Multiplex polymerase chain reaction of genetic markers for detection of potentially pathogenic environmental Legionella pneumophila isolates

Abstract

Background & objectives: Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment.

Methods: Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at p H 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR.

Results: The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers.

Interpretation & conclusions: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.

Fig. 1

Flowchart showing processing of water samples. BCYE-GVPC, buffered charcoal-yeast extract with Glycine, Vancomycin, Polymyxin B, and Cycloheximide; PCR, polymerase chain reaction; REA, restriction enzyme analysis.

Fig. 2

16S rRNA gene polymerase chain reaction (PCR) and restriction enzyme analysis (REA) of standard strains. Lane 1, negative control; lane 2, low range Marker (25-700 bp); lanes 3 & 4, PCR & REA of ATCC 33152 Legionella pneumophila, respectively; lanes 5 & 6, PCR & REA of ATCC 33153 L. pneumophila, respectively.

Fig. 3

(A) Sensitivity of PCR for individual markers: (i) 11A2 - Lane 1, negative control; lane 2, 1:1000 dilution (0.17 ng/μl); lane 3, 1:500 dilution; lane 4, 1:100 dilution; lane 5, 1:50 dilution; lane 6, 1:10 dilution; lane 7, ATCC 33153 DNA (177.4 ng/μl); lane 8, 100 bp ladder. (ii) 19H4 - Lane 1, 100 bp ladder; lane 2, ATCC 33153 DNA (177.4 ng/μl); lane 3, 1:10 dilution; lane 4, 1: 50 dilution; lane 5, 1:100 dilution; lane 6, 1:500 dilution, lane 7, 1:1000 dilution (0.17 ng/μl); lane 8, negative control. (iii) 10B12 - Lane 1, Blank; lane 2, 100 bp ladder; lane 3, ATCC 33153 DNA (177.4 ng/μl), lane 4, 1:10 dilution; lane 5, 1: 50 dilution; lane 6, 1:100 dilution; lane 7, 1:1000 dilution (0.17 ng/μl), lane 8, 1:500 dilution; lane 9, negative control. (iv) 24B1 - Lane 1, Blank, lane 2, 100 bp ladder; lane 3, ATCC 33153 DNA (177.4 ng/μl), lane 4, 1:10 dilution; lane 5, 1: 50 dilution; lane 6, 1:100 dilution; lane 7, 1:500 dilution; lane 8, 1:1000 dilution (0.17 ng/μl); lane 9, negative control. (v) 8D6 - Lane 1, Blank; lane 2, 100 bp ladder; lane 3, ATCC 33153 DNA (177.4 ng/μl); lane 4, 1:10 dilution; lane 5, 1: 50 dilution; lane 6, 1:100 dilution; lane 7, 1:500 dilution; lane 8, 1:1000 dilution (0.17 ng/μl); lane 9, negative control. (B) Specificity of PCR for individual markers: (i) 11A2, (ii) 19H4, (iii) 10B12, (iv) 24B1, (v) 8D6 - Lane 1, 100 bp ladder; lane 2, Escherichia coli DNA; lane 3, Klebsiella pneumoniae DNA; lane 4, Proteus mirabilis DNA; lane 5, Pseudomonas sp. DNA; lane 6, Haemophilus influenzae DNA; lane 7, Neisseria meningitidis DNA; lane 8, Staphylococcus aureus DNA; lane 9, Mycobacterium tuberculosis DNA; lane 10, Legionella pneumophila ATCC 33152 DNA; lane 11, L. pneumophila ATCC 33153 DNA; lane 12, negative control; lane 13, Blank; lane 14, 100 bp ladder.

Fig. 4

Multiplex PCR of five markers in Legionella pneumophila standard strains ATCC 33152 and ATCC 33153 - Lane 1, Blank; lane 2, low range marker 25-700 bp size; lane 3, Legionella pneumophila ATCC 33152 DNA; lane 4, L. pneumophila ATCC 33153 DNA; lane 5, negative control.

Fig. 5

Multiplex PCR of environmental Legionella pneumophila isolates (isolates 27 to 45 represented here). (A) Isolate 27 to 37 - Lane 1: low range Marker 25-700 bp, lane 2: isolate 27 (2B); lane 3, isolate 28 (5B); lane 4, isolate 29 (11A2); lane 5, isolate 30 (11B); lane 6, isolate 31 (6B); lane 7, isolate 32 (6C); lane 8, isolate 33 (7A); lane 9, isolate 34 (7C); lane 10, isolate 35 (12B); lane 11, isolate 36 (15B); lane 12, isolate 37 (15C); lane 13, positive control (ATCC 33153 Legionella pneumophila); lane 14, negative control; lane 15, 100 bp ladder. (B) Isolate 38 to 45 - Lane 1, low range Marker 25-700 bp; lane 2, isolate 38 (11B); lane 3, isolate 39 (11C), lane 4, isolate 40 (15A); lane 5, isolate 41 (15B); lane 6, isolate 42 (15C); lane 7, isolate 43 (17B); lane 8, isolate 44 (17C); lane 9, isolate 45 (19B); lane 10, positive control (ATCC 33153 L. pneumophila); lane 11, negative control; lane 12, 100 bp ladder.