Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR

Abstract

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.

Figures

FIG. 1

Generation of the standard curve for the 16S rRNA gene PCR. (A) Serial 10-fold dilutions of L. pneumophila serogroup 1 DNA were amplified with the primers JFP and JRP and detected by the hybridization probes Leg FL and Leg LC (results from 1 representative experiment out of 10 are shown). (B) The standard curve was generated by linear regression of the threshold cycle where the fluorescence signal first exceeds the level of background noise versus the logarithm of the sample concentration, using the second-derivative maximum method of the LightCycler software (see Results ). Fluorescence intensity is in units obtained by the LightCycler.

FIG. 2

Dual-color multiplex PCR of the Legionella 16S rRNA gene with external standards and internal inhibitor control. Standards (L. pneumophila serogroup 1 DNA; amounts per PCR assay are shown) and water samples were amplified according to the protocol described in Materials and Methods. The internal inhibitor control (1 fg of DNA/PCR assay) was added to each reaction mixture. Amplifications of the Legionella DNA and the inhibitor control were detected in fluorescence channels F2 (A) and F3 (B), respectively. A positive signal from the control signifies the absence of PCR inhibitors in the sample. The lack of signals from standards 1, 2, and 3 is caused by inhibition of the control DNA by the large amounts of standard DNA. Results from one representative experiment with 4 water samples out of a total of 77 samples are shown; fluorescence intensity is in units obtained by the LightCycler.

FIG. 3

Comparison of culture results with the 16S rRNA gene PCR results. Water samples (n = 76 [one sample contained PCR inhibitors and was disregarded]) were investigated by culture and 16S rRNA gene PCR as described in Materials and Methods. The detection limit for culture was 1 CFU/100 ml, and the lowest limit for quantification in the PCR was 10 fg of Legionella DNA/PCR, i.e., 23 CFU/100 ml (detection limit of 1 fg of DNA/PCR, i.e., 2.3 CFU/100 ml). The ratios between the CFU counts detected by culture and those calculated by 16S rRNA gene PCR are indicated by the dotted lines. Symbols on the line indicating the lowest limit for quantification (i.e., 23 CFU/100 ml) represent samples in which the number was less than or equal to this limit but different from zero.

FIG. 4

Comparison of 16S rRNA gene PCR results with mip gene PCR results. Water samples (n = 75 [one sample contained PCR inhibitors and was disregarded, and one sample was mip PCR negative]) were investigated by 16S rRNA gene PCR and mip gene PCR as described in Materials and Methods. CFU counts per 100 ml were calculated from the PCR results. The dotted line represents a 1:1 ratio between the CFU counts per 100 ml detected by the 16S rRNA gene PCR and the mip gene PCR. Symbols on the lines indicating the lowest limit for quantification (i.e., 23 CFU/100 ml) represent samples in which the number was less than or equal to these limits but different from zero.