerae that enter the VBNC state at low temperatures (below 10°C) can be resuscitated to normal growth by a simple upshift of temperature to 25°C.

           

In cholera endemic areas like Bangladesh, V. cholerae could be isolated easily from aquatic environment during summer, but not during winter. The difficulty in isolating V. cholerae during winter was explained on the basis of the ability of the organism to enter VBNC state. Thus, during winter, when the temperature is below 10°C, bacteria enter VBNC state and become non-culturable. However, during summer, they resuscitate and regain their culturability. Moreover, during summer they multiply rapidly due to ‘phytoplankton bloom’ which provides nutrients for growth of the organism.

           

However, the resuscitation of VBNC and its significance has been questioned by many researchers (this aspect has been reviewed in detail in my book). Whereas a number of researchers failed to observe the resuscitation of VBNC both in vitro and in vivo, others could notice resuscitation only in vitro and not in vivo, thus questioning the significance of VBNC in causing an outbreak of a disease. According to many researchers, the growth recovery noticed in a number of experiments was not due to true resuscitation of VBNC but only due to regrowth of a few culturable bacteria. Other researchers consider VBNCs are sublethally injured cells or cells nearing the death that do not have the capability to resuscitate even though they may remain viable for some time. Moreover, in a river microbial community, bacteria may be ingested by predators even before entry to VBNC state, thus reducing the importance of VBNC in natural aquatic systems.

           

Scientists continue to report the entry of different bacteria to VBNC state without considering the above criticisms (they repeat the same in vitro experiments to prove the resuscitation of VBNCs i.e. incubating a bacterial culture in water or sea water microcosms at low temperature and then ‘resuscitate’ them at high temperatures soon after the entry of organism to VBNC state). Our knowledge on VBNC state has not grown much since it was first reported in 1982. Thus, even after 30 years of research on VBNCs, it has not been conclusively proved that the resuscitation of V. cholerae VBNCs is responsible for seasonal cholera outbreak in endemic areas. How much more time and money need to be invested to prove a hypothesis?

Next- The fantasy theory on the ecology of V. cholerae