Aims:  To assess chlorine susceptibility of Legionella pneumophila grown from two amoebic hosts, Acanthamoeba castellanii and Hartmannella vermiformis. Methods and Results:  After being released from amoebae, Leg. pneumophila were chlorinated at 2 and 5 mg l−1 for 5 min–24 h. Bacterial culturability and cytoplasmic membrane deterioration were quantified by culture assay on BCYEα agar and BacLight stains coupled with a fluorescent microscope, respectively. Chlorination reduced the culturability of Leg. pneumophila by 2·93–4·59 log CFU ml−1 and damaged cellular membrane by 53·8–99·2%. Moreover, cells released from H. vermiformis exhibited significantly lower degrees in culturability reduction (P = 0·0008) and membrane deterioration (P < 0·0001) when compared with those from A. castellanii. The amoebic genus is the most significant parameter affecting cytoplasmic membrane integrity of chlorinated Legionella (P < 0·0001), followed by free chlorine concentration (P = 0·042). Conclusions:  Legionella pneumophila replicated from H. vermiformis possess greater chlorine resistance than the cells from A. castellanii. Significance and Impact of the Study:  This study shows the heterogeneity of amoebae‐grown Leg. pneumophila in chlorine susceptibility, which should be considered in the control of legionellae proliferation, particularly in the systems where H. vermiformis is dominant, e.g. hot water plumbing.

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