Inhibition effects of Gorge Fischer plumbing system on growth and amplification of Legionella Pneumophila with culture and PCR based on 16sRNA and mip genes


 Background

Legionella is an aquatic bacterium that causes Legionnaires' fever.
Methods

Fifty-six samples of the hot and cold-water system were collected. Characteristics of water samples were measured. Culture was performed in BCYE agar. Molecular identification was performed by PCR.
Results

The mean residual chlorine were 0.73 to 0.88 mg / l. Culture results were positive 58.8% of George Fischer and 23.5% of Ray Ho and 60% Taleghani hospital samples. PCR results based on 16sRNA were 35.2% of the George Fischer system, 45.4%, from Ray Ho piping and 54.5% from Taleghani hospital. the results of the mip gene, 82.3% of George Fischer, 54.5% of RayHo and 20% of Taleghani hospital were positive.
Conclusion

George Fischer’s Legionella stopper pipes and fittings have a good ability to control the growth Legion density in the indoor water supply system and can be considered as a suitable option for use in indoor plumbing and water supply.

Introduction of bacteria. This relationship creates conditions for the bacterium that can easily resist adverse environmental conditions and disinfection (20,21).
Due to the health problems caused by Legionella and the fact that water in various centers and places is the most important cause of transmission and disease, it is necessary to take effective measures to remove this bacterium from water sources, especially water used in medical centers.

Materials And Methods
This descriptive cross-sectional study was conducted for three months (from the beginning of autumn to the end of autumn of 2020) in the water supply system of two commercial centers with three different types of piping (George Fischer pipes and ttings, Ray Ho pipe, and ordinary polyethylene). The G1 Center used George Fischer Legionella Stopper pipes and ttings for indoor water supply, the G2 Center used Ray Ho pipes and ttings, and Center No. 3 had regular polyethylene pipes. All three centers were located close to each other and in area one of Tehran Municipality, and due to the lack of distance in the geographical location, the chemical quality of water entering the urban network in both centers was no different. 56 samples (36 samples of cold water and 20 samples of hot water) from the gallerycommercial center of Galleria, in which 34 samples of the system with pipes and ttings of George Fischer and 22 samples of the ward equipped with pipes and ttings of Ray Ho, as well as 5 samples of the hospital Taleghani was collected with a conventional metal piping system in sterile containers made of polyethylene with a volume of 1.5 liters from different parts of the o ce-commercial complex of Taleghani Gallery and Hospital according to Table 2-1 and was collected and analyzed to examine the  presence of Legionella. 3% sodium thiosulfate was used to remove residual chlorine from water samples. Residual chlorine, pH, and temperature were measured at the sampling site according to standard methods. Samples were immediately transferred to the laboratory in the vicinity of ice boxes for a maximum of 8 hours and concentrated using membrane ltration and vacuum pump (model VE115N) and polycarbonate micron carbon lter (U ow Membrane Filters, pore size: 0.22-0.45 μm). An autoclave (temperature 121 ° C, pressure 15 ppm, and time 15 minutes) was used to sterilize the components of the ltration system. After ltration, the lter was separated and ltered in 50 ml of water and crushed into sterile glass containers, and suspended. To ensure the separation of bacteria from the water lter containing the lter particles was mixed for 24 hours using an orbital shaker (GFL-3017) at a speed of 230 rpm and stored in a refrigerator at + 4 ° C until culture.
Culture BCYE agar medium (manufactured by Biomark) contains L-cysteine supplements (manufactured by Merck Germany), pyrophosphate (manufactured by Sigma Aldrich), and GVPC (Glycine, Vancomycin, Polymyxin B, Cycloheximide) (manufactured by Biomark) according to the instructions used for bacterial culture. To ensure the control of other bacteria, the samples were heat-treated using a water bath (56 ° C for 12 minutes) before planting. 100 μl of the sample was inoculated on the culture medium and the plates were placed in a candle jar containing 2-5% carbon dioxide for 7-14 days at 37 ° C in a humid environment ( Figure 1). Legionella colonies were identi ed based on size, color, and biochemical properties (catalase, oxidase, hydrolyze Hippurate, and gram staining tests). To be sure, the grown colonies were re-inoculated on blood agar medium (base material made by German Merck company and Iranian sheep spring de brinated blood) and incubated at 35 ° C and after ensuring no growth, they were assigned to Legionella was con rmed (22,23). Data were analyzed and reported using Excel 2010 software and descriptive statistics.

Detection of Legionella
Legionella growth was monitored and recorded on the third, fth, seventh, tenth, and fourteenth days. The colonies that appear on the rst and second days are not Legionella, and the colonies that appear on the third and later days should be thoroughly examined. Also, 0.5 ml of sodium-containing sample was stored in sterile Eppendorf tubes for PCR in a -70 ° C freezer.
Legionella colonies were detected under ultraviolet light by their size, color, type, and special and their uorescence properties. In addition, the grown colonies were re-cultured on BCYE agar and their appointment to Legionella was con rmed after ensuring that they did not grow.
Legionella colonies are white-gray or blue-green, convex, shiny with a diameter of 2-4 mm. The central part of the young colonies appears as light gray and granular like a glass background, while the peripheral part of the colony is light pink or blue.

Primer preparation
To select and design speci c primers, some cases should be considered. Due to the diversity of primer sequences, to design the best primer, the sequence of identi ed genes in genome databases is collected and then analyzed. The primers were compared with the sequences recorded in the Genome Bank (Blast) by a set of analysis programs and sequence comparisons. (Tables 1, 2). The quantitative examination of extracted DNA In this method, using a spectrophotometer, light absorption of all samples is read at 260 nm, and using the following equation, was obtained the DNA concentration of sample C (/g / ml) = 50 d.A260.

Gel electrophoresis PCR product
To view the PCR product, amplicon electrophoresed on agarose gel 1% n and observed in the presence of UV light.

Sequencing of 16srRNA and mip gene
To con rm the genes, the PCR product of each of the mip and 16srRNA genes sent to the pishgam company for sequencing, and the sequence results were BLAST.

Results
Culture results and diagnostic tests Suspected colonies on BCYE agar gram were stained with gram technique. Legionella organisms are gram-negative bacilli with a length of approximately 2-20 μm. Catalase and oxidase tests were performed.

Morphology of Legionella
Legionella is found in fresh culture as bacilli and in old culture as lamentous.

Sodium Hippurate hydrolysis test
If the bacterium is Legionella pneumophila, it releases the enzyme hypuurase into the medium, which breaks down the Hippurate to glycine and benzoic acid. The addition of ferric chloride reagent results in a reaction between the reagent and benzoic acid and a white precipitate.
By adding Chloride Ferric reagent to the culture medium containing 1% sodium hypochlorite, the formation of a white precipitate is a positive sign of the test.
Culture on blood agar medium In this experiment, the bacterium was isolated from the suspected colony cultured on BA. After 48 hours of incubation, if the bacteria did not grow in this medium, the test result will be negative for Legionella.

Discussion
The wide and increasing range of Legionella diseases, as well as the isolation and identi cation of species and serogroups of this bacterium from environmental sources and patients, indicates the global spread of Legionella species in different countries.
Numerous reports of the epidemic and sporadic occurrence of Legionnaires' disease and its resulting deaths have attracted the attention of researchers and specialists in lung and respiratory diseases, infectious diseases, microbiology, immunology, environment, genetics, etc. (12) Early diagnosis of Legionellosis and epidemiological conditions in the hospital is necessary not only for correct and effective treatment but also to control and prevent the subsequent onset of the disease. It seems that due to the high mortality rate of Legionella disease, more effective measures should be taken to prevent the spread of Legionella species from the hospital environment to patients susceptible to infection. (7,12).
Legionella infections in immunocompromised individuals, corticosteroid recipients, organ transplants, the elderly and children, alcohol and drug addicts, smokers, patients with diabetes mellitus, underlying diseases, chronic obstructive pulmonary disease, intubation, intubation and it poses a serious threat to heart patients more than anyone else.
Legionella alone causes a high percentage of nosocomial pneumonia. This bacterium causes 1 to 40% of hospital-acquired pneumonia. The mortality rate in legionellosis is 5-30%. About 5% of cases of Legionnaires' disease are reported from hospitals. (26) Legionella pneumophila was the cause of 3.8% of fatal cases of nosocomial pneumonia. Bacteria of the Legionella genus are isolated from natural water sources, public water reservoirs, water pipes, and even bath showers.
Because various factors are effective in the growth and pathogenicity of Legionella bacteria, the factors affecting the growth and survival of bacteria can be largely identi ed and their ndings can be used to design an appropriate control method. (!2,7,26) Today, various methods to control Legionella in hospital and community aquatic environments are being studied and performed. Therefore, identifying pathogenic strains in that area can provide speci c ideas for the use of effective and e cient disinfectants to control Legionella.
In a hospital population, there are always patients who are susceptible to infection and are at high risk for Legionella. Water is one of the common sources of Legionella transmission in hospitalized patients.
Early diagnosis of legionellosis and epidemiological conditions in the hospital is necessary not only for correct and effective treatment but also to control and prevent the subsequent onset of the disease. It seems that due to the high mortality rate of Legionella disease, more effective measures should be taken to prevent the spread of Legionella species from the hospital environment to patients susceptible to infection. (12) Many types of research have been done using different methods in removing Legionella from water, some of which are mentioned, and the advantages and disadvantages of this research are examined.
Loret et al. reported in 2005 studying a variety of disinfectants such as ozone, chlorine dioxide, chlorine, monochloramine, and copper and silver ionization to remove amoebae and plankton. Marches et al.
Chlorine oxide was used to remove Legionella bacteria (24). Sodium hypochlorite was used to remove Legionella from water, which of course has a higher oxidation rate than other disinfectants and affects a wide range of microorganisms, but has disadvantages such as carcinogenicity and side effects (26).
Other methods used to remove Legionella bacteria were photocatalytic oxidation (using 365 nm UV) with titanium dioxide. Chen et al. used the heat shock method in 2005.
In a 2008 study by Cheng et al., They used the ionization process of copper and silver as a disinfectant to remove Legionella pneumophila. This procedure took 4 to 7 months and studies have shown that this method is effective in the short term and the pollution has not been eliminated (27).
Diamond electrode electrolysis is used to remove and inactivate Legionella bacteria (28).
In general, the advantages of this research over other research in this eld are as Long-term bactericidal stability of George Fischer tube, do not use chemicals, No carcinogenicity, No corrosion of pipes, No side effects, No need for preparation, Low investment cost.
The results showed that George Fischer pipes and ttings are much less contaminated and only out of 34 samples related to the George Fischer pipe and ttings system, 2 samples (samples number (20 and 10) showed contamination with Legionella, based on which it can be said that the pipe and George Fischer connections in more than 95% of the growth of Legionella pneumophila in water, which showed the role and impact of using this system in controlling infections associated with this bacterium, which causes very acute and important problems in the health system, especially in Medical centers, hotels and other administrative, commercial and recreational centers, so that in case of contact with this bacterium and infection and lack of proper treatment can cause the death of infected people.
The results showed that the use of Ray ho pipe and ttings system has higher contamination than George Fischer pipe and ttings system, so that out of 22 samples collected from Ray ho pipe and ttings, 12 samples showed positive and Legionella contamination. These results showed that Ray ho pipes and ttings in more than 55% of Legionella contamination, which con rms the superiority of George Fischer pipes and ttings over Ray ho pipes and ttings.
Examination of the results in terms of Legionella bacterial density in these systems also shows that George Fischer pipes and ttings are not only less contaminated but also in terms of contamination density than Ray ho pipes and ttings and metal pipes. As the system in George Fischer pipes and ttings, only 2 samples with 2 bacteria per liter of water have been observed, while in Ray ho and metal pipes, the probability of contamination and bacterial density is high, which indicates a good ability George Fischer pipes and ttings are in control of Legionella and the inability of these pipes to control Legionella.
The results showed that the average bacterial density in Ray ho pipe and ttings was more than twice that of George Fischer pipe and ttings, so that the range of changes in Legionella bacterial density in Ray ho pipe and ttings was in the range of 2-16 colonies per liter, while this density in pipe George Fischer's connections are only 2 bacteria per liter, which is even lower than the recommended standards for Legionella density in the piping system of developed countries. Comparison of Legionella density in metal pipes also shows that these pipes are not comparable to George Fischer pipes in terms of both probabilities of contamination and density so that in these pipes, 3 out of 5 samples had contamination in which bacterial density. On average, there are more than 43 colonies per liter of water.
The presence of positive results and densities of Legionella bacteria in water samples and their negation in PCR method based on mip gene indicates the presence of contaminants in water that contact with the pipe ttings of George Fischer has prevented its growth or caused their destruction. The only evidence of the presence of Legionella pneumophila DNA in water, which con rms the effect of George Fischer pipes and ttings in controlling and removing Legionella, which shows that this system is suitable for use in the plumbing of medical centers, hotels, leisure centers, etc. Because in such centers, people are more likely to be exposed to acquired lung pneumonia, which not only has a high cost of treatment but also causes death if not treated properly. The results showed that 28 of the 34 samples of George Fischer pipes and ttings in the culture method were negative but based on positive mip gene, which indicates the absence of culturable bacteria but also shows the presence of Legionella pneumophila DNA as a dangerous species of Legionella in water and ability the effect of the tube on the control of Legionella. This is the case for 6 out of 22 Ray ho pipes and ttings.
Abbreviations Gorge Fischer plumbing system: Unique properties of polybutylene pipes in the IFIT system All-plastic polyethylene pipes used in the latest building plumbing system, or IFIT system, are produced exactly in accordance with BS 15876 standard.

Declarations
Ethics approval and consent to participate Not applicable. Consent for publication All of the authors agree with publication manuscript. Availability of data and materials All data generated or analyzed during this study are included in this published article and are available from the corresponding author on reasonable request. Funding The authors don't receive funding for this research. Authors' contributions All authors read and approved the nal manuscript. Declaration of competing interest The authors declare that they have no competing interests. Acknowledge: We thank the Iranian Plumbing Systems Technology Development Company as the only representative of the Swiss company George Fischer in Iran.

Figure 1
Image of Legionella culture on BCYE agar.

Figure 2
Image A is primary culture and image B is old culture.

Figure 3
Sodium Hippurate hydrolysis test to con rm Legionella pneumophila.    Total number of samples collected from hot and cold-water systems of George Fischer piping system, Ray Ho piping system, and metal pipe. Total number of samples collected from hot and cold-water systems of George Fischer piping system, Ray Ho piping system, and metal pipes. Comparison of mip and 16srRNA gene results in George Fischer, Ray Ho, and metal piping systems.