Salmonella Biofilms and Chlorine Dioxide: Serovar Variation, Mature-Biofilm Tolerance and the Risk of Under-Dosing

Serovar and isolate variation is real, and it is not tidy

Salmonella enterica comprises more than 2,600 serovars, and their biofilm phenotypes differ. The regulator CsgD drives the classic 'rdar' (red, dry and rough) morphotype in which curli fibres and cellulose bind cells into a matrix, but expression of that programme varies by serovar, by isolate within a serovar and by conditions. A 2026 study in Foods (Seres-Steinbach et al., 3 May 2026; doi 10.3390/foods15091574) grew 16 isolates of S. Enteritidis, S. Infantis and S. Typhimurium under four media, three temperatures and three surface types and found that medium and temperature changed biofilm output dramatically, with wide variation among isolates of the same serovar; a low-nutrient medium at low temperature, conditions closer to a chilled processing area than to an incubator, strongly supported biofilm formation.

A 2026 survey in the Journal of Applied Poultry Research (Kuyucuklu-Kazan and Polat; doi 10.1016/j.japr.2026.100760) tested 56 Salmonella isolates of 16 serotypes from commercial layer houses in Türkiye and found that all formed biofilm by 72 hours, with biomass rising significantly from 24 to 48 to 72 hours, but with no significant difference between the serotypes that were represented by five or more isolates. Taken together, the two studies caution against the shorthand that some serovars are 'biofilm formers' and others are not. Isolate, surface, temperature and nutrient state can matter as much as the serovar name, which is why a hygiene programme should be verified against the organisms actually present on a site rather than assumed from typing.

Mature biofilm is a different target from fresh attachment

The poultry-house isolates above illustrate a general rule: Salmonella biofilm biomass increases with age over the first days, and older biofilm is harder to remove and harder to kill through. The matrix thickens, cells deep in the structure slow their metabolism, and the disinfectant is consumed at the outer layers before it reaches the interior. Any efficacy figure is therefore tied to the age of the biofilm it was measured on.

The clearest chlorine dioxide data on mature Salmonella biofilm come from gas-phase work. A 2022 study (Kim et al.; PMC9579236) grew five-day-old biofilms of S. Typhimurium, E. coli O157:H7 and Listeria monocytogenes on stainless steel and high-density polyethylene and exposed them to chlorine dioxide gas at 60 and 90 per cent relative humidity for up to 20 minutes. Reductions rose with concentration, time and humidity; at 90 per cent relative humidity the S. Typhimurium biofilm cells on both materials fell below the detection limit (0.48 log CFU/cm²) within 20 minutes, whereas at 60 per cent humidity the same exposure achieved roughly 2 to 4.6 log reductions. Humidity mattered because chlorine dioxide has to dissolve into a water film to act on the biofilm. The study is about gaseous chlorine dioxide in a chamber, not about a liquid PT4 disinfectant, and it tested one Typhimurium strain; it is cited for what it shows about mature biofilm, contact conditions and material, not as a claim for any product.

The under-dosing problem: stress that stimulates biofilm

Bacteria within biofilms sense sub-lethal stress and respond to it, and one documented response in Salmonella is to make more biofilm. Lories et al. (Current Biology, 2020) showed that S. Typhimurium uses general stress responses, including the oxidative-stress regulon, to detect competitors and respond by increasing biofilm formation. Reviews of food-surface disinfection cite reports of Salmonella strains forming more durable biofilm on polystyrene and stainless steel after sub-lethal chlorine or hydrogen peroxide exposure, and sub-inhibitory concentrations of sodium hypochlorite and other food-grade biocides have been shown to alter Salmonella biofilm formation in laboratory models. The direction of the effect is not universal (some strains and some agents reduce biofilm at sub-lethal levels), but the risk is well enough established to be treated as a design principle: an oxidant exposure too weak to kill can leave a biofilm that is larger and more tolerant than before.

This guide could not verify a published study that measured increased S. Typhimurium biofilm formation after low-level chlorine dioxide exposure specifically, and it does not claim one. Whether chlorine dioxide triggers the same response at sub-lethal levels is untested in the studies reviewed. For a product authorised or supported by applicable transitional evidence for PT3 or PT4, follow its label: the stated concentration for the stated contact time on a cleaned surface. Off-label dilution or extended weak residuals are not supported by the label and, on the chlorine and peroxide evidence, carry a plausible risk of selecting for tolerant, matrix-rich survivors.

What this means for PT3 and PT4 hygiene programmes

The evidence points to sequence and verification rather than to a number. Cleaning first: Salmonella biofilm forms readily in low-nutrient conditions at chilled temperatures, and organic soil both feeds it and consumes oxidant, so detergent cleaning and rinsing precede disinfection. Label dose, label contact time: on the chlorine and peroxide evidence, sub-lethal exposure is the failure mode to design out. Surface and age awareness: plastics and worn or lined surfaces tend to carry more attached Salmonella than sound stainless steel, and the longer the interval between effective treatments, the more mature and tolerant the biofilm. Verification on the site's own isolates: because serovar and isolate variation is large, swabbing and culture (or a validated rapid method) after cleaning and disinfection is the evidence that the programme works for the organisms present.

In Great Britain, disinfectants used in animal housing are PT3 and those used in food and feed areas are PT4 under GB BPR; products for notifiable-disease situations must additionally be Defra-approved at the stated dilution. Dosing, dilution and contact time come from the product label and applicable lawful route. None of the concentrations, humidities or times quoted from the studies above is a dosing instruction for any ChloroKlean product, and none of the studies tested one.

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, lawful GB BPR route 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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