Collection and measurement of temperature, pH and residual chlorine of water samples
During one year period, from December 2018 to December 2019, a total of 100 water samples were collected from nine hospitals in Tehran, Iran. Samples were from oxygen humidifier bottle (43%), shower head (46%) and water bath (11%). All samples had residual chlorine between 0.3 to 0.6 mg/ml (Fig. 1). The pH of the water samples were between 5.6 to 6 (Fig. 1). Temperature of the samples was between 25–30 C in 43%, 31–36 C in 9% and 37–41 C in 48% of the cases (Fig. 1).
Concentration And Treatment Of Water Samples
For this step, we examined two different concentrations (centrifugation and filtration) and then used two different treatments methods for each concentration (heat and acid). Water samples were concentrated with centrifuge at 3000 rpm for 10 minutes at 4º C. The deposits were resuspended in 5 ml of original water sample as concentrates. In concentration by filtration, at first all water samples were passed through 0.45 µm pore size nitrocellulose membranes. Then the membranes were aseptically removed, put into sterilized 50 ml tube and resuspended in 10 ml of the original water samples. Each concentrated water samples was shaken for 30 min to get out bacterial cells from the membrane. To exclusion of non-legionella bacteria, the concentrated water samples were diluted (1:10) in Kcl-Hcl solution (pH: 2.2), mixed and incubated at room temperature for 4 minutes. In an alternative treatment for concentrated water, heating at 50ºC for 30 min was used.
Culture of water samples on GVPC (Glcine, Vancomycin, Polymyxin and Cycloheximide)
A 0.1 ml volume of concentrated and treated water samples were spread onto the surface of BCYE supplemented with glycin, vancomycin Hcl (1 µg/ml), polymyxin B (79.2 IU/ml), and cycloheximide (80 µg/ml) (GVPC agar). Plates were incubated in candle jar (3–5% Co2) at 37ºC in a humidified atmosphere.
Identification of Legionella pneumophila colony
For verification of suspected colonies with the typical ground glass appearance on GVPC, these colonies were inoculated on BCYE with or without L-cysteine and onto non selective media such as blood agar. Identification of Legionella pneumophila were performed with gram stain and biochemical tests. Strains unable to grow on media without L-cycteine and blood agar were further analyzed by PCR with specific primers for Legionella genus (16srRNA gene) and pneumophila specie (mip gene).
DNA extraction from Legionella pneumophila colonies
Freshly grown legionella colonies on GVPC medium were suspended in distilled water and 200 µl of the suspension was used for DNA extraction and purification according to a commercial kit manufacture instruction. 50 µl of lysis solution was added into each microcentrifuge tube containing legionella suspension for breakage of the cell membrane. The tubes were vortexed and then incubated at 95 °C for 10 min in a hot plate and then were left to equilibrate at room temperature for 5 minutes. The tubes were then vortexed and centrifuged at 6,000 rpm for 2 minutes and the eluted DNA was transferred to an eppendorf tube. Quality of the extracted DNA was assessed by optical density at 260 nm and electrophoresis on agarose gel. Extracted DNA from legionella colonies was stored at − 20 °C for a maximum of 2 days.
Biochemical Analysis, Culture And Pcr Methods On Water Samples
None of the 12 suspected colonies grown on GVPC with a negative reaction in gram staining when cultured on blood agar and BCYE without L-cystein showed any growth. Identification of these suspected colonies were performed by biochemical test such as positive oxidate test and weak reaction in catalase test. Additional molecular confirmation was performed by PCR method with specific primers for Legionella genus (16srRNA) and pneumophila species (mip). All of the isolates were positive as Legionella pneumophila. Using culture and PCR methods for isolation and detection of Legionella in water samples shown that 12 and 42% of the hospital water samples were colonized by Legionella, respectively. All of the samples that were positive in culture methods (12%) were also positive by PCR. Legionella was isolated with a rate of 8%, 3% and 1% from shower heads; oxygen humidifier bottle and water bath respectively (Fig. 2). Fifty (5%) samples from 12 with positive culture have a colony forming unit higher than 1000 cfu/100 ml and the rest had values. A correlation was found between Legionella culture positivity rate and temperature of water samples in analysis by chi-square and likehood test (p value = 0.000, r = 0.493). No significant correlation was found between residual chlorine of water samples and the presence of Legionella with chi-square and Fisher test (p value = 0.313). A correlation was detected between the presence of Legionella and pH (p = 0.000; r = 0.546). Since sample collection continued for a period of one year, the isolation rate in summer and spring was 10 cases of 12 ( 83.33% ) as compared to winter and autumn which was 2 cases (16.66%).
Detection of virulence factors in the Legionella pneumophila isolates
To estimate whether Legionella isolates are pathogenic for human the presence of virulence genes including mip, dot, hsp, rtx and lvh were detected among the isolates. Findings showed that 12 (100%) isolates were positive for mip genes, 9 (75%) were positive for dot gene, 8 (66.66%) were positive for hsp, 6(50%) were positive for lvh and 4(33.33%) for rtx (Fig. 3, Fig. 4). Twelve cases showed eight virulence patterns that were reported in Table 2 and Fig. 5. All of the isolates had at least two of these virulence factors.
Biofilm Formation Assay Among Isolates
The ability of Legionella pneumophila for biofilm formation were estimated and results revealed that two isolates in first day have a higher ability to form biofilm in reference to the standard strain and this ability increased to eight and ten isolates compare to standard strain in third and ninth days (Table 3).
Table 2
Different patterns of virulence genes among isolated Legionella strains.
Strains | Lvh | Rtx a | Hsp60 | Dot | Mip | 16srRNA |
Lp1 | + | + | + | + | + | + |
Lp2 | - | - | + | - | + | + |
Lp3 | + | + | + | - | + | + |
Lp4 | + | - | + | + | + | + |
Lp5 | - | - | + | + | + | + |
Lp6 | - | - | + | + | + | + |
Lp7 | - | - | - | + | + | + |
Lp8 | + | + | - | + | + | + |
Lp9 | + | - | - | + | + | + |
Lp10 | - | - | - | + | + | + |
Lp11 | - | - | + | + | + | + |
Lp12 | + | + | + | - | + | + |
Table 3
Results of OD mean for biofilm formation among isolated Legionella strains
Legionella strains | First day | Third day | Ninth day |
Negative control | 0.009 ± 0.001 | 0.009 ± 0.001 | 0.009 ± 0.001 |
Positive control | 0.123 ± 0.004 | 0.644 ± 0.028 | 1.297 ± 0.070 |
LP1 | 0.139 ± 0.001 | 0.982 ± 0.081 | 2.008 ± 0.020 |
LP2 | 0.101 ± 0.001 | 0.975 ± 0.223 | 1.767 ± 0.042 |
LP3 | 0.145 ± 0.001 | 0.905 ± 0.014 | 1.463 ± 0.026 |
LP4 | 0.099 ± 0.001 | 0.935 ± 0.002 | 1.841 ± 0.131 |
LP5 | 0.93 ± 0.001 | 0.851 ± 0.093 | 1.397 ± 0.035 |
LP6 | 0.105 ± 0.001 | 0.804 ± 0.042 | 1.785 ± 0.0151 |
LP7 | 0.098 ± 0.001 | 0.608 ± 0.013 | 1.917 ± 0.084 |
LP8 | 0.103 ± 0.004 | 0.500 ± 0.023 | 1.536 ± 0.082 |
LP9 | 0.105 ± 0.001 | 0.646 ± 0.046 | 1.188 ± 0.082 |
LP10 | 0.103 ± 0.001 | 0.804 ± 0.042 | 1.767 ± 0.042 |
LP11 | 0.096 ± 0.001 | 0.608 ± 0.013 | 1.841 ± 0.131 |
LP12 | 0.093 ± 0.001 | 0.851 ± 0.093 | 1.297 ± 0.035 |