Objectives: Application of noncultural assay methods for the evaluation of the residual risk represented by viable but nonculturable (VBNC) Legionella during monochloramine (MC) disinfection of hot water network in a hospital building. Methods: Following the start of the MC water disinfection system (with a set concentration ³ 1.5 mg/L of MC) in December 2010, samples were taken monthly from hot water taps in six sites at various distance from the MC generator. Legionella spp. was cultured in accordance with ISO-11731 and typed following the Sequence Based Typing protocol for epidemiological typing (ELDSNet). Water samples negative for Legionella on culture were assayed for VBNC forms by real-time PCR according to the published reference method (AFNOR XP T90-471, 2006) and by ATP-bioluminescence analysis (Quench-Gone Aqueous, Aqua-tools) (IMS-ATP) following immunomagnetic separation (IMS, Dynabeads anti-Legionella, Invitrogen). Samples positive to any of the above described methods were submitted to 'resuscitation' test into Acanthamoeba polyphaga (García M.T., 2007). Results: Throughout the 30 months of study all samples taken from sites previously positive for presence of Legionella pneumophila ST 269 (mean count 7.2x103CFU/L) were negative at culture, with the exception of three instances temporally linked to a failure of the MC generator. Among the 106 negative samples, 69 (65%) gave positive results at PCR analysis (mean load 3.7x103UG/L). PCR positive samples, tested by IMS-ATP, yielded high values of ATP (mean 8.2x102pg/L), confirming the presence of VBNC cells. Only two samples, associated to MC concentration ≤ 1.5 mg/L, were positive at 'resuscitation' test, while samples collected in correspondence to higher MC levels were negative. Conclusions: Our results suggest the usefulness of high sensitivity non-conventional methods for testing presence of resuscitable VBNC cells, not detectable by traditional cultural tests, to evaluate the residual risks represented by VBNC cells.Our study suggest the importance of keeping an appropriate and uninterrupted MC dosage (more than 1.5 mg/L) to ensure control of Legionella-related risk.

DETECTION OF VIABLE BUT NONCULTURABLE LEGIONELLA IN HOSPITAL WATER NETWORK FOLLOWING MONOCHLORAMINE DISINFECTION

CASINI, BEATRICE;PORRETTA, ANDREA DAVIDE;VALENTINI, PAOLA;PRIVITERA, GAETANO PIERPAOLO
2014

Abstract

Objectives: Application of noncultural assay methods for the evaluation of the residual risk represented by viable but nonculturable (VBNC) Legionella during monochloramine (MC) disinfection of hot water network in a hospital building. Methods: Following the start of the MC water disinfection system (with a set concentration ³ 1.5 mg/L of MC) in December 2010, samples were taken monthly from hot water taps in six sites at various distance from the MC generator. Legionella spp. was cultured in accordance with ISO-11731 and typed following the Sequence Based Typing protocol for epidemiological typing (ELDSNet). Water samples negative for Legionella on culture were assayed for VBNC forms by real-time PCR according to the published reference method (AFNOR XP T90-471, 2006) and by ATP-bioluminescence analysis (Quench-Gone Aqueous, Aqua-tools) (IMS-ATP) following immunomagnetic separation (IMS, Dynabeads anti-Legionella, Invitrogen). Samples positive to any of the above described methods were submitted to 'resuscitation' test into Acanthamoeba polyphaga (García M.T., 2007). Results: Throughout the 30 months of study all samples taken from sites previously positive for presence of Legionella pneumophila ST 269 (mean count 7.2x103CFU/L) were negative at culture, with the exception of three instances temporally linked to a failure of the MC generator. Among the 106 negative samples, 69 (65%) gave positive results at PCR analysis (mean load 3.7x103UG/L). PCR positive samples, tested by IMS-ATP, yielded high values of ATP (mean 8.2x102pg/L), confirming the presence of VBNC cells. Only two samples, associated to MC concentration ≤ 1.5 mg/L, were positive at 'resuscitation' test, while samples collected in correspondence to higher MC levels were negative. Conclusions: Our results suggest the usefulness of high sensitivity non-conventional methods for testing presence of resuscitable VBNC cells, not detectable by traditional cultural tests, to evaluate the residual risks represented by VBNC cells.Our study suggest the importance of keeping an appropriate and uninterrupted MC dosage (more than 1.5 mg/L) to ensure control of Legionella-related risk.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11568/655870
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