Comparison of two molecular methods used for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply

Abstract

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

Optimization of Pulsed-Field Gel Electrophoresis forLegionella pneumophilaSubtyping

“…There are several reports comparing different molecular typing methods for L. pneumophila subtyping (6,23,25). Their results show that PFGE has a high degree of consistency with other molecular typing methods for L. pneumophila subtyping, such as RAPD analysis, ribotyping, and SBT.…”

“…Strain differentiation is necessary for the identification of sources of contamination and determination of routes of transmission; this could in turn enable us to more accurately detect outbreaks and limit the spread of L. pneumophila infections. A variety of subtyping techniques have been used to identify and characterize L. pneumophila strains, including monoclonal antibody (MAb) analysis (16,19), ribotyping (4), amplified fragment length polymorphism (AFLP) analysis (9, 22), PCR-based methods (15, 24), sequence-based typing (SBT) (9, 16), and pulsedfield gel electrophoresis (PFGE) (1,6).Preliminary reports demonstrated that PFGE is a highly discriminative epidemiological marker for subtyping of L. pneumophila (6,11,23,25), and a number of L. pneumophila PFGE protocols have been described in the literature (1, 2, 4, 14); however, most laboratories that use PFGE to subtype L. pneumophila cannot compare their results because the protocols differ from each other in critical parameters, such as the restriction enzymes and electrophoresis conditions used to generate the DNA fingerprints. To enhance our ability to monitor this pathogen, there is an urgent need for a standardized L. pneumophila PFGE protocol which can readily be implemented in different laboratories for information interpretation.…”

“…Strain differentiation is necessary for the identification of sources of contamination and determination of routes of transmission; this could in turn enable us to more accurately detect outbreaks and limit the spread of L. pneumophila infections. A variety of subtyping techniques have been used to identify and characterize L. pneumophila strains, including monoclonal antibody (MAb) analysis (16,19), ribotyping (4), amplified fragment length polymorphism (AFLP) analysis (9, 22), PCR-based methods (15, 24), sequence-based typing (SBT) (9, 16), and pulsedfield gel electrophoresis (PFGE) (1,6).…”

“…Preliminary reports demonstrated that PFGE is a highly discriminative epidemiological marker for subtyping of L. pneumophila (6,11,23,25), and a number of L. pneumophila PFGE protocols have been described in the literature (1,2,4,14); however, most laboratories that use PFGE to subtype L. pneumophila cannot compare their results because the protocols differ from each other in critical parameters, such as the restriction enzymes and electrophoresis conditions used to generate the DNA fingerprints. To enhance our ability to monitor this pathogen, there is an urgent need for a standardized L. pneumophila PFGE protocol which can readily be implemented in different laboratories for information interpretation.…”

Validation of IRS PCR, a molecular typing method, for the study of the diversity and population dynamics of Legionella in industrial cooling circuits

“…; Casini et al . ). Those studies have shown that IRS PCR, PFGE and SBT were both able to differentiate unrelated strains and to cluster related strains in a same group.…”