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Int J Env Health Eng 2015,  4:20

Phenotypic characterization of Nocardia spp. isolated from Iran soil microflora

1 Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Bacteriology and Virology, School of Medicine, Alborz University of medical Sciences, Karaj, Iran

Date of Web Publication08-Jun-2015

Correspondence Address:
Seyyed Saeed Eshraghi
Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Qods St., Poursina St., Tehran
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Source of Support: Tehran University of Medical Sciences, Conflict of Interest: None

DOI: 10.4103/2277-9183.158388

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Aims: The present study was conducted to identify Nocardia spp. from Iran soil by various phenotypic tests.
Materials and Methods: A total of 300 soil samples were collected of five different geographical regions in Iran. Nocardia isolation was performed by paraffin baiting technique. The colonies that were similar to be Nocardia spp. were stained with Gram, partially acid fast and acid-fast. Phenotypic tests were used for identification of Nocardia spp.
Results: After analysis of phenotypic tests, the identified species are as follow: Nocardia asteroides (49.12%), Nocardia cyriacigeorgica (24.56%), Nocardia otitidiscaviarum (38.6%), N. asteroides complex (19.29%), Nocardia africana (3.5%), and Nocardia pseudobrasiliensis, Nocardia coubleae, Nocardia ignorata (each species; 1.75%). In this study, some of isolates (8.77%) remained undetected.
Conclusion: Due to great number of Nocardia spp., high similarities among biochemical characteristics of species and the variability of some these characters, a wide range of biochemical tests should be used to identify Nocardia spp. to gain more accurate results.

Keywords: Iran, Nocardia, paraffin baiting technique, phenotypic tests

How to cite this article:
Habibnia S, Nasab MR, Heidarieh P, Bafghi MF, Pourmand MR, Eshraghi SS. Phenotypic characterization of Nocardia spp. isolated from Iran soil microflora. Int J Env Health Eng 2015;4:20

How to cite this URL:
Habibnia S, Nasab MR, Heidarieh P, Bafghi MF, Pourmand MR, Eshraghi SS. Phenotypic characterization of Nocardia spp. isolated from Iran soil microflora. Int J Env Health Eng [serial online] 2015 [cited 2021 Apr 17];4:20. Available from:

  Introduction Top

Nocardia are filamentous Gram-positive bacilli, aerobic, and partially acid-fast. The genus Nocardia is caused opportunity infections in immunosuppressive disorder, AIDS, cancer, diabetes and organ transplantation. They cause infections in the respiratory and extra respiratory tract, skin and brain. Pulmonary nocardiosis frequently is acquired through inhalation of aerosols containing the bacteria. [1],[2],[3] Nocardia spp. is soil saprophytic that can play an important role in the turnover of soil organic materials. This bacterium is significant organism in industries, agricultural, and biotechnology. In recent years, classification of the genus Nocardia is changed. Until date, 103 species of bacteria have been identified which some of them have been known as Nocardia asteroides complex and Nocardia nova complex. [4]

Identification of Nocardia spp. from each region is very important because the distribution of these species due to climatic variations is different in each region. A few literatures have been reported identification of Nocardia spp. in Iran soil. [5],[6] The aim of this study is significant for two reasons: First, the investigation of Nocardia recognition in different geographical regions is important; second, the utilization of various phenotypic tests can help us to identify more accurate Nocardia spp. This study was not to evaluate the epidemiology, but isolation and evaluation of Nocardia spp. from all regions in Iran. [5] According to the environmental condition, each region has special species of Nocardia, and, on the other hand, treatment is different for each species. Therefore, identification species any area is important to follow-up treatment. [7]

There are several methods to isolate Nocardia from soil such as paraffin baiting method, humic acid vitamin B agar and paraffin agar and sucrose gradient centrifugation. Based on recent studies, paraffin baiting technique is more effective than other methods for Nocardia isolation of soil because it is cost-effective, and also this method prevents the growth of other organisms. [2],[8],[9],[10],[11] Hence, we investigated Nocardia spp. in Iran soil by various phenotypic tests.

  Materials and Methods Top

Soil samples were randomly collected from 4 cm deep within 15 months (from March 2011 until July 2013). In this study, 300 samples were collected from various soils from five geographical regions (North, West, East, South, and central) of Iran [Figure 1]. The samples were collected in sterile plates and were transferred to actinomycetes lab (Tehran University of Medical Sciences, School of Public Health) within 24-48 h.
Figure 1: Nocardia isolates of geographical regions from Iran

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Isolation method

In this study, isolation of Nocardia performed by paraffin baiting technique. A total of 1 g of soil mixed into a sterile tube in 10 ml of distilled water and shacked for 5 min. About 1 ml of the supernatant is added to the carbon-free broth tube with paraffin sticks and incubated at 35°C for 20-30 days (every 3 days, the paraffin sticks were examined). The white colonies similar to Nocardia on paraffin stick were cultured on nutrient agar and sabouraud dextrose agar (contain cycloheximide, Sigma-Aldrich, USA) for purification. [12]

Phenotypic tests

The colonies that suspected to Nocardia spp. were stained with Gram, partially acid-fast and acid-fast. Lysozyme broth was used for the identification of the genus Nocardia. Colonies were investigated with a stereo microscope. Nocardia spp. identification was according to biochemical tests that are including: Growth in lysozyme broth, hydrolysis of amino acids such as hypoxanthine, xanthine, tyrosine, casein, gelatin and urea, production of nitrate reductase (Sigma-Aldrich, USA), Growth at 45°C, producing acid from carbohydrates such as glucose, maltose, lactose, galactose, salicine, xylose, raffinose, arabinose, rhamnose, sorbitol, and sucrose (Merck, Germany). [1]

  Results Top

Among 300 soil samples cultured with paraffin baiting method, 65 isolates were suspected to be similar colonies to Nocardia spp. (white, cream-colored and chalky) (21.66%). Colonies stained and eight isolates eliminated because they were contaminated by fungal and Bacillus spp. 57 isolates (19%) were identified with initial tests. Colonies were positive with Gram and partially acid-fast and they were negative for acid-fast. The genus Nocardia was confirmed by the growth in lysozyme broth. Then, various biochemical tests were performed for these strains of Nocardia [Figure 2], [Figure 3] and [Figure 4]. The analysis of phenotypic tests were identified that 19 isolates of N. asteroides (49.12%), 14 isolates of Nocardia cyriacigeorgica (24.56%), 3 isolates of Nocardia otitidiscaviarum (38.6%), 11 isolates of N. asteroides complex (19.29%), 2 isolates of Nocardia africana (3.5%), 1 isolate of Nocardia pseudobrasiliensis, Nocardia coubleae, and Nocardia ignorata (each species; 1.75%). In our study, five isolates remained (8.77%) undetected. Most isolates belonged to the N. asteroides complex [Table 1].
Figure 2: (a) Hydrolysis of casein; (b) hypoxanthine; (c) tyrosine

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Figure 3: (a) Production acid of carbohydrates; (b) urea hydrolysis; (c) nitrate reduction; (d) Utilization of citrate

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Figure 4: (a) Hydrolysis of gelatine, (b) growth at 45°C

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Table 1: Physiological characteristics of Nocardia isolates

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  Discussion Top

Nocardia is soil saprophytes that widespread all over the world. Thus, it can potentially transmitted to human with inhalation in contaminated soil or skin injury. Nocardia isolation can vary in different geographical areas based on soil type. [12] We recognized of Nocardia in different regions by various phenotypic tests.

Aghamirian and Ghiasian in 2006-2007, studied 300 soil samples of different areas of Qazvin, Iran. Samples were cultured on brain-heart infusion agar and Sabouraud's dextrose agar contains cycloheximide. Overall, were isolated N. asteroides (15.7%), N. otitidiscaviarum (9.4%), and Nocardia brasiliensis (7.3%) that were cultured on brain-heart infusion agar and Sabouraud's dextrose agar contain cycloheximide. [5] Kachuei et al. isolated Nocardia spp. of soil in different regions from Isfahan province in the center of Iran. Identification of species was done with kanamycin and conventional biochemical tests such as tyrosine, casein, hypoxanthine, xanthine, starch and gelatin. From 153 isolates, N. asteroides complex (45.5%), Nocardia brasiliensis (24.7%), N. otitidiscaviarum (2.2%), Nocardia transvalensis (1.1%), Nocardiopsis dassonvillei, Actinomadura madura (each one 1.7%) were identified. Also 23.0% of total remained undetected. [6] Another study by Wauters et al. from clinical samples with phenotypic methods in 2005 reported Nocardia farcinica 44%, Nocardia nova 22%, and N. cyriacigeorgica 15%. [13]

According to the results obtained in this study using a range of phenotypic tests, more species of Nocardia was identified. In addition to identification of N. asteroides, another species, for example, N. africana, N. coubleae, N. ignorata, and Nocardia psudobrasiliansis were determined. Wide range of biochemical tests for identification of Nocardia spp. from clinical samples were used. This investigate is the first study were gathered soil samples in different climatic regions of Iran to isolation species of Nocardia. Most of Nocardia spp. were isolated from north of Iran [Figure 5]. In the present study, the highest percentage of Nocardia spp. were isolated from forest and garden zones. Also, percent of Nocardia isolates in different areas is: Gardens (15.78%), cultivated lands (35%), parks (20%), forest (31.42%), desert regions (15.38%), green spaces and boulevards (14.28%), greenhouse (17.24%), and sludge (7.69%). The positive samples were not isolated from coastal areas [Table 2]. Therefore, probably conditions for the growth of Nocardia are favorable due to the presence of organic matter and nutrients in different soil, suitable moisture, and pH.
Figure 5: Percentage of Nocardia isolates in Iran soil

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Table 2: Rate of Nocardia isolated from different regions of Iran

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Due to the increasing number of new species in the genus Nocardia and limited biochemical tests so cannot identify precisely and reliably. The use of a wide range of biochemical tests can be helpful in more accurate identification of species. In the present study, we used various phenotypic tests to identify of Nocardia spp.

  Conclusion Top

Due to great number of Nocardia spp., high similarities among biochemical characteristics of species and the variability of some of these characteristics, a wide range of biochemical tests should be used to identify Nocardia spp. to gain more accurate results. We recommend usage of molecular methods such as polymerase chain reaction (PCR) sequencing and PCR-restriction fragment length polymorphism to confirm phenotypic tests results. [14],[15],[16]

  Acknowledgment Top

This study was supported by Tehran University of Medical Sciences, Deputy of Research.[18]

  References Top

Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ Jr. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev 2006;19:259-82.  Back to cited text no. 1
Shawar RM, Moore DG, LaRocco MT. Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens. J Clin Microbiol 1990;28:508-12.  Back to cited text no. 2
Bafghi MF, Eshraghi SS, Heidarieh P, Habibnia S, Rasouli Nasab MR. Nocardiosis in immune disorder disease. Malays J Med Sci 2014;21:75-6.  Back to cited text no. 3
Available from:   Back to cited text no. 4
Aghamirian MR, Ghiasian SA. Isolation and characterization of medically important aerobic actinomycetes in soil of Iran (2006-2007). Open Microbiol J 2009;3:53-7.  Back to cited text no. 5
Kachuei R, Emami M, Mirnejad R, Khoobdel M. Diversity and frequency of Nocardia spp. in the soil of Isfahan province, Iran. Asian Pac J Trop Biomed 2012;2:474-8.  Back to cited text no. 6
Rodríguez-Nava V, Khan ZU, Pötter G, Kroppenstedt RM, Boiron P, Laurent F. Nocardia coubleae sp. nov. isolated from oil-contaminated Kuwaiti soil. Int J Syst Evol Microbiol 2007;57:1482-6.  Back to cited text no. 7
Khan ZU, Neil L, Chandy R, Chugh TD, Al-Sayer H, Provost F, et al. Nocardia asteroides in the soil of Kuwait. Mycopathologia 1997;137:159-63.  Back to cited text no. 8
Singh M, Sandhu RS, Randhawa HS. Comparison of paraffin baiting and conventional culture techniques for isolation of Nocardia asteroides from sputum. J Clin Microbiol 1987;25:176-7.  Back to cited text no. 9
Hayakawa M, Nonomura H. Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. J Ferment Technol 1987;65:501-9.  Back to cited text no. 10
Yamamura H, Hayakawa M, Iimura Y. Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil. J Appl Microbiol 2003;95:677-85.  Back to cited text no. 11
Khan Z, Al-Sayer H, Chugh TD, Chandy R, Provost F, Boiron P. Antimicrobial susceptibility profile of soil isolates of Nocardia asteroides from Kuwait. Clin Microbiol Infect 2000;6:94-8.  Back to cited text no. 12
Wauters G, Avesani V, Charlier J, Janssens M, Vaneechoutte M, Delmée M. Distribution of Nocardia species in clinical samples and their routine rapid identification in the laboratory. J Clin Microbiol 2005;43:2624-8.  Back to cited text no. 13
Rodríguez-Nava V, Couble A, Devulder G, Flandrois JP, Boiron P, Laurent F. Use of PCR-restriction enzyme pattern analysis and sequencing database for hsp65 gene-based identification of Nocardia species. J Clin Microbiol 2006;44:536-46.  Back to cited text no. 14
Conville PS, Fischer SH, Cartwright CP, Witebsky FG. Identification of Nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene. J Clin Microbiol 2000;38:158-64.  Back to cited text no. 15
Bafghi MF, Heidarieh P, Habibnia S, Rasouli Nasab M, Neyestanaki DK, Afshar D, et al. Phenotypic and molecular properties of the Nocardia species. Avecinna J Clin Microbiol Infect 2014;1:e19215.  Back to cited text no. 16
Yassin A, Rainey F, Burghardt J, Brzezinka H, Mauch M, Schaal K. Nocardia paucivorans sp. nov. International journal of systematic and evolutionary microbiology. 2000;50:803-9.  Back to cited text no. 17
Kiska DL, Hicks K, Pettit DJ. Identification of medically relevant Nocardia species with an abbreviated battery of tests. Journal of clinical microbiology. 2002 Apr;40:1346-51.  Back to cited text no. 18


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]

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