Imagine that you want to test the efficiency of an antimicrobial treatment in inhibiting a certain bacterial pathogen. As part of the experiment, you expose the bacteria to the treatment and monitor the cultivability of the microorganism by counting the number of colony forming units (CFU) formed on culture media. If the microorganism is sensitive to the treatment, CFU numbers will decrease steadily over time until eventually no CFUs are observed. You rejoice because it appears that the bacteria are inhibited, thus confirming the hypothesis that the treatment is effective. But is it too early for celebrations?
It has been shown that under a number of adverse environmental conditions such as UV radiation, nutrient starvation, and antibiotic treatment, several species of bacteria can enter the viable but non-culturable (VBNC) state. VBNC bacteria, first recognized by environmental microbiologist Rita Colwell and her collaborators, have very low metabolic activity and don’t grow on nutrient culture media. Transitioning into this dormant state is a strategy for cells to survive lethal stresses, until they are resuscitated when conditions become more favourable. VBNC bacteria are zombies of the bacterial world!
Why Should We Be Concerned About These Bacterial Zombies?
VBNC bacteria are usually more resistant to antibiotics and other environmental stresses when compared to culturable bacteria, allowing them to persist and be reactivated later. As VBNC bacteria fail to grow on nutrient culture media, their survival rates are often underestimated by culture-based methods. Pathogenic bacteria in the VBNC state pose a threat to public health and food safety as they may escape routine microbiological detection. Some pathogenic bacteria are capable of initiating infections in the VBNC state while others are not virulent but resume their pathogenicity when revived. Indicator bacteria such as fecal indicator bacteria (FIB) may also not be quantified accurately by culture-based methods when they enter the VBNC state in response to stresses in the environment. This affects the accuracy of environmental monitoring processes such as the detection of fecal contamination by routine microbiological methods.
How Can We Detect the Presence of VBNC Bacteria?
- By quantitative real-time PCR: Molecular methods such as PCR and quantitative real-time PCR, which measure changes in RNA or DNA, or metabolic assays/stains are most commonly used to enumerate VBNC bacteria due to their availability and ease of usage. Instead of DNA, mRNA is a preferred marker for viability because it is synthesized during cellular processes and has a short half-life. mRNA is extracted from bacteria and transcribed into cDNA by reverse transcription. Subsequently, qPCR is performed to quantify the expression of essential housekeeping genes such as 16S rDNA and rpoS which codes for the Sigma S transcription factor. If the bacteria are in the VBNC state, these genes will be expressed at similar levels as in viable bacteria, even if no visible growth is observed when the bacteria are inoculated in culture media. The number of viable bacteria in the sample is quantified using a standard curve which correlates gene expression to bacteria numbers. When bacterial viability is compromised, these genes will be expressed at significantly lower levels. A sufficient amount of mRNA needs to be extracted at the start to get a good qPCR readout. For samples with very low bacteria numbers (e.g. environmental samples), sufficient mRNA can be obtained by filtering and concentrating the bacteria before extraction.
- By assessing metabolic activity: Assays/stains which distinguish between viable and dead bacteria do so by measuring metabolic activity such as respiration or cellular characteristics like membrane integrity. An example of a widely used stain which discriminates viable and dead bacteria is the Live/Dead BacLight Bacterial Viability Kit (Molecular Probes). Green-fluorescent SYTO® 9 and red-fluorescent propidium iodide are used to stain nucleic acids in bacterial cells. SYTO® 9 penetrates both live and dead bacteria while propidium iodide enters only dead bacteria with damaged membranes. When both stains are used, the SYTO® 9 fluorescence in dead bacteria is reduced by propidium iodide. Hence, bacteria with intact membranes fluoresce green while dead bacteria with damaged membranes fluoresce red. Viable and dead bacteria can then be directly enumerated by fluorescent microscopy, quantitative assays using a fluorescence microplate reader or flow cytometry. VBNC bacteria which are still alive will fluoresce green. Some important factors to take note of which may affect the results of the stain are the differential binding of SYTO® 9 to live and dead cells, bleaching of SYTO® 9 and background fluorescence. Nonetheless, with the right controls and precautions, SYTO® 9 and propidium iodide staining is still a useful technique for quantifying VBNC bacteria.
So the next time you notice that your bacterial culture isn’t growing, don’t assume that it is dead just yet!
Coutard F, Pommepuy M, Loaec S and Hervio-Heath H. mRNA detection by reverse transcription-PCR for monitoring viability and potential virulence in a pathogenic strain of Vibrio parahaemolyticus in viable but nonculturable state. J Appl Microbiol (2005). 98(4): 951-961.
Kumar A, Ng D and Cao B. Fate of Enterococcus faecalis in stormwater matrices under ultraviolet-A (365 nm) irradiation. Environmental Science: Water Research & Technology (2018). 4: 639-643.
Oliver JD. The Viable but Nonculturable State for Bacteria: Status Update. Microbe (2016). 11(4): 159-164.
Stiefel P, Schmidt-Emrich S, Maniura-Weber K and Ren Q. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol (2015). 15:36.
Ramamurthy T, Ghosh A, Pazhani GP and Shinoda S. Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria. Front Public Health (2014). doi: 10.3389/fpubh.2014.00103.