Viable but Non-Culturable (VBNC) Bacteria After Disinfection

What the VBNC state is

Bacteria exposed to sub-lethal stress, including oxidising disinfectants, cold, starvation and salinity, can enter a dormant state in which they stop dividing on laboratory media but keep an intact membrane and some metabolic activity. The state was first described in 1982 and has since been reported for well over a hundred species. VBNC cells are, by definition, invisible to the plate-count methods that most routine compliance monitoring relies on, and some can resuscitate when conditions improve.

This matters for how results are read. Culture answers the question 'did anything grow?'. It does not answer 'is anything alive?'. Methods that assess membrane integrity or metabolic activity, such as flow cytometry with viability stains, ATP measurement or viability-PCR, are needed to see the difference.

The 2026 Enterococcus faecalis drinking-water study

Zhang and colleagues, publishing in the Journal of Hazardous Materials (Volume 512, 1 July 2026; doi 10.1016/j.jhazmat.2026.142416), collected water from four stages of a working drinking-water treatment train (source water, pre-filtered water, filtered water and pipe-network water) and exposed Enterococcus faecalis in those waters to chlorine dioxide at 0.6 to 2.0 mg/L. Flow cytometry showed that the cells entered a VBNC state in all four water types. The VBNC cells kept their membranes intact but changed their metabolism, with raised ATP and reduced esterase activity and respiration.

Crucially, the non-culturable cells were not harmless. They still adhered to human intestinal (Caco-2) and blood-brain-barrier (HBMEC) cells at 49 to 69% of the untreated control, triggered release of inflammatory cytokines, and survived and recovered inside macrophages and in the presence of sheep red blood cells, regrowing to 7 to 8 log CFU/mL. Gene-expression analysis linked VBNC entry to shut-down of energy metabolism and linked resuscitation to reactivation of pyruvate metabolism, sugar transport and ribosomes. Water chemistry, notably calcium deficiency, influenced both entry into the state and recovery from it.

Loss of culturability is not confirmed elimination

The practical distinction is between three outcomes that a plate count cannot separate: cells that are dead, cells that are alive but temporarily non-culturable, and cells that were never captured by the sample. Only the first is elimination. A disinfection programme that reports zero colonies has demonstrated the first outcome only if the method used can also exclude the other two, which routine culture cannot.

This is a property of disinfection in general, not of chlorine dioxide specifically. The study's own introduction cites VBNC induction by chlorine, acids, salinity, heat and antibiotics. Chlorine dioxide was the stressor tested here because it is a common drinking-water disinfectant, and the concentrations used bracket those found in practice. The right response is not to abandon a disinfectant but to interpret its monitoring data with the VBNC possibility in mind, particularly after shock treatments or where results swing from positive to negative and back.

The PT5 drinking-water dimension

Under the Biocidal Products Regulation, disinfection of drinking water for humans or animals falls under product-type 5 (PT5). UK public-supply compliance monitoring for microbiological quality relies largely on culture of indicator organisms such as Escherichia coli and enterococci. That framework is appropriate for its regulatory purpose; it is not a viability census. The study's finding that E. faecalis, itself an enterococcal indicator, can become non-culturable under chlorine dioxide stress while retaining infectivity is therefore directly relevant to how PT5 verification data should be read, although it does not change what the regulations require.

For anyone operating or advising on a PT5 application, the implication is procedural: pair culture results with residual records, flow and temperature data, and, where risk justifies it, culture-independent methods; treat a sudden fall to zero after a dose change as a prompt to confirm rather than a conclusion.

What this study does and does not establish

It establishes that chlorine dioxide at 0.6 to 2.0 mg/L induced a VBNC state in a laboratory strain of E. faecalis suspended in real treatment-train waters, that those cells retained pathogenic traits in cell-culture models, and that some resuscitated. It supports the general principle that culture negativity after disinfection is not proof of elimination.

It does not establish that VBNC E. faecalis occurs at meaningful levels in any supplied water; the organisms were added to the water samples, not detected in them. Infectivity was shown in cell lines and blood cells, not in people or animals. The water came from one treatment works, and no ChloroKlean product was involved. Nor does it show that chlorine dioxide performs worse than other disinfectants in this respect, because no comparison was run.

Evidence and uncertainty

Published biofilm studies are valuable for understanding mechanisms, but their conditions may not match a particular installation. Species, surfaces, deposits and operating conditions should be recorded when interpreting evidence.

For safety-critical systems, decisions should be documented through the relevant risk assessment and management plan.

Choosing the next question

A useful next step is to identify what is known, what is inferred and what needs verification. This avoids treating a general reference as a site diagnosis.

Where a product is considered, confirm the intended use, authorisation and label directions independently of this educational guide.

Use this sequence to frame investigation and control; it is not a dosing protocol.

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