Molecular typing of nosocomial isolates of Legionella pneumophila serogroup 3

Abstract

Paris, France, an outbreak of pneumonia due to Legionella pneumophila serogroup 3 was observed in Necker (four cases) and Pitié (six cases) hospitals. Neither the 10 clinical isolates nor 5 tap water isolates from Necker Hospital harbored plasmids. Clinical and environmental serogroup 3 isolates and serogroup 3 reference strain Bloomington 2 were analyzed by chromosomal probe fingerprinting. rRNA, 16S and 23S from Escherichia coli and a randomly cloned 15-kilobase-pair nucleotide sequence from L. pneumophila serogroup 3 were used as probes. All strains tested showed a single pattern after HindlI digestion of DNA and hybridization with the 32P-end-labeled rRNA probe, whereas three patterns were obtained after hybridization with the 32P-labeled 15-kilobase-pair DNA probe. One pattern was given by all clinical and tap water isolates from Necker Hospital, another one was given by all clinical isolates from Pitié Hospital, and a last one was given by reference strain Bloomington 2. Thus, molecular analysis showed that the two hospital outbreaks of legionellosis were unrelated and could link the outbreak in Necker Hospital to contaminated tap water.

Molecular epidemiology of Legionella pneumophilia serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting

“…acetate buffer (0.04 M containing 0.002 M EDTA, pH 8). DNA fragments were transferred to nitrocellulose membrane (Schleicher and Schuell) as previously described [3]. The transfer membrane was prehybridized at 60 °C for 2 h (solution of 6 X saline sodium citrate (SSC), 5 X Denhardt, 0.5% w/v SDS, and 100 /xg ml i of shear-denatured herring sperm DNA); hybridization solution was added (6 x SSC, 5 x Denhardt, 100 txg ml-1 denatured herring sperm DNA and 250 ng m1-1 of denatured acetylaminofluorene (AAF)-labelled ribosomal 16 + 23S RNA from E. coli, purchased from Eurogentec, Belgium) and the membrane was incubated overnight at 60°C with shaking.…”

“…Although many Legionella species are found in the natural environment, L. pneumophila is responsible for more than 90% of cases of legionnaires' disease [1]. Outbreaks of community-acquired and nosocomial L. pneumophila infections have been described [2,3]. Epidemiological studies were undertaken during * Corresponding author.…”

“…Study of DNA digests with restriction endonucleases and standard or pulse electrophoresis was proposed [4,7]. Restriction fragments of L. pneumophila DNA were used as probes for molecular typing of nosocomial iso-SSDI 0928-8244(94)00011-H 24 lates from the same serogroup (serogroup 3) which had no plasmids [3]. Ribotyping was reported for the epidemiology of legionnaires' disease [3,7] with contradictory results for its value.…”

“…Restriction fragments of L. pneumophila DNA were used as probes for molecular typing of nosocomial iso-SSDI 0928-8244(94)00011-H 24 lates from the same serogroup (serogroup 3) which had no plasmids [3]. Ribotyping was reported for the epidemiology of legionnaires' disease [3,7] with contradictory results for its value. In previous studies, only radiolabelled probes were used.…”

“…2. PCR fingerprinting of L. pneumophila serogroup 1 strains by using an arbitrary primer.Lanes 1,2,3,4,5,9,12, 13, 14, 15, 16, 17: unrelated strains. Lanes 6, 7, 8 and 10: related strains.…”