Free-living amoebae and Acanthamoebae are known to harbour a range of opportunistic microbial pathogens such as Legionellae, sequestering them from antimicrobial agents as well as environmental stresses. Less is known however of the interaction between the thermotolerant free-living amoebae and Legionellae. In the current study, such phenomena were investigated between an environmental and clinical thermotolerant Acanthamoebae isolate and 6 Legionellae; L. anisa, L. birminghamiensis, L. bozemanii, L. dumoffii, L. erythra and L. pneumophila. All Legionellae could be located within either Acanthamoeba isolate, with L. erythra, and L. pneumophila found located within vacuoles. At concentrations exceeding 2 mg/l, free chlorine was a better disinfectant than combined chlorine against Acanthamoebae-bound Legionellae, though thermal treatment was the most effective of the treatment types investigated. While the interaction with free-living Acanthamoebae increased the resistance of Legionellae to thermal treatment, it increased the sensitivity of Legionellae to free and combined chlorine. Interaction with biofilms did not affect the sensitivity of sessile and intracellular Legionellae to disinfection, caused in part by the thin coverage of biofilm on coupon surfaces. Acanthamoebae cysts remained viable after treatment with 100 mg/l chlorine (free and combined) for 10 min, as well as 80 degrees C, implying that conventional hyper-disinfection may be insufficient for long-term control of Acanthamoebae-bound Legionellae in water distribution systems.
Transmission routes of Helicobacter pylori remain poorly understood. The finding of bacterial DNA in water suggests the involvement of environmental factors. Here we report successful co-cultivation of H. pylori with Acanthamoeba castellanii, which circumvents the requirement of this bacterium for precise microaerobic conditions and a large supply of nutrients in order to grow. H. pylori was able to propagate and remain viable for several weeks in the presence of amoebae under experimental conditions. Intact, metabolically active bacteria could be demonstrated in vacuoles. The putative dependence of H. pylori on free-living amoebae in nature could be important with respect to transmission and prevalence, as shown for some other pathogenic bacteria.
The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.
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