Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C

Abstract

Legionella pneumophila proliferates in freshwater environments at temperatures ranging from 25 to 45°C. To investigate the preference of different sequence types (ST) for a specific temperature range, growth of L. pneumophila serogroup 1 (SG1) ST1 (environmental strains), ST47, and ST62 (disease-associated strains) was measured in buffered yeast extract broth (BYEB) and biofilms grown on plasticized polyvinyl chloride in flowing heated drinking water originating from a groundwater supply. The optimum growth temperatures in BYEB were approximately 37°C (ST1), 39°C (ST47), and 41°C (ST62), with maximum growth temperatures of 42°C (ST1) and 43°C (ST47 and ST62). In the biofilm at 38°C, the ST47 and ST62 strains multiplied equally well compared to growth of the environmental ST1 strain and an indigenous L. pneumophila non-SG1 strain, all attaining a concentration of approximately 10⁷ CFU/cm^-2 Raising the temperature to 41°C did not impact these levels within 4 weeks, but the colony counts of all strains tested declined (at a specific decline rate of 0.14 to 0.41 day^-1) when the temperature was raised to 42°C. At this temperature, the concentration of Vermamoeba vermiformis in the biofilm, determined with quantitative PCR (qPCR), was about 2 log units lower than the concentration at 38°C. In columns operated at a constant temperature, ranging from 38 to 41°C, none of the tested strains multiplied in the biofilm at 41°C, in which also V. vermiformis was not detected. These observations suggest that strains of ST47 and ST62 did not multiply in the biofilm at a temperature of ≥41°C because of the absence of a thermotolerant host.

Importance: Growth of Legionella pneumophila in tap water installations is a serious public health concern. The organism includes more than 2,100 varieties (sequence types). More than 50% of the reported cases of Legionnaires' disease are caused by a few sequence types which are very rarely detected in the environment. Strains of selected virulent sequence types proliferated in biofilms on surfaces exposed to warm (38°C) tap water to the same level as environmental varieties and multiplied well as pure culture in a nutrient-rich medium at temperatures of 42 and 43°C. However, these organisms did not grow in the biofilms at temperatures of ≥41°C. Typical host amoebae also did not multiply at these temperatures. Apparently, proliferation of thermotolerant host amoebae is needed to enable multiplication of the virulent L. pneumophila strains in the environment at elevated temperatures. The detection of these amoebae in water installations therefore is a scientific challenge with practical implications.

FIG 1

Specific growth rates (μ h⁻¹) of L. pneumophila SG1 strains in buffered yeast extract broth at a range of temperatures. (A) Sequence type (ST) 1 strains LP25 (○) and LP26 (●). (B) ST47 strain LP7. (C) ST62 strains LP3 (●) and LP8 (○).

FIG 2

Growth (optical density [OD]) of strains of L. pneumophila ST1 (white bars; n = 6), ST47 (dashed bars; n = 3), and ST62 (black bars; n = 4) in BYEB after 72 h or 96 h (ST1) of incubation at different temperatures. All strains were tested only at 37, 42, 43, and 44°C. Bars show the average values ± standard deviations for strains of the indicated STs. The origins of the strains are shown in Table 1.

FIG 3

Concentration of attached (A and C) and planktonic (B and D) biomass in the columns with pieces of plasticized PVC (pPVC) operated at 38°C (○) and at various temperatures (●), as indicated. Columns were operated at 25°C from day 0 to day 9.

FIG 4

Growth of L. pneumophila ST1 (strain LP25) and ST62 (strain LP3) in the biofilm on pPVC (A and C) and water (B and D) in the columns operated at 38°C (○) and in the columns operated at the indicated temperatures (●). The columns were operated at 25°C from day 0 to day 9. The L. pneumophila concentration determined by qPCR (number of gene copies per square centimeter) is shown for the columns operated at 38°C (△) and the columns operated at the other indicated temperatures (▲). Colony counts below the detection level in water (3.3 CFU ml⁻¹) are shown as 1 CFU ml⁻¹.

FIG 5

Colony counts of L. pneumophila in the biofilm on pPVC (A) and in the water (B) in the columns operated at a constant temperature ranging from 38°C to 41°C. Colony counts below the detection level in water (3.3 CFU ml⁻¹) are shown as 1 CFU ml⁻¹. Symbols are as follows: ○, 38°C; ●, 39°C; □, 40°C; ■, 40.5°C; △, 41°C.