THE INTRODUCTION OF 3+1 IMPORTANT METHOD FOR THE ISOLATION OF ENVIRONMENTAL MYCOBACTERIA FROM DRINKING WATERS

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1 Acta Medica Mediterranea, 2017, 33: 909 THE INTRODUCTION OF 3+1 IMPORTANT METHOD FOR THE ISOLATION OF ENVIRONMENTAL MYCOBACTERIA FROM DRINKING WATERS MEHDI ROSHDI MALEKI 1, HOSSEIN SAMADI KAFIL 2, NASER HARZANDI 1, SEYYED REZA MOADDAB 3* 1 Department of Microbiology, College of Sciences, Karaj Branch, Islamic Azad University, Alborz, Iran - 2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran - 3 Department of Laboratory Sciences, Faculty of Paramedicine, Tabriz University of Medical Sciences, Tabriz, Iran ABSTRACT Water is one of the important reservoirs of environmental mycobacteria (EM). The resistance against chlorine and biofilm formation is among the important factors involved in colonization of these bacteria in water. Environmental mycobacteria cause different types of diseases especially pulmonary diseases due to their transfer to humans. The isolation and identification of these bacteria and the knowledge of how species are spread out as well as the identification of the dominant species in different geographical regions are necessary to be paid attention to because of differences in strategies for treating diseases caused by them. Different methods for isolating environmental mycobacteria have been employed by different researchers, but no standard method has been suggested for this isolation yet. Therefore, in the present study, in addition to introducing an ideal protocol, other studies related to the isolation of these bacteria from water resources were investigated, among which three top methods were selected and introduced. In addition, effective factors such as the type and concentration of disinfectants, the type of water processing, the type of culture medium, their ph, and incubation temperature involved in isolating bacteria are mentioned. Conclusion: using the filtration method instead of centrifuge one, using disinfectant of the cetylpyridinium chloride (CPC) instead of other disinfectants, using the Löwenstein-Jensen culture medium with an acidic ph, incubation in temperature of 30 C and incubation duration are among factors affecting in the isolation of environmental mycobacteria from the water sample. Keywords: isolation, environmental mycobacteria, drinking water. DOI: / _2017_1s_135 Received November 30, 2016; Accepted March 20, 2017 Introduction Mycobacteria are a large family including obligate and environmental pathogens which in recent years have caused deadly diseases in humans particularly in immunocompromised patients. Environmental mycobacteria are also called nontuberculous mycobacteria (NTM), mycobacterium other than tuberculosis (MOTT), and atypical mycobacteria (1). So far, more than 150 species of these bacteria have been identified most of which are opportunistic pathogens (2). Environmental mycobacteria are ubiquitous and their important reservoirs include natural water, drink water, and soil. They have been isolated from drinking water, water reservoirs, bathtubs, faucets in hospitals, dental unit, water coolers, mineral water, and also ice (3). The role of drinking water in transfering diseases has been investigated and the relationship between species isolated from diseases and drinking water has been confirmed using phenotypic methods (4). Environmental mycobacteria are colonized in drinking water resources and cause skin diseases, especially soft tissue infections and

2 910 Mehdi Roshdi Maleki, Hossein Samadi Kafil et Al respiratory diseases by transmission to humans. A large number of them are resistant against high temperatures and wide range of ph. It has been observed that the environment of bathroom shower heads is filled with organisms constructing biofilms mycobacteria (5). Therefore, the inhalation of aerosols generated during bathing is a confirmed path for getting infections caused by NMT and probably the use of showers is the factor of the global increase in pulmonary infections caused by the bacteria (6). Children, adults, immunocompromised patients, and patients with chronic pulmonary diseases are at the exposure of higher risks of infections (7). Isolation and identification of mycobacteria and knowledge of how these species are spread out as well as identification of the dominant species in different geographical areas due to the different strategies for treatment of diseases caused by them are necessary; therefore, it is of utmost importance that they should be seriously taken into consideration (8). Factors affecting the colonization of mycobacteria in tap water pipes Water is one of the important reservoirs of environmental mycobacteria (9). Studying drinking water indicates that some of the EM species such as M. xenopi and M. kansasii are only isolated from drinking water. Older systems of water distribution have higher degrees of EM colonization, but the prevalence of hospital infections out of newer water distribution systems have been reported in relation with the EM as well. Investigation of a wide range of water distribution systems indicates that regardless of the duration of connection, plastic pipes have higher density of EM than other materials. In a study, the resistance of environmental mycobacteria isolated form water distribution systems against chlorine has been evaluated in which M. fortuitum and M. chelonae were reported as the most resistant and M. gordonae and M. aurum were considered as the most sensitive mycobacteria. However, M. aurum and M. grodonae were 100 times as resistant as and 330 times as resistant as E. coli against chlorine (10). Accordingly, this issue can confirm this hypothesis that the growth of bacteria in protozoa provides conditions for the infection of human macrophages. In other words, the ability of growth of the EM in macrophages is the result of protozoa and amoeba (11). Isolation of environmental mycobacteria Isolation of environmental mycobacteria from drinking water and environmental water samples can be traced back to the early 1900, but this mycobacteria have been known as pathogens for human beings in recent three decades (12). Isolation of these bacteria is generally difficult due to long split time. If no appropriate method is selected for their selection, related organisms (contaminants) contaminate the environment before mycobacteria have grown (13). However mycobacteria are resistant against some disinfectants and this property can be used in isolating them from other bacteria, it should be pointed out firstly all mycobacteria are not resistant against disinfectants to the same extent. Secondly, some contaminants such as pseudomonas are present in the environment which are resistant against antimicrobial substances and can be problematic in isolation of EM and contaminate environments. A number of disinfecting methods such as Petroff and NALC-NaOH (N- acetyl cysteine L- sodium hydroxide) methods for isolating the causative agent of tuberculosis (Mycobacterium tuberculosis) from clinical samples routinely (14). This study is part of a research project in Tabriz University of Medical Sciences in which it has been tried to investigate the best method of isolation of mycobacteria out of water samples. Furthermore, in the present study, articles related to isolation of mycobacteria in valid databases such as PubMed and Scopus were investigated. Searching was conducted using English keywords such as NTM, environmental mycobacteria (EM), and MOTT. Then, all valid articles and related to the present study were selected. From among 87 articles, a number of 24 articles presented the isolation method of these bacteria and from among them, 3 articles which reported the most appropriate isolation method of mycobacteria with high percentage were selected. Materials and methods The First Method Sampling water as 50 ml using falcon sterile tube Centrifuging with 8000 rpm for 15 minutes to obtain pellet Adding 20 ml the 3% SDS solution of the 4% NaOH to pellet and doing vortex The solution is divided into two parts (A and B)

3 The introduction of 3+1 important method for the isolation Incubation at room temperature (RT); 15 minutes for part A and 30 minutes for part B Centrifuging with 8000 rpm for 15 minutes to obtain pellet Adding 20 ml of 2% Cetrimide on pellet Incubation at RT; 15 minutes for part A and 30 minutes for part B. Centrifuging for 15 minutes and adding 2 ml sterile distilled water (SDW) to sediments and doing vortex Again centrifuging for 15 minutes and adding 2 ml SDW to pellet and doing vortex Centrifuging for 15 minutes to obtain pellet Adding 0.5 ml SDW to pellet and culture in the Löwenstein-Jensen medium (LJ). Incubation at two temperature ( 30 and 37 C) for 4 to 8 weeks. In this method, part A isolates Rapidly Growing Mycobacteria (RGM) and part B isolates Slowly Growing Mycobacteria (SGM). This method was presented by Parashar et al. (15). In this method, the degree of samples contamination has been reported as 0% and a large number of mycobacteria have been isolated in such a way that for working a sample, at least a period of 2.5 hours is needed. Secondly, a refrigerator-equipped centrifuge is need which can centrifuge 50 ml water with 8000 rpm and such a centrifugation may not be accessible in each laboratory. The third deficit is the use of three disinfectants instead of one disinfectant which is relatively costly. The Second Method Sampling water using sterile flasks Disinfecting with 0.04% cetylpyridinium chloride (CPC) for 30 minutes Filtering of water samples through membranes with 0.45-µm pore size Washing filters in a few ml of SDW Culture in the Middlebrook 7H10 agar and incubation in the temperature range 35 to 37 C for 6 to 10 weeks Doing the second culture from positive Middlebrook 7H10 agar in LJ culture media and incubation in the temperature range 35 to 37 C for 6 to 10 weeks Controlling the growth of mycobacteria once a week. The Tertiary Method Sampling drinking water as 1000 ml using sterile flasks Neutralizing chlorinated water (0.45 ml of 1.8 % sodium thiosulphate) Add 0.05% CPC to water with the ratio of 1 to 9 to 30 minutes for decontamination Filtering with 0.45-µm pore size filters Washing filters with 4 ml of SDW and doing vortex Centrifuging with 6,000 rpm for 15 minutes Culture of pellets in 4 L-J media (each 0.2 ml) and incubation at 25, 30 and 37 C Controlling the growth of colonies weekly in terms of growth rate, morphology, or not pigment) Using this method, Sebakova et al. (16) studied 120 samples of warm water in hospitals and from among this samples, 56 samples (46.7%) were reported to contain positive EM. This study was conducted merely on samples of warm water and isolated mycobacteria belonged to 4 species. The Fourth Method Sampling water after 10 seconds of flowing water as the 500 ml using sterile flasks Filtration through membranes with 0.45-µm pore size and 30 mm diameter (Millipore, PES Syringe Filter, Orange Scientific, Belgium). Removing the filter and washing it in sterile distilled water as 2 ml Doing vortex for 5 minutes for isolating matters collected in the filter Removing the filter using sterile forceps Adding 2 ml of 0.01 % CPC and incubation at RT temperature for 20 minutes Centrifuging for 10 minutes with 6000 rpm 3 ml of the supernatant was discarded and 1mL of SDW was added to it (for neutralization of residual CPC) Doing vortex in and culture in 4 LJ tubes containing culture medium (each 0.5 ml) Two media in temperature 30º C and two others in temperature 37º C incubated for 8 weeks. Mycobacteria growth was controlled on a weekly. The Ziehl-Neelsen staining technique confirmed that the suspected colonies were acid alcohol-resistant bacilli (AARB). This method which is part of a research project in Tabriz University of Medical Sciences, was conducted. In doing this research, disinfectants such as 4% NaOH, 2% Cetrimide, 3% SDS + 4% NaOH, and CPC with concentrations as 0.01, 0.05, and 0.005% were used. Concentration of 0.01% CPC resulted in the best results.

4 912 Mehdi Roshdi Maleki, Hossein Samadi Kafil et Al In this method and using the 0.01% CPC, not only the degree of contamination of samples was very low, but also the growth of mycobacteria was not prevented so that different types of mycobacteria such as RGM and SGM were isolated. Pseudomonas and Nocardias were the only contaminants of media which were observed only in a low percentage of samples. Discussion Water is one of the important reservoirs of environmental mycobacteria (11). Most environmental mycobacteria, except in a few cases, are opportunistic pathogens which result in a variety of skin and pulmonary diseases, lymphadenitis, soft tissue infection, etc. (3). In recent decades, isolation and identification of these bacteria from different resources such as water have been considered important. Dispersion of these bacteria in different geographical areas have been different and due to the difference in strategies of treatment of diseases caused by them, isolation and identification of the dominant species in each geographical area is necessary. Methods used by different researchers in different parts of the world for isolating environmental mycobacteria have their own advantages and disadvantages. In the present study, all methods presented by different researchers were investigated and those methods enjoying high productivity whether in terms of disinfection or the number of isolated species were introduced. Using the Löwenstein- Jensen medium with ph as about 6.5, the use of incubation temperature with 30 C, the use of the filtration method instead of the centrifuge method, the use of disinfectants with appropriate concentration are effective factors in isolating mycobacteria from environmental samples such as water. By observing the above cases and selecting the 0.01% CPC as an ideal disinfectant, most species of environmental mycobacteria can be isolated. Although many methods are used by different researchers for isolation of EM from water reservoirs, no standard method or protocol has been presented yet. Conclusion The results obtained from the present study as well as the study conducted on different studies and articles conducted on isolation of environmental mycobacteria from water reservoirs indicate that isolation conducted using the disinfectant CPC is better than other disinfectants. The disinfectant CPC, in addition to being effective for reducing the growth of contaminants, causes the significant reduction in the growth of the number of mycobacteria in high concentrations (as 0.05%). The concentration of 0.01% CPC is appropriate for contamination of drinking water and the concentration of 0.04% is appropriate for contamination of surface water. In isolating of environmental mycobacteria from drinking water samples, addition of sodium thiosulfate to water for neutralization of residual chlorine is not necessary and may interfere with mycobacteria growth (12). Environmental mycobacteria, contrary to the set of Mycobacterium tuberculosis which like neutral ph (ph 7), prefer ph about (17). Therefore, it is better that ph of thee Löwenstein- Jensen culture medium be regulated with some acid which is normalized by HCL (18). - Although the optimal temperature of the growth of some of species including M. xenopi is between 40 to 45 C, in general states, incubation in the 30 C is the best result. The filtration method (using membrane filters with a 0.45-µm pore size) have higher sensitivity than the centrifuge method. To isolate mycobacteria from water resources, the performance of the LJ culture medium is better than other media. The application of CPC is facilitated by easy handling and by its nontoxic nature. We recommend a 30-min exposure to 0.01% CPC as the method of choice for decontaminnting drinkin water samples. References 1) Vasconcellos SE, Huard RC, Niemann S, Kremer K, Santos AR, Suffys PN, Ho JL Distinct genotypic profiles of the two major clades of Mycobacterium africanum. BMC Infect Dis 10: 80. 2) Johnson MM, Odell JA Nontuberculous mycobacterial pulmonary infections. J Thorac Dis 6: ) Halstrom S, Price P, Thomson R. Review: Environmental mycobacteria as a cause of human infection. International Journal of Mycobacteriology 4: ) Whiley H, Keegan A, Giglio S, Bentham R Mycobacterium avium complex-the role of potable

5 The introduction of 3+1 important method for the isolation water in disease transmission. J Appl Microbiol 113: ) Feazel LM, Baumgartner LK, Peterson KL, Frank DN, Harris JK, Pace NR Opportunistic pathogens enriched in showerhead biofilms. Proc Natl Acad Sci U S A 106: ) van Ingen J, Boeree MJ, Dekhuijzen PN, van Soolingen D Environmental sources of rapid growing nontuberculous mycobacteria causing disease in humans. Clin Microbiol Infect 15: ) Asgharzadeh M, Kafil HS, Khakpour M Comparison of mycobacterial interspersed repetitive unit-variable number tandem repeat and IS6110-RFLP methods in identifying epidemiological links in patients with tuberculosis in Northwest of Iran. Ann Microbiol 58: ) Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ, Jr., Winthrop K An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 175: ) Collins CH, Grange JM, Yates MD Mycobacteria in water. J Appl Bacteriol 57: ) Le Dantec C, Duguet JP, Montiel A, Dumoutier N, Dubrou S, Vincent V Occurrence of mycobacteria in water treatment lines and in water distribution systems. Appl Environ Microbiol 68: ) Falkinham JO, 3rd Nontuberculous mycobacteria from household plumbing of patients with nontuberculous mycobacteria disease. Emerg Infect Dis 17: ) Thomson R, Carter R, Gilpin C, Coulter C, Hargreaves M Comparison of methods for processing drinking water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare. Appl Environ Microbiol 74: ) Roland S-R, Weber A, Fischeder R Comparison of decontamination methods for the isolation of mycobacteria from drinking water samples. Journal of Microbiological Methods 14: ) Peres RL, Maciel EL, Morais CG, Ribeiro FC, Vinhas SA, Pinheiro C, Dietze R, Johnson JL, Eisenach K, Palaci M Comparison of two concentrations of NALC-NaOH for decontamination of sputum for mycobacterial culture. Int J Tuberc Lung Dis 13: ) Parashar D, Das R, Chauhan DS, Sharma VD, Lavania M, Yadav VS, Chauhan SV, Katoch VM Identification of environmental mycobacteria isolated from Agra, north India by conventional & molecular approaches. Indian J Med Res 129: ) Sebakova H, Kozisek F, Mudra R, Kaustova J, Fiedorova M, Hanslikova D, Nachtmannova H, Kubina J, Vraspir P, Sasek J Incidence of nontuberculous mycobacteria in four hot water systems using various types of disinfection. Can J Microbiol 54: ) Iivanainen E, Martikainen PJ, Katila ML Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters. J Appl Microbiol 82: ) Leylabadlo HE, Kafil HS, Yousefi M, Aghazadeh M, Asgharzadeh M Pulmonary Tuberculosis Diagnosis: Where We Are?. Tuberc Respir Dis 79: Corresponding author REZA MOADDAB Department of Laboratory Sciences, Faculty of Paramedicine, Tabriz University of Medical Sciences Tabriz (Iran)