This research compared the numbers and types of different Mycobacterium species in soil samples taken from 2 areas of Golestan province, Islamic Republic of Iran, 1 with a high prevalence of tuberculosis and 1 with a low prevalence. From 220 samples, 91 grew positive cultures (41.4%) and 161 different strains were diagnosed. The most common species isolated were Mycobacterium fortuitum, M. flavescens and M. chelonae. The frequencies of environmental Mycobacterium in the low-prevalence area were much higher than in the high-prevalence area, perhaps due to different environmental factors

Ghaemi, E.

Ghazisaidi, K.

Khodabakhshi, B.

Koohsari, H.

Mansoorian, A.

English

Golestan University of Medical Sciences Repository

   280 La Revue de Santé de la Méditerranée orientale, Vol. 12, No 3/4, 2006 Environmental mycobacteria in areas of high and low tuberculosis prevalence in the Islamic Republic of Iran E. Ghaemi,1 K. Ghazisaidi,2 H. Koohsari,3 B. Khodabakhshi4 and A. Mansoorian4 ABSTRACT This research compared the numbers and types of different Mycobacterium species in soil samples taken from 2 areas of Golestan province, Islamic Republic of Iran, 1 with a high prevalence of tuberculosis and 1 with a low prevalence. From 220 samples, 91 grew positive cultures (41.4%) and 161 different strains were diagnosed. The most common species isolated were Mycobacterium fortuitum, M. flavescens and M. chelonae. The frequencies of environmental Mycobacterium in the low-prevalence area were much higher than in the high-prevalence area, perhaps due to different environmental fac­tors. Mycobactéries environnementales dans des zones à forte et faible prévalence tuberculeuse en République islamique d’Iran RÉSUMÉ La présente recherche a comparé le nombre et les types des différentes espèces de Myco­bacterium dans des échantillons de sol prélevés dans deux zones de la province de Golestan (Répu­blique islamique d’Iran), l’une ayant une forte prévalence tuberculeuse et l’autre une faible prévalence. Sur les 220 échantillons, 91 ont produit des cultures positives (41,4 %) et 161 souches différentes ont été diagnostiquées. Les espèces les plus couramment isolées étaient Mycobacterium fortuitum, M. flavescens et M. chelonae. La fréquence des mycobactéries environnementales dans la zone à fai­ble prévalence était beaucoup plus élevée que dans la zone à forte prévalence, ce qui tient sans doute à différents facteurs environnementaux. 1Department of Microbiology, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Islamic Republic of Iran (Correspondence to E. Ghaemi: eghaemi@yahoo.com). 2Department of Pathobiology, Health Faculty, University of Tehran, Tehran, Islamic Republic of Iran. 3Department of Microbiology, Lahijan Free University, Islamic Republic of Iran. 4Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Islamic Republic of Iran. Received: 24/03/04; accepted: 05/10/04 ﺔﻠﺠﳌ ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣                           281Eastern Mediterranean Health Journal, Vol. 12, Nos 3/4, 2006 Introduction There are currently 71 recognized or pro­posed species of Mycobacterium [1], all of which, except M. tuberculosis complex and M. leprae, are considered as environmental mycobacteria and can usually be isolatedfrom environmental samples including wa­ter, soil and dust [2]. The different environ­mental mycobacteria are very similar andfor many years were mistakenly assumed to be M. tuberculosis in patients [1,2]. Nowa­days these bacteria are believed to havean important role in infections, allergies,immunity to other pulmonary infectionsand the efﬁcacy of bacille Calmette-Guerin (BCG) vaccination [3]. These facultative pathogens can causeinfection, especially among immunosup­pressed or immunodeﬁcient patients [4].Four types of opportunistic mycobacterial disease of humans have been described:skin lesions (following traumatic inocula­tion of bacteria), localized lymphadenitis,tuberculosis (TB)-like pulmonary lesionsand disseminated disease [5]. However,the relative incidence of the environmental Mycobacterium spp. involved in humandisease varies from region to region and is related to the occurrence of the species in the environment [6,7]. These bacteria areubiquitous, so the nature, route and dose of exposure to environmental saprophytes are variables that depend on where and how an individual lives. Another reason for the medical impor­tance of environmental Mycobacteriumis the potential impact on the immune re­sponse. Exposure to environmental myco­bacteria has been suggested to inﬂuenceimmune responsiveness to the pathogenicmycobacteria and to sensitize individuals to skin test reagents; it can also affect the ef­ﬁcacy of BCG vaccination [3,7–9]. Contact with different species of environmental My­cobacterium can cause acquired immunity to M. tuberculosis or increase the efﬁcacy of BCG vaccine protection (M. vaccae, M. microti), although some species of thesebacteria reduce the efﬁcacy of BCG vaccine (M. scrofulaceum) [8,10–13]. Golestan province, which is located in north of the Islamic Republic of Iran, near the Caspian Sea, has one of the highest rates of incidence of TB in the country (45.5 per 100 000). However, the distribution of TB is not homogenous and the incidence ishigher in the western than the eastern part of the province: mean incidences are 54.6 to 28.1 per 100 000 respectively (provincial health centre, personal communication).Our objective in this research was tocompare the number and types of different species of Mycobacterium in the soil from these 2 areas of the province. Methods The study took place in 2001–02. During the summer of 2001, a total of 220 soil samples were taken from humid and wet mud from a depth of 3–5 cm; 120 samples from 20 loca­tions in an area of high TB prevalence in the western part of Golestan province and the remainder from 24 locations in an area of low TB prevalence in the eastern part of the province. The locations, mostly agricultural areas, were chosen randomly and 5 samples were taken from each. Samples were taken from upper layers of soil, at a depth of 5 cm, and placed in separate sterile glass tubes. Samples were transferred directly to the MicrobiologyLaboratory of Golestan University of Medi­cal Sciences. A suspension was made using 1 g of soil in 10 mL of sterile distilled water containing 2–3 drops of Tween 80 [14].The prepared suspensions were shaken for 30 min, left for undisturbed for 30 min,ﺔﻠﺠﳌ ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣                      282 La Revue de Santé de la Méditerranée orientale, Vol. 12, No 3/4, 2006then 3 mL of supernatant was transferred to another test tube and 3 mL of solution (11.7 g/L NaOH and 5% NaHCl, ratio 6:1) added to decontaminate the samples. The testtubes were centrifuged for 20 min at 2000 rpm, then the supernatant was decanted and a few drops of sterile distilled water were added to the sediment to neutralize the sam­ple, 200 μL of sample were inoculated inLowenstein–Jensen media and growthswere assessed every 2–3 days for a period of 1 month. The isolated colonies were as­sessed by growth rate, morphology of colo­ny, pigmentation in dark and light, and the results of a battery of standard biochemical tests: catalase production test at 25 ºC and 68 ºC, growth on MacConkey agar plate,NaCL tolerance, niacin production test, tel­lurite and nitrate reduction test, urease test and Tween 80 hydrolysis [6,15]. Results From 220 soil samples, environmental my­cobacteria were isolated from 91 samples(41.4%). Overall 161 strains of Mycobac­terium belonging to 12 species were iso­lated. The isolation rate of environmentalmycobacteria in the high-prevalence areawas 20.8% (25 samples) but in the low-prevalence area was 66.0% (66 samples).This difference was statistically signiﬁcant (P < 0.05) (Table 1). In the high-prevalence area, 47 strains of environmental mycobacteria were isolatedfrom 25 positive samples and M. fortuitum,M. ﬂavescens and M. chelonae were themost common species. In 9 samples2–5 strains were isolated. All the isolated Mycobacterium from this area belonged to 8 species (Table 2). In the low-prevalence area, 114 strains belonging to 11 species were isolated from 66 positive soil cultures, and M. ﬂavescens, M. chelonae and M. fortuitum were moreprevalent than other strains (23, 21 and 19 cases, respectively). M. phlei were isolated from 14 soilsamples in the low-prevalence area butwere not isolated from the high-prevalence area. M. terrae, M. gordonae, M. kansasiiwere also isolated in limited numbers from the low-prevalence but not from the high-prevalence area. M. gastri was only isolated from the high-prevalence area (Table 2). In 35 samples 2–6 strains were isolated. Discussion In this study in Golestan province 41.4%of soil samples contained environmentalmycobacteria. This ﬁgure is similar to the results from other provinces in the north of the Islamic Republic of Iran bordering the Caspian Sea [16–18], presumably due to the  Table 1 Partial distribution of positive soil culture of environmental Mycobacterium spp. in a Caspian Sea province in areas of high and low tuberculosis prevalence    Variable High- Low- Total  prevalence area prevalence area   No. % No. % No. % Samples taken 120  100  220 Positive cultures 25 20.8 66 66.0 91 41.4 Isolated species 8  11  12 ﺔﻠﺠﳌ ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣                                   Table 2 Partial distribution of different environmental Mycobacterium spp.isolated from soil in a Caspian Sea province in areas of high and low tuberculosis prevalence   Species High- Low- Totalprevalence area prevalence area No. % No. % No. % M. fortuitum  16 34.0 19 16.6 35 21.8M. flavescens 10 21.2 23 20.1 33 20.5M. chelonae 6 12.8 21 18.4 27 16.8M. thermoresistibile 5 10.6 15 13.1 20 12.4M. phlei – – 14 12.3 14 8.7M. triviale  4 8.5 6 5.3 10 6.2M. terrae – – 8 7.1 8 4.9M. gordonae – – 4 3.5 4 2.5M. fallax 2 4.3 2 1.8 4 2.8M. gastri 3 6.4 – – 2 1.9M. marinum 1 2.1 1 0.9 2 1.2M. kansasii – – 1 0.9 1 0.6Total 47 100.0 114 100.0 161 100.0     283Eastern Mediterranean Health Journal, Vol. 12, Nos 3/4, 2006           similarity in climate (Mediterranean-type), humidity and plant ﬂora in these areas. In the present study, the isolation rateof Mycobacterium spp. in the area of high TB prevalence (20.8%) was signiﬁcantlylower than in the area of low TB prevalence (66.0%). Environmental factors are themost likely explanation for this differ­ence (high-prevalence areas are warmer,dryer, further from the sea with less herbal coverage than low-prevalence areas). Itsuggests, however, that people living inlow-prevalence areas have more contactwith environmental Mycobacterium strains than residents of high-prevalence areas. The most prevalent species isolatedfrom the soil in this study was M. fortuitumcomplex. This is similar to Kampala’s study from south India [19]. Some of the isolated environmental Mycobacterium, such asM. marinum, M. kansasii and some rapid-growing species such as M. chelonae and M. fortuitum, can be pathogenic for humans [1,6]. They are almost always acquireddirectly from the environment. A majorfactor determining the occurrence of infec­tions caused by the various species is their distribution in the environment, especially in water pipes [6]. As for the high preva­lence of 2 species of M. fortuitum and M. chelonae in this region, physicians should be aware of opportunistic Mycobacteriumwhen encountering patients with pulmonary and soft tissue diseases [5,6]. Geographical variation in the efﬁcacyof Mycobacterium bovis BCG vaccinationagainst TB is well recognized [12]. Dif­ferent studies in human populations andexperimental studies with animals showthat infection with some environmentalmycobacteria can offer a level of protection against TB similar to that offered by BCG and that, depending on the nature of theenvironmental mycobacteria and the tim­ﺔﻠﺠﳌ ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣                                                                      284 La Revue de Santé de la Méditerranée orientale, Vol. 12, No 3/4, 2006ing, this exposure can also enhance, mask or interfere with the effect of subsequentBCG vaccination. Thus, regional differ­ences in environmental mycobacteria ﬂora along with several other mechanisms could be responsible for the widely varying results of BCG trials or could inﬂuence the course of subsequent infection with virulent TBbacilli [6,11,13,19]. A study in Karongadis­trict in Northern Malawi (a region in which BCG vaccination has no effect againstpulmonary TB but provides over 50%protection against leprosy) demonstratedthat prior sensitization with environmental mycobacteria can inhibit BCG multiplica­tion and thereby prevent the induction of an efﬁcient BCG-mediated immune responseand protection against TB challenge [8].Geographic gradients of leprosy prevalence are reported in Karonga District, with thedisease more common in the north than in the south, a pattern that may be related to protective mycobacterial exposure in thelow-incidence area [9]. The data presented in the above studies provide strong evidences that exposure tovarious environmental mycobacteria can inﬂuence the immune response to BCGvaccination and subsequent prevalence ofTB [9]. However, there have been few stud­ies on the effect of repeated environmental mycobacteria contact on those individuals whom received earlier BCG vaccine (ininfancy) and in its efﬁcacy on inhibitionof TB. BCG vaccination is given to all infants at the time of birth in Golestan province,and the prevalence of environmental my­cobacteria is higher in locations of highTB incidence than locations of low TBincidence. This raises the question whether these mycobacteria can act as booster dose of BCG vaccine to continually stimulate the immune system and keep it as active form in case of contact with M. tuberculosis. Further studies are required to answer this question. References 1.	 Collier L, Balows A, Sussman M, eds. Topley and Wilson’s microbiology and mi­crobial infections, 9th ed. London, Edward Arnold, 1998. 2.	 Zyazarifi A. Biology and bacteriology ofmycobacteria. Tehran, Aboryhan Press,1987:10–5 [in Farsi]. 3.	 Primm TP, Lucero CA, Falkinham JO.Health impacts of environmental myco­bacteria. Clinical microbiology reviews, 2004, 17(1):98–106. 4.	 Covert TC et al. Occurrence of nontu­berculous mycobacteria in environmen­tal sample. Applied and environmentalmicrobiology, 1999, 65:2492–6. 5.	 Wallace RJ et al. Spectrum of diseasedue to rapidly growing mycobacteria.Reviews of infectious diseases, 1983, 5: 657–79. 6. 	 Murray PR et al. Manual of clinical microbi­ology, 7th ed. Washington DC, American Society for Microbiology Press, 1999. 7.	 Howard CJ. Exposure to Mycobacteri­um avium primes the immune system of calves for vaccination with Mycobacteri­um bovis BCG. Clinical and experimental immunology, 2002, 130:190–5. 8.	 Brandt L et al. Failure of the Mycobacte­rium bovis BCG vaccine: some species of environmental mycobacteria block multi­plication of BCG and induction of protec­tive immunity to tuberculosis. Infectionand immunity, 2002, 70(2):672–8. 9. 	 Black GF et al. Patterns and implications of naturally acquired immune responses to environmental and tuberculous mycobac­terial antigens in northern Malawi. Journal of infectious diseases, 2001, 184:322–9. ﺔﻠﺠﳌ 	ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣           285Eastern Mediterranean Health Journal, Vol. 12, Nos 3/4, 2006 10. Stanford JL, Shield MJ, Rook GA. How environmental mycobacteria may prede­termine the protective efficacy of BCG. Tubercle, 1981, 62:55–62. 11. Palmer CE, Long MW. Effects of infection with atypical mycobacteria on BCG vacci­nation and tuberculosis. American review of respiratory disease, 1966, 94:553–68. 12. Fine PE. Variation in protection by BCG: implications of and for heterologous im­munity. Lancet, 1995, 346:1339–45. 13. Lozes E et al. Cross-reactive immune responses against Mycobacterium bovis BCG in mice infected with non-tuberculous mycobacteria belonging to the MAIS-group. Scandinavian journal of immunology, 1997, 46(1):16–26. 14. Donoghue HD, Overend E, Stanford JL. A longitudinal study of environmental mycobacteria on a farm in South-West England. Journal of applied microbiology, 1997, 82:57–67. 15. Isenberg HD. 	Essential procedures forclinical microbiology, 1st ed. Washington DC, American Society for MicrobiologyPress, 1998:187–97. 16. Bashiri AR. Environmental mycobacte­ria isolation from soil in Gilan Province[thesis]. Tehran, Islamic Republic of Iran, Tehran University, 1998:69–75 [in Farsi]. 17. Rastegar M. Environmental mycobacteria of soil in Mazenderan Province [thesis].Tehran, Islamic Republic of Iran, Tehran University, 1992:85–91 [in Farsi]. 18. Mohammadi M. Environmental mycobac­teria in fish pools in Gilan and Mazendran Provinces of Iran [thesis]. Tehran, Is­lamic Republic of Iran, Tehran University, 1994:78–89 [in Farsi]. 19.	 Kamala T et al. Isolation and identifica­tion of environmental mycobacteria in the Mycobacterium bovis BCG trial area ofSouth India. Applied and environmentalmicrobiology, 1994, 60(6):2180–3. ﺔﻠﺠﳌ 	ﺮﺸﻟ ﺔﻴﺤﺼﻟ ﺔﻤﻈﻨﻣ ،ﻂﺳﻮﺘﳌ ﺔﺤﺼﻟ ،ﺔﻴﳌﺎﻌﻟ ﺪﻠﺠﳌ ﺎﺜﻟ ،ﺮﺸﻋ ﺪﻌﻟ ٢٠٠٦ ،٤-٣ View publication stats 

Environmental mycobacteria in areas of high and low tuberculosis prevalence in the Islamic Republic of Iran

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Environmental mycobacteria in areas of high and low tuberculosis prevalence in the Islamic Republic of Iran

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