The Efficacy of Heat and Chlorine Treatment against Thermotolerant Acanthamoebae and Legionellae

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°C, implying that conventional hyper-disinfection may be insufficient for long-term control of Acanthamoebae-bound Legionellae in water distribution systems.

[1]  M. R. Brown,et al.  Unexplored reservoirs of pathogenic bacteria: protozoa and biofilms. , 1999, Trends in microbiology.

[2]  S. Falkow,et al.  Growth of Legionella pneumophila in Acanthamoeba castellanii enhances invasion , 1994, Infection and immunity.

[3]  M V Storey,et al.  Biofilms, thermophilic amoebae and Legionella pneumophila--a quantitative risk assessment for distributed water. , 2004, Water science and technology : a journal of the International Association on Water Pollution Research.

[4]  H. Scaife,et al.  Intraphagocytic growth induces an antibiotic-resistant phenotype of Legionella pneumophila , 1995, Antimicrobial agents and chemotherapy.

[5]  M. R. Brown,et al.  Relationship between Legionella pneumophila and Acanthamoeba polyphaga: Physiological Status and Susceptibility to Chemical Inactivation , 1992, Applied and environmental microbiology.

[6]  S. Kilvington,et al.  Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. , 1990, The Journal of applied bacteriology.

[7]  John N. Dowling,et al.  Impact of Chlorine and Heat on the Survival of Hartmannella vermiformis and Subsequent Growth of Legionella pneumophila , 1993, Applied and environmental microbiology.

[8]  M. Heinemann,et al.  Infection of Acanthamoeba castellanii by Chlamydia pneumoniae , 1997, Applied and environmental microbiology.

[9]  C. Ruef.  Nosocomial Legionnaires' disease — strategies for prevention , 1998 .

[10]  R. J. Gibson,et al.  Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila , 1979, Journal of clinical microbiology.

[11]  Michael Steinert,et al.  Legionella pneumophila: an aquatic microbe goes astray. , 2002, FEMS microbiology reviews.

[12]  H. Bergmann,et al.  [Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, after their isolation from a microbiologically contaminated drinking water system in a hospital]. , 1995, Zentralblatt fur Hygiene und Umweltmedizin = International journal of hygiene and environmental medicine.

[13]  T. Fritsche,et al.  Occurrence of bacterial endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses , 1993, Journal of clinical microbiology.

[14]  S A Mueller,et al.  Literature review--efficacy of various disinfectants against Legionella in water systems. , 2002, Water research.

[15]  K. Schleifer,et al.  In Situ Detection of Novel Bacterial Endosymbionts of Acanthamoeba spp. Phylogenetically Related to Members of the Order Rickettsiales , 1999, Applied and Environmental Microbiology.

[16]  S Falkow,et al.  Interaction of Mycobacterium avium with environmental amoebae enhances virulence , 1997, Infection and immunity.

[17]  T. E. Cloete,et al.  Biofouling and Biocorrosion in Industrial Water Systems , 2005, Critical reviews in microbiology.

[18]  D V Seal,et al.  Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization. , 1995, The Journal of applied bacteriology.

[19]  P. Edelstein.  Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens , 1981, Journal of clinical microbiology.

[20]  R. Atlas.  Legionella: from environmental habitats to disease pathology, detection and control. , 1999, Environmental microbiology.

[21]  P. Stewart,et al.  Direct measurement of chlorine penetration into biofilms during disinfection , 1994, Applied and environmental microbiology.

[22]  K. Schleifer,et al.  Phylogenetic Diversity among Geographically Dispersed Chlamydiales Endosymbionts Recovered from Clinical and Environmental Isolates of Acanthamoeba spp , 2000, Applied and Environmental Microbiology.

[23]  J. Darelid,et al.  Control of nosocomial Legionnaires' disease by keeping the circulating hot water temperature above 55 degrees C: experience from a 10-year surveillance programme in a district general hospital. , 2002, The Journal of hospital infection.

[24]  M. Storey,et al.  A comparison of methods and models for the analysis of water distribution pipe biofilms , 2002 .

[25]  M V Storey,et al.  The fate of legionellae within distribution pipe biofilms: measurement of their persistence, inactivation and detachment. , 2004, Water science and technology : a journal of the International Association on Water Pollution Research.

[26]  Janet E. Stout,et al.  Legionella in water distribution systems , 1998 .

[27]  F. Schuster.  Cultivation of Pathogenic and Opportunistic Free-Living Amebas , 2002, Clinical Microbiology Reviews.

[28]  Barry S. Fields,et al.  Legionella and Legionnaires' Disease: 25 Years of Investigation , 2002, Clinical Microbiology Reviews.

[29]  B. Drasar,et al.  Association of Vibrio cholerae with fresh water amoebae. , 1992, Journal of medical microbiology.

[30]  R. Amann,et al.  Obligate intracellular bacterial parasites of acanthamoebae related to Chlamydia spp , 1997, Applied and environmental microbiology.

[31]  K. Birkness,et al.  Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls , 1998, Applied and Environmental Microbiology.

[32]  K. Schleifer,et al.  The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae. , 1992, FEMS microbiology letters.

[33]  B. Fields,et al.  The molecular ecology of legionellae. , 1996, Trends in microbiology.

[34]  V L Yu,et al.  Comparative assessment of chlorine, heat, ozone, and UV light for killing Legionella pneumophila within a model plumbing system , 1987, Applied and environmental microbiology.

[35]  L. Engstrand,et al.  Free-living Amoebae Promote Growth and Survival of Helicobacter pylori , 2002, Scandinavian journal of infectious diseases.

[36]  G. McFeters,et al.  Physiological responses of bacteria in biofilms to disinfection , 1994, Applied and environmental microbiology.

[37]  K. Schleifer,et al.  In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. , 1995, Microbiology.

[38]  T. Ford,et al.  Microbiological safety of drinking water: United States and global perspectives. , 1999, Environmental health perspectives.