Impact of Acanthamoeba Cysts on Stress Resistance of Salmonella enterica Serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a, and Escherichia coli O:26

Abstract

The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H 2 O 2 , benzalkonium chloride, 55°C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i) Acanthamoeba castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34-to 4.72-log 10 cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H 2 O 2 (Ͼ92% decrease in median log 10 CFU/ml reduction) and 70% ethanol (Ͼ99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of Yersinia enterocolitica (74% decrease in median log 10 reduction), Escherichia coli (58%), and Salmonella enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%), and Listeria monocytogenes (99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria. IMPORTANCEThe results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens.KEYWORDS Acanthamoeba, bacteria, cyst, resistance, stress, survival F ree-living amoebae are ubiquitous in soil, air, and water and are part of the in-house microbiota of food-related environments (1). Besides being bacterial predators, free-living amoebae, such as Acanthamoeba, can also host grazing-resistant bacteria and are therefore regarded as a potential reservoir, vector, shelter, and virulence training ground for pathogenic bacteria. It is hypothesized that these free-living

Trapping the Enemy: Vermamoeba vermiformis Circumvents Faustovirus Mariensis Dissemination by Enclosing Viral Progeny inside Cysts

“…Faustovirus infected cysts fail excystment. Studies demonstrated that several intracellular bacteria are able to survive and take advantage of amoebal encystment (11,17). It has been suggested this feature would guarantee protection of bacteria during adverse environmental conditions while inside the cyst.…”

“…We have demonstrated that, once infected by mimivirus, Acanthamoeba trophozoites are no longer able to encyst because mimivirus blocks the expression of a serine proteinase gene, a canonical element involved in the encystment process (10). Although it has been described that Acanthamoeba trophozoites are able to encyst in the presence of some intracellular bacteria (11), this phenomenon has never been described during amoebal virus infections.…”

SARS-CoV-2 surrogate (Phi6) environmental persistence within free-living amoebae

“…Since amoebic cysts are highly resistant to various harsh environmental stressors, thereby protecting their intracellular microbes and readily aerosolized to enhance dispersal from engineered water systems (Aksozek et al 2002;Lambrecht et al 2017;Borges et al 2019), we also investigated the stability of Phi6 phage inside V. vermiformis cysts. TEM analysis showed that at 2 months after the infection vacuoles still contained infectious viruses in V. vermiformis cysts (Figure 3(a) and 3(b)).…”

Listeria monocytogenes in food businesses: From persistence strategies to intervention/prevention strategies—A review

“…Various pathogens like Salmonella spp., L. monocytogenes (Fieseler et al, 2014;Lambrecht et al, 2017;Minh et al, 1990aMinh et al, , 1990bZhou et al, 2007), Legionella spp., Yersinia enterocolitica (Lambrecht et al, 2013), pathogenic E. coli, and Campylobacter jejuni can be protected and/or obtain an increased tolerance by protozoa like Acanthamoeba castellanii, Acanthamoeba polyphaga, and/or Tetrahymena pyriformis toward various treatments like hydrogen peroxide, benzalkonium chloride, sodium hypochlorite, and NaCl (Lambrecht et al, 2015). More pathogen-protozoa relationships are given in Amaro and Martín-González (2021).…”