Decontamination of equipment using chemicals

In Summary

• Cleaning and sanitising surfaces and equipment are important in preventing the formation of biofilms. Biofilms are communities of bacteria which encase themselves in a sticky matrix on surfaces, which can make chemical sanitisers less effective.
• If not managed, L. monocytogenes can persist in plants for many years.
• Heat is the preferred method for the removal of L. monocytogenes, compared to chemical treatments. However higher than usual concentrations of chemicals treatments may be considered (after checking the risk to cleaning staff).
• One sanitiser formulation has been reported as effective against L. monocytogenes biofilms, peroxyacetic acid (PAA), although other chemicals may be sufficient.

Cleaning and disinfection

When L. monocytogenes persists in food processing environments, it is often in the form of a biofilm (Tomkin 2002; Klaeboe et al, 2010). The biofilm formation gives a degree of protection to environmental stresses for L. monocytogenes. For example, greater numbers of L. monocytogenes cells survived drying on stainless steel surfaces in biofilms compared with unattached cells (Truelstup et al 2011). Although the majority of L. monocytogenes are attached to the biofilm, the cells in the upper layer can move around and were observed to transfer from the biofilms onto smoked or fresh salmon on contact (Truelstup et al 2011).

Routine cleaning and sanitation can be effective in preventing the establishment of biofilms (Aarnisalo et al 2006). However, there are some studies that suggest that effective cleaning and sanitation may not always occur for all surfaces (Salvat et al 1995). When environmental surface samples were taken from a factory after cleaning and sanitation, but before the commencement of processing, Listeria was isolated from some of the samples. When the study was expanded to determine if the original factory was typical, Listeria was isolated from 15 of the 23 factories specifically sampled (65%). In those processing facilities that were Listeria positive, 7.2% of all the surfaces sampled tested positive (Miettinen et al, 2001).

Heat is the preferred method for the removal of L. monocytogenes when it is isolated from processing equipment and environments. However, some sanitising chemicals used at above the usual concentration (after checking the risk to cleaning staff) also have merit. Pan et al, (2006) report increasing resistance for biofilm L. monocytogenes cells to sanitisers. However, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitising agents. Thus, there may be changes in the composition of polysaccharides and other materials used to construct the biofilm that is the basis of increasing resistance to chemicals by biofilm L. monocytogenes.

One sanitiser formulation that has been reported as effective against L. monocytogenes biofilms is peroxyacetic acid (PAA; an active oxygen-based sanitiser composed of hydrogen peroxide, peracetic acid, and acetic acid in combination; Stopforth et al., 2002). Stopforth created an artificial biofilm onto stainless steel tiles under laboratory conditions. The Stopforth study concluded that PAA, in contrast to a number of other sanitisers that were assessed, was more effective in killing attached cells compared with cells treated in suspension.

Later work by Bagge-Ravn and colleagues (2003) attempted to build on the findings of Stopforth et al. (2002) by determining the effectiveness of PAA in a commercial fish smoking environment. As part of their studies, the Bagge-Ravn team applied a fog of PAA to the slicing area at a salmon smokehouse and compared its effectiveness with that of a foam sanitizer that used sodium hypochlorite as the active agent (the established sanitation process routinely performed at the smokehouse). The effect of each procedure on L. monocytogenes populations was assessed. Environmental samples (n=223) were collected with sponges and swabs after each of the sanitization procedures, and 68 samples were collected post clean during production. Using a selective isolation method, strains of L. monocytogenes were isolated and subsequently genetically characterized by DNA fingerprinting. Following chlorine foam treatment, 14-42% of the samples contained <10 cfu L. monocytogenes per site, whereas 29-78% of the samples collected after fog sanitisation contained the same proportion of undetected L. monocytogenes. Although a higher proportion of samples had lowered numbers of L. monocytogenes for PAA, the overall prevalence of L. monocytogenes was unchanged. For both treatments, L. monocytogenes was found only in poorly cleaned areas such as drains. The authors make specific note that, in keeping with established dogma, effective cleaning is a pre-requisite for effective sanitation (Bagge-Ravn et al, 2003). The L. monocytogenes types from every single positive drain sample were identical to the type that had persisted in the smokehouse over a seven-year period, emphasising the importance of drains as a persistent L. monocytogenes niche. The Bagge-Ravn study is further notable because it demonstrates that the method of sanitiser application can influence the effectiveness of sanitation. The original Stopforth et al (2002) study applied PAA directly to L. monocytogenes-contaminated films and demonstrated effective killing of the bacteria. Although more convenient for commercial premises, when the PAA was applied as a fog by the Bagge-Ravn study, the kill effectiveness was significantly reduced.

Harrand et al. (2020) investigated L. monocytogenes evolution in a cold-smoked salmon processing facility over a 17-year period between 1998 and 2015. The historical isolates over this period were typed by WGS and the results used to classify isolates into one of three clusters. The majority of the interesting findings were from cluster 3. The average mutation rate for cluster 3 was estimated as 1.15 x 10-7 changes per nucleotide per year (roughly a single base change per genome every three years). There were no isolates from before 1998, but backtracking using the WGS-established mutation rate, the most recent common ancestors of two subclusters of cluster 3 (3a and 3b) were estimated to have occurred around 1958 and 1974, respectively. The timeframe was after the establishment of the facility. L. monocytogenes is well known for persistence lasting years or exceptionally decades. The estimation suggests exceptionally long-term persistence in the fish plant of around 70 years. There was evidence of multiple phage infections within cluster 3. In total there were seventeen different prophages (phage DNA integrated into a host bacterial genome) identified, with a single prophage widely dispersed amongst many of the cluster 3 isolates. There was plasmid-based resistance to quaternary ammonium (QA) with a transposon-borne sanitizer tolerance gene qacH in one cluster 1 isolate and a plasmid-borne sanitizer tolerance transporter bcrABC found in all cluster 2 and 3 isolates. The presence of these resistance genes was correlated with the ability to survive increased concentrations of sanitisers. Selected isolates showed significant variation in the ability to attach to surfaces, with persistent isolates attaching better than transient isolates at 21oC.

A summary of the published literature regarding the effective removal of L. monocytogenes by commonly encountered sanitising agents is provided as Table 1. It is important to note the effect of ineffective cleaning prior to the application of a sanitiser on the number of L. monocytogenes cells killed.

Table 1 The efficacy of sanitisers in removing L. monocytogenes contamination from poorly and properly cleaned surfaces or suspensions (reproduced from Hoelzer et al 2012)