In Summary
- L. monocytogenes can exist in two forms; as free-swimming cells, and as stationary cells in a biofilm. Studies suggest that biofilms are a route for L. monocytogenes to become persistent in a plant.
- The free-swimming cells are more susceptible to cleaning and sanitisation, and can therefore be controlled more easily. L. monocytogenes in a biofilm require 10-20 times more chemical to achieve the same level of control, with strategies such as prolonged scrubbing needed to remove biofilm.
- As biofilms protect bacteria, like L. monocytogenes, chemical sanitisers may not reach the recommended concentration in a biofilm. These sub-lethal conditions may promote L. monocytogenes becoming resistant against the chemical.
- Biofilms are complex, normally involving many species of bacteria, sometimes the other bacteria inhibit growth of L. monocytogenes, however these will vary on a case-by-case basis.
L. monocytogenes is known to exist in two forms: as planktonic cells (i.e. as free-swimming, mobile single cells) or as a sessile biofilm (i.e. as part of a group of cells, frequently mixed species, living in close proximity and enmeshed within a web of protective polymers). Planktonic forms of bacteria are susceptible to most mainstream cleaning and sanitization agents and so can be effectively controlled (Chapman, 2003). However, as a biofilm, L. monocytogenes can be resistant to the action of cleaners and sanitisers (Charalambia-Eirini et al, 2011). Compared with planktonic forms, biofilm L. monocytogenes required 10-20 times more chemical to achieve the same degree of kill (Robbins et al, 2005). Different cleaning and sanitation strategies such as prolonged scrubbing are therefore required for the removal of L. monocytogenes biofilms.
When growing as a biofilm, L. monocytogenes is exposed to a range of sanitising chemicals in food processing areas. Biofilms may provide a mechanism whereby the chemical exposure is at concentrations below those recommended by manufacturers (Allen et al. 2016). Such sublethal exposures may last for long periods, are frequently repeated, and are potentially a route that may promote increased resistance over time to quaternary ammonium and phenolic based sanitisers. There is some evidence that sometimes (but not always) biofilm growth may result in plant residency and persistence. In general, sanitiser resistance does not correlate with the ability of L. monocytogenes strains to persist in a processing plant environment (Ferreira et al. 2011). Carpentier and Cerf (2011) asserted that L. monocytogenes persistence is primarily a random accident because there have been no loci identified within L. monocytogenes that have an association with plant residency and persistence, but niches exist in food industry premises and equipment where L. monocytogenes do persist for extended periods of up to several years (Wang et al. 2015). Having comprehensively reviewed a large body of pertinent literature, Martínez-Suárez et al. (2016) concluded that commonly local environmental conditions in plants that create persistent niches fortuitously by blocking full exposure to disinfectants. Biofilm growth may be one of those fortuitous blocking mechanisms. Repeated imperfect cleaning, which leaves organic residues, can also interfere with effective sanitation (Martínez-Suárez et al. 2016).
Heir et al. (2018) assessed the ability of L. monocytogenes to compete for their niche in biofilms individually, in combination with other strains of L. monocytogenes, with other Listeria species, and with mixtures of Gram-positive and Gram-negative cells. The reason for the Hier et al. (2018) study was a belief that biofilms are the primary route to plant residency, which, given the discussions above; might be a little off target. The strains used for the study were isolated originally from processing surfaces in fish and meat processing plants. L. monocytogenes was able to form biofilms in all of the strain mixtures assessed. Biofilm growth was restricted in strain mixtures that contained more than just L. monocytogenes. Pseudomonas and L. innocua most effectively competed with L. monocytogenes and sometimes inhibited the growth of L. monocytogenes. Overall, L. monocytogenes inhibition that was further enhanced by the presence of a background Gram-negative microbiota. In keeping, with the pre-amble to the review of this paper, and taking into account the original sources of strains used for the work, the Heir et al. (2018) study indicated there were variations among L. monocytogenes strains in their competitiveness under multi-bacterial culture conditions.
The abilities of six strains of L. monocytogenes to form biofilms on stainless steel under simulated fish processing conditions was assessed by (Papaioannou et al. 2018). The biocide tolerance of the sessile cells was also assessed. L. monocytogenes biofilms were created by incubating stainless steel coupons at 15oC for up to 10 days in a liquor prepared by extracting sea bream flesh with salt water. The L. monocytogenes strains were assessed in combination with four Pseudomonads, or in combination with a natural fish microbiota. The Pseudomonas dominated all of the biofilms throughout the entire incubation period. L. monocytogenes was able to co-exist in a sessile community with the Pseudomonas, albeit with slight suppression compared with a pure culture, single strain, L. monocytogenes biofilm. L. monocytogenes survived but was even more suppressed by the indigenous fish microbiota in a mixed species biofilm. The sessile colonies were exposed to two commercial sanitisers - Hypofoam VF6 (chlorinated alkali), and Divosan SU 319/VT8w (quaternary ammonium), at manufacturer recommended concentrations and conditions. Neither of the sanitisers were completely effective against single strain, sessile L. monocytogenes. There was resistance to the sanitisers observed from day 3 through day 7. In contrast, L. monocytogenes grown in mixed culture biofilms containing indigenous fish microflora was destroyed by either of the sanitisers. The authors speculated that the low numbers of L. monocytogenes in the mixed biofilms might be the reason for the entire destruction. The conclusions from the paper were that L. monocytogenes can form biofilms more effectively in monoculture, struggled to form biofilms when forced to compete with indigenous fish microbiota and was susceptible to the commercial sanitisers tested only when grown in mixed culture. The final conclusion was “The seafood industry should consider [these findings] when designing and applying effective anti-biofilm strategies” (Papaioannou et al. 2018), although no guidance on how to approach such considerations was provided by the authors.
Brauge et al. (2020) also investigated the efficiency of commercially available sanitisers on biofilms containing single strains or cocktails of L. monocytogenes. In summary, there were two sanitisers evaluated, based on hydrogen peroxide or quaternary ammonium. Neither was able to completely destroy L. monocytogenes, growing as biofilms. There was some conversion of some cells to a VBNC (viable but non culturable) state. An overall message of the paper was sanitised surfaces can test negative for L. monocytogenes using classic microbiological testing, but VBNC cells might be present and if such cells are ingested, they may be resurrected and potentially cause illness. The work used robust laboratory testing for the differentiation of VC and VBNC cells and included sampling undertaken in four commercial processing labs to confirm that the lab-based studies accurately reflected the situation under commercial operating conditions.
