Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants

International Journal of Food Microbiology

et al. 2014

In the present study, the occurrence of free-living protozoa (FLP) and foodborne bacterial pathogens on dishcloths was investigated. Dishcloths form a potentially important source of cross-contamination with FLP and foodborne pathogens in food-related environments. First various protocols for recovering and quantifying FLP from dishcloths were assessed. The stomacher technique is recommended to recover flagellates and amoebae from dishcloths. Ciliates, however, were more efficiently recovered using centrifugation. For enumeration of freeliving protozoa on dishcloths, the Most Probable Number method is a convenient method. Enrichment was used to assess FLP diversity on dishcloths (n = 38). FLP were found on 89% of the examined dishcloths; 100% of these tested positive for amoebae, 71% for flagellates and 47% for ciliates. Diversity was dominated by amoebae: vahlkampfiids, vannellids, Acanthamoeba spp., Hyperamoeba sp. and Vermamoeba vermiformis were most common. The ciliate genus Colpoda was especially abundant on dishcloths while heterotrophic nanoflagellates mainly belonged to the genus Bodo, the glissomonads and cercomonads. The total number of FLP in used dishcloths ranged from 10 to 10 4 MPN/cm 2 . Flagellates were the most abundant group, and ciliates the least abundant. Detergent use was identified as a prime determinant of FLP concentrations on used dishcloths. Bacterial load on dishcloths was high, with a mean total of aerobic bacteria of 7.47 log 10 cfu/cm 2 . Escherichia coli was detected in 68% (26/38) of the used dishcloths, with concentrations up to 4 log 10 cfu/cm 2 . Foodborne pathogens including Staphylococcus aureus (19/38), Arcobacter butzleri (5/38) and Salmonella enterica subsp. enterica ser. Halle (1/38) were also present. This study showed for the first time that FLP, including some opportunistic pathogens, are a common and diverse group on dishcloths. Moreover, important foodborne pathogens are also regularly recovered. This simultaneous occurrence makes dishcloths a potential risk factor for cross-contamination and a microbial niche for bacteria-FLP interactions.

Section: Discussionmentioning

confidence: 99%

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: Implications for food safety

Chavatte

International Journal of Food Microbiology

et al. 2014

“…The morphology and exact composition of the cyst wall can vary between species and strains and, under in vitro conditions, also depends on the encystation medium used (2). Amoebal cysts are resistant to adverse physical and chemical conditions, such as desiccation (6), freezing-thawing cycles, radiation (7), heat (10 min at 80°C, 30 min at 70°C, or 60 min at 60°C [8]), and various biocides (9)(10)(11). It has been shown that mature Acanthamoeba cysts are more resistant than trophozoites to disinfectants and biocides (10,12).…”

mentioning

confidence: 99%

“…Several studies investigated the survival of protozoan trophozoites and cysts after exposure to chemical compounds, such as biocides used in water treatment systems and lens disinfectant solutions (10,(14)(15)(16)(17). Although these studies are relevant for the prevention and treatment of infections by pathogenic or opportunistic free-living protozoa, they do not take into account the fate of the internalized (pathogenic) bacteria.…”

mentioning

confidence: 99%

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

Sabbe

et al. 2017

Appl Environ Microbiol

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

“…Cysts usually possess a thick, often double or multilayered protective wall, consisting of lipids, (glyco)proteins, and carbohydrates like chitin and cellulose. This protects the protozoon against unfavorable environmental conditions, such as freezing (19), gamma and UV radiation (20), and chemicals used for disinfection in health care settings (21) and drinking water production (22,23). Some cysts can withstand desiccation for more than 20 years (24).…”

mentioning

confidence: 99%

“…Cysts may thus enable internalized foodborne pathogens to survive physical and chemical cleaning and disinfection methods (21,23).…”

mentioning

confidence: 99%

Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria

Chavatte

et al. 2015

Appl Environ Microbiol

The production of cysts, an integral part of the life cycle of many free-living protozoa, allows these organisms to survive adverse environmental conditions. Given the prevalence of free-living protozoa in food-related environments, it is hypothesized that these organisms play an important yet currently underinvestigated role in the epidemiology of foodborne pathogenic bacteria. Intracystic bacterial survival is highly relevant, as this would allow bacteria to survive the stringent cleaning and disinfection measures applied in food-related environments. The present study shows that strains of widespread and important foodborne bacteria (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, and Listeria monocytogenes) survive inside cysts of the ubiquitous amoeba Acanthamoeba castellanii, even when exposed to either antibiotic treatment (100 g/ml gentamicin) or highly acidic conditions (pH 0.2) and resume active growth in broth media following excystment. Strain-and species-specific differences in survival periods were observed, with Salmonella enterica surviving up to 3 weeks inside amoebal cysts. Up to 53% of the cysts were infected with pathogenic bacteria, which were located in the cyst cytosol. Our study suggests that the role of free-living protozoa and especially their cysts in the persistence and epidemiology of foodborne bacterial pathogens in food-related environments may be much more important than hitherto assumed. Foodborne pathogenic bacteria are a major cause of foodborne illness and have important implications for human public health along with economic consequences (1). Despite thorough disinfection protocols and hygiene monitoring during food production and processing, pathogenic bacteria often persist in foodrelated environments and on food, suggesting that our knowledge about the transmission routes and epidemiology of foodborne pathogenic bacteria is still incomplete.Recent studies have shown that bacteria can benefit from intracellular associations with free-living protozoa (FLP) (2, 3), heterotrophic eukaryotic microorganisms that are common in natural aquatic and terrestrial ecosystems (4). Although FLP feed on bacteria, some bacteria resist digestion. These so-called "digestion-resistant bacteria" can survive and even grow inside their FLP hosts (5). These hosts thus effectively act as a reservoir, shelter, and vector for the bacteria and can as such play an important role in their ecology (3, 6). Intracellular association with FLP has also been demonstrated for human-pathogenic bacteria (7, 8), including food-related pathogens (e.g., see references 5, 9, and 10). As FLP have been isolated from diverse food-related habitats, such as broiler houses (11-13), meat cutting plants (14), domestic refrigerators (15), and vegetables (16,17), this suggests that FLP may be implicated in the epidemiology of foodborne pathogens.Many FLP have two life cycle stages: the trophozoite and the dormant cyst. The former is the actively feeding stage, preying on bacteria, algae, viruses, yeast, and ...