Background: Understanding legionella distribution and relatedness is essential for determining the epidemiology of nosocomial infections and for aiding in the choice of effective control methods. Molecular typing also allows the assessment of strain interrelationship, so as to evaluate the evolutionary response of the isolates to the environment when sanitizing procedures are undertaken. Chlorine-tolerant types may occur in hospital water-system when chlorination is applied, since the disinfection fails to completely eliminate legionella colonisation. Purpose: To assess the circulation of Legionella pneumophila serogroup 1 in different wards of the Azienda Ospedaliero-Universitaria Pisana by the study of the genetic variability and the chlorine-susceptibility, prior to and throughout the application of continuous chlorine dioxide treatment. Rapid enumeration and screening of chlorine-susceptibility was also performed by quantitative ATP detection. Materials and methods: A total of 61 Legionella pneumophila serogroup 1 isolates, collected during a five-year monitoring programme applied to the hospital water-system between March 2002 and December 2006, were selected on spatial and temporal criteria in order to follow the evolution of the colonisation. Continuous chlorine dioxide treatment of the warm water network was started in April 2003. The species identification was carried out by the mip gene nucleotide sequence analysis and the genotyping was performed by sequence-based typing (SBT), using the EWGLI standard schemes. Moreover, genomic profile of DNA macrorestriction digests were analysed applying pulsed-field gel electrophoresis (PFGE), according to De Zoysa protocol. Isolates representative of the prevalent genotypes, isolated prior to and following the start of hyperchlorination, were studied for their chlorine-susceptibility according to the BS EN 1040:1997. Feasibility of the rapid assessment of chlorine-susceptibility using quantitative ATP detection was also evaluated. Results: The sequences of the mip gene of all the 61 amplicons of the selected isolates gave a single amplification product, which aligned with L. pneumophila strain Wadsworth. Both PFGE and SBT indicated the circulation in the hospital water-system of three types of L. pneumophila Wadsworth, showing allelic and electrophoretic characteristic profiles. Type 1 and 2 were randomly distributed in space and time, whereas Type 3 was isolated only after two years of water hyperchlorination and only in one building. The dendrogram of the PFGE patterns demonstrated a greater similarity (64%) between Type 1 and 3, as well as SBT showed among them only two different genes. The assay of chlorine-susceptibility showed that Type 2 was promptly killed at low chlorine concentrations, while Type 1 was inactivated only at concentrations equal or greater than 5mg/L, a 105 fold viability reduction being achieved after 60 minutes of contact. As expected on the basis of its genetic relatedness the susceptibility of the newly emerging Type 3 was comparable to that of Type 1. Following exposure to chlorine, ATP levels correlated well with the measured number of viable cells of the three Types, but the luminescence signals were significantly decreased only for Type 2 and after 60-minute of contact, compared to untreated controls. Conclusion: Chlorine-susceptibility of Type 1 and 2, as well as their relative prevalence among environmental isolates, remained unchanged over time and were not influenced by long-term exposure to hyperchlorination despite their different susceptibilities. Chlorine-tolerance of Type 1 was pre-dated chlorine dioxide treatment and cannot therefore be considered an evolutionary response to the disinfection treatment. Following these observations, our hospital water safety plan relying on chlorine dioxide for sanitation has been modified to include point-of-use filtration as an additional measure in selected wards.
Long term colonisation by chlorine-tolerant types of Legionella pneumophila serogroup 1 strain Wadsworth of a University Hospital water system
CASINI, BEATRICE;BAGGIANI, ANGELO;PRIVITERA, GAETANO PIERPAOLO
2008-01-01
Abstract
Background: Understanding legionella distribution and relatedness is essential for determining the epidemiology of nosocomial infections and for aiding in the choice of effective control methods. Molecular typing also allows the assessment of strain interrelationship, so as to evaluate the evolutionary response of the isolates to the environment when sanitizing procedures are undertaken. Chlorine-tolerant types may occur in hospital water-system when chlorination is applied, since the disinfection fails to completely eliminate legionella colonisation. Purpose: To assess the circulation of Legionella pneumophila serogroup 1 in different wards of the Azienda Ospedaliero-Universitaria Pisana by the study of the genetic variability and the chlorine-susceptibility, prior to and throughout the application of continuous chlorine dioxide treatment. Rapid enumeration and screening of chlorine-susceptibility was also performed by quantitative ATP detection. Materials and methods: A total of 61 Legionella pneumophila serogroup 1 isolates, collected during a five-year monitoring programme applied to the hospital water-system between March 2002 and December 2006, were selected on spatial and temporal criteria in order to follow the evolution of the colonisation. Continuous chlorine dioxide treatment of the warm water network was started in April 2003. The species identification was carried out by the mip gene nucleotide sequence analysis and the genotyping was performed by sequence-based typing (SBT), using the EWGLI standard schemes. Moreover, genomic profile of DNA macrorestriction digests were analysed applying pulsed-field gel electrophoresis (PFGE), according to De Zoysa protocol. Isolates representative of the prevalent genotypes, isolated prior to and following the start of hyperchlorination, were studied for their chlorine-susceptibility according to the BS EN 1040:1997. Feasibility of the rapid assessment of chlorine-susceptibility using quantitative ATP detection was also evaluated. Results: The sequences of the mip gene of all the 61 amplicons of the selected isolates gave a single amplification product, which aligned with L. pneumophila strain Wadsworth. Both PFGE and SBT indicated the circulation in the hospital water-system of three types of L. pneumophila Wadsworth, showing allelic and electrophoretic characteristic profiles. Type 1 and 2 were randomly distributed in space and time, whereas Type 3 was isolated only after two years of water hyperchlorination and only in one building. The dendrogram of the PFGE patterns demonstrated a greater similarity (64%) between Type 1 and 3, as well as SBT showed among them only two different genes. The assay of chlorine-susceptibility showed that Type 2 was promptly killed at low chlorine concentrations, while Type 1 was inactivated only at concentrations equal or greater than 5mg/L, a 105 fold viability reduction being achieved after 60 minutes of contact. As expected on the basis of its genetic relatedness the susceptibility of the newly emerging Type 3 was comparable to that of Type 1. Following exposure to chlorine, ATP levels correlated well with the measured number of viable cells of the three Types, but the luminescence signals were significantly decreased only for Type 2 and after 60-minute of contact, compared to untreated controls. Conclusion: Chlorine-susceptibility of Type 1 and 2, as well as their relative prevalence among environmental isolates, remained unchanged over time and were not influenced by long-term exposure to hyperchlorination despite their different susceptibilities. Chlorine-tolerance of Type 1 was pre-dated chlorine dioxide treatment and cannot therefore be considered an evolutionary response to the disinfection treatment. Following these observations, our hospital water safety plan relying on chlorine dioxide for sanitation has been modified to include point-of-use filtration as an additional measure in selected wards.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
