Incubation of premise plumbing water samples on Buffered Charcoal Yeast Extract agar at elevated temperature and pH selects for Legionella pneumophila

Abstract

Worldwide, over 90% of the notified cases of Legionnaires' disease are caused by Legionella pneumophila. However, the standard culture medium for the detection of Legionella in environmental water samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH 6.9 ± 0.4 with or without antimicrobial agents incubated at 36 ± 1 °C, supports the growth of a large diversity of Legionella species. BCYE agar of elevated pH or/and incubation at elevated temperature gave strongly reduced recoveries of most of 26 L. non-pneumophila spp. tested, but not of L. pneumophila. BCYE agar of pH 7.3 ± 0.1, incubated at 40 ± 0.5 °C (BCYE pH 7.3/40 °C) was tested for selective enumeration of L. pneumophila. Of the L. non-pneumophila spp. tested, only L. adelaidensis and L. londiniensis multiplied under these conditions. The colony counts on BCYE pH 7.3/40 °C of a L. pneumophila serogroup 1 strain cultured in tap water did not differ significantly from those on BCYE pH 6.9/36 °C when directly plated and after membrane filtration and showed repeatability's of 13-14%. By using membrane filtration L. pneumophila was detected in 58 (54%) of 107 Legionella-positive water samples from premise plumbing systems under one or both of these culture conditions. The L. pneumophila colony counts (log-transformed) on BCYE pH 7.3/40 °C were strongly related (r² = 0.87) to those on BCYE pH 6.9/36 °C, but differed significantly (p < 0.05) by a mean of - 0.12 ± 0.30 logs. L. non-pneumophila spp. were detected only on BCYE pH 6.9/36 °C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40 °C can facilitate the enumeration of L. pneumophila and their isolation from premise plumbing systems with culturable L. non-pneumophila spp., some of which, e.g. L. anisa, can be present in high numbers.

Keywords: Culture method; Elevated incubation temperature; Elevated medium pH; L. non-pneumophila species; Legionella pneumophila detection.