Annals of Tropical Medicine and Public Health
Home About us Ahead Of Print Instructions Submission Subscribe Advertise Contact e-Alerts Editorial Board Login 
Users Online:138
  Print this page  Email this page Small font sizeDefault font sizeIncrease font size
 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 6  |  Issue : 2  |  Page : 211-214
Isolation and molecular identification of actinomycetes from mycetoma patients in Sudan


1 Department of Microbiology, College of Medical Laboratory Science, Sudan University of Science and Technology, Khartoum, Sudan
2 Department of Microbiology, Soba University Hospital, Khartoum, Sudan
3 Department of Clinical Microbiology and Parasitology, College of Medicine, King Khalid University, Abha, Saudi Arabia

Click here for correspondence address and email

Date of Web Publication14-Aug-2013
 

   Abstract 

Introduction: In order to minimize chance of amputation due to actinomycetoma, it is important to correctly identify the causative agents. Microscopic examination of grains is not definite and further confirmatory diagnostic tests are needed. This study aims to investigate the prevalence of actinomycetoma and to explore the usefulness of strb1 gene in the diagnosis of the disease. Materials & Methods: The present study is a prospective cross-section laboratory-based study in which clinical samples (n = 100) from patients with mycetoma lesions were collected. The samples were cultured on Lowenstein Jensen and glucose yeast extract agar media. Grown colonies were initially identified using Gram's stain, Ziehl Neelsen stain, and selected biochemical reactions. Confirmation was done by the analysis of polymerase chain reaction amplified strb1 gene. Results: Actinomycetoma was represented by a high ratio (12%) among the study population. Nine out of the 12 isolates (75%) were found to belong to the genus Streptomyces; whereas three isolates (25%) were identified as Nocardia spp. on the basis of phenotypic and mycolic acid contents. Conclusion: It could be concluded that actinomycetoma exists with significant prevalence (12%) among patients investigated in the present study. Streptomyces is the most important etiological agent of actinomycetoma compared to Nocardia.

Keywords: Actinomycetoma, mycolic acids, Nocardia, strb1 gene, Streptomyces, Sudan

How to cite this article:
Elhassan MM, Yousif AM, Elmekki MA, Hamid ME. Isolation and molecular identification of actinomycetes from mycetoma patients in Sudan. Ann Trop Med Public Health 2013;6:211-4

How to cite this URL:
Elhassan MM, Yousif AM, Elmekki MA, Hamid ME. Isolation and molecular identification of actinomycetes from mycetoma patients in Sudan. Ann Trop Med Public Health [serial online] 2013 [cited 2020 Apr 4];6:211-4. Available from: http://www.atmph.org/text.asp?2013/6/2/211/116523

   Introduction Top


Mycetoma is a chronic specific granulomatous subcutaneous inflammatory disease that is produced by various causative agents that can be true fungi or aerobic actinomycetes of the genera Actinomadura, Nocardia, and Streptomyces. Actinomadura spp. commonly occurs in some countries and can produce as much as 50% of the total cases of mycetoma. [1]

Actinomycetoma is one of the most neglected diseases and is considered a major health problem in several parts of Sudan. [2],[3],[4] Geographical distribution of mycetoma causative agents show considerable variations which could be explained by several environmental factors, specially the rainfall. [1],[5] The major agents of aerobic actinomycetes are: Nocardia brasiliensis, Actinomadura madurae, and Streptomyces somaliensis. [6],[7] Streptomyces sudanensis has recently being described as one of the etiological agents of actinomycetoma. [8] Many isolates belong to the genus Streptomyces were described to cause actinomycetoma in Sudan and were found to have variable antibiotic profiles which make in vivo treatment very difficult. [9]

People at risk are mainly males and farmers in rural areas due to their nature of contact with microorganism, which lives as saprophyte in soil and other natural ecological niches. Mycetoma is not considered to be transmissible from person to person or from animal to person. There are some conflicting reports about the role of the immune status of susceptible population. Some investigators reported partial impairment of the cell-mediated immune response in patients severely infected or not responding to medical treatment. [10] The infection progresses slowly over a long period of time without painful manifestation and ultimately may extend to deep tissues and bones leading to deformity of the affected site and disability. [2],[11]

It is important to identify the causative agent in order to develop a correct plan of treatment. Direct examination of grains may be useful in determining the type of mycetoma. Unlike fungal grains, crushed bacterial grain show fine filament, which can be stained according to Gram's. Isolation of mycetoma agents can be difficult, especially in case of actinomycetoma. Culture required deep surgical biopsy containing grain. Biopsies without grains are not suitable for diagnosis. [12]

It is important to identify the causative agents of actinomycetoma and to conduct regular hospital-associated surveillance of actinomycetoma infections. Also in vitro sensitivity testing of the causal agents is needed to prompt the treatment alone or in conjunction with surgery to reduce chance of amputation and prolong patient hospitalization due to actinomycetoma. This study aims to investigate the prevalence of actinomycetoma and to explore the usefulness of strb1 gene in the diagnosis of the disease.


   Materials & Methods Top


Ethical clearance

The ethical approval of this study was obtained from the Ethical Committee of Federal Ministry of Health and the Ethical Committee of Research Council of the Sudan University of Science and Technology. Written consent was obtained from every patient before being enrolled in the study.

Study design

The study was a cross-sectional study. Clinical specimens (n = 100) including grains from sinuses discharges and incision biopsies were collected. Mycetoma patients attending Mycetoma Research Center, Soba Teaching Hospital, Khartoum; Wad Madeni Teaching Hospital, and Um Rawaba Hospital from November 2009 to December 2010 were included in this study.

Grains were taken from sinuses (lesions) and washed several times with sterile normal saline, and were then inoculated onto Lowenstein Jensen (LJ) slopes and glucose yeast extract agar (GYEA) media. Plates were incubated at 37°C and examined after 7, 14, and 21 days for the presence of microbial growth.

Identification of isolates

Typical and well-isolated actinomycetes-like colonies from the primary cultures were purified by sub-culturing on LJ, trypticase soya agar (TSA), and GYEA. Isolates were identifies on the basis of colony morphology and confirmed using selected phenotypic and mycolic acids analysis.

Phenotypic characterization

Isolates were tentatively identified as member of the genus Streptomyces on the basis of selected phenotypic criteria. [13] The cultural and microscopic features of the genus Streptomyces are: aerobic growth, Gram-positive, non-acid-alcohol-fast, non-motile actinomycete which forms extensively branched, light-yellow substrate mycelia on a variety of media with or without aerial hyphae, with or without diffusible pigments on the media. In the present study, phenotypic clusters of isolates were identified mainly on colony color and presence of diffusible pigments.

Mycolic acid profiles

The extraction of mycolic acids and thin layer chromatography (TLC) analysis of extracted mycolates was done as described by previously. [14]

Amplification of strb1 gene

DNA extraction from cultures was done by the standard phenol chloroform method according to Chomczynski and Sacchi. [15] Amplification of strb1 region (490 bp) [16] was performed with a set of primers having the following sequence: Forward primer: (5-TGAGCCTTGTAAGCGTCCAC-3) and reverse primer: (3-TTCATGCCGTGCTTCTCCAG-5). Master mix was prepared for 10 reactions according to Huddleston et al. [16] S. somaliensis DSM 41607 isolate was used as positive control. The negative control contained a reaction mixture without template DNA.

PCR program was conducted with an initial 10 min denaturation step at 95°C for 1 cycle, followed by repeating cycles of denaturation (94°C for 30 s), annealing (30 s at 55°C), and extension (30 s at 72°C) for 40 cycle, followed by 5 min of final extension step at 72°C.

The PCR products were electrophoresed on 1.5% agarose gel, containing ethidium bromide (10 μg/ml) to ensure that a fragment of the correct size had been amplified.


   Results Top


Demographic data

In the present study, 100 patients who were presented with signs of mycetoma were enrolled. The majority of the patients were males (73%), compare with females (17%). Patients were categorized into three age groups: Group 1 (<20 years; 17%), group 2 (21-49 years; 57%), and group 3 (>50 years; 26%). The majority of the patients were farmers (64%) followed by pastoralists (23%), students (4%), and people with other minor jobs (13%).

Phenotypic properties

Eighty eight of the samples were tentatively identified as eumycetoma (88%) depending on the texture of the grains (course and black grains). The rest of the isolates (n = 12) appeared with different coloration and were tentatively classified as actinomycetoma. Colony morphology and different biochemical tests were used to identify these isolates. Nine out of the 12 actinomycete isolates (75%) showed typical appearance of Streptomyces spp.

The results confirmed that three of the 12 isolates fit in the genus Nocardia given their phenotypic properties after 3-4 days aerobic growth on GYEA medium at 37°C. The distinguishing features are: formation of glabrous, rough, adherent, waxy, or dry chalky colonies. Colony colors range from cream-yellow, orange to gray-white with or without diffusible brown back pigments [Figure 1]. Microscopically they were characterized by the formation of branching filamentous gram positive bacteria which can fragments into short rods and coccobacillary forms.
Figure 1: Colony morphology features of Nocardia spp. isolated from cases of actinomycetoma exhibiting glabrous, rough, adherent, waxy or dry chalky colonies ranging in color from cream-yellow, orange to gray-white

Click here to view


Thin layer chromatography analysis

TLC analysis of mycolic acids extracted from the three suspected Nocardia spp. revealed characteristic nocardo-mycolates with Rf values equivalent to references Nocardia species [Figure 2].
Figure 2: Thin layer chromatography analysis of mycolic acids extracted from three suspected Nocardia spp. (lanes 1, 2, and 3) isolated from cases of actinomycetoma. Note the characteristic nocardo-mycolates (M) with Rf values equivalent to references Nocardia africana DSM 44502 (lane +ve) in comparison to a negative control Staphylococcus aureus (lane −ve)

Click here to view


PCR analysis

Seven of the nine Streptomyces spp. showed 490 bp bands equivalent to strb1 region sequence [Figure 3]. The remaining two strains were identified on the basis of phenotypic characters.
Figure 3: 1.5% Agarose gel electrophoresis of PCR amplified 490 bp amplicon of strb1 gene of some actinomycetoma isolates. Abbreviations: Lane 1 - Molecular weight marker (250 bp), Lanes 2, 3, 4, 5, and 6 - Samples showing positive results (490 bp), Lane 7 - Positive control, Lane 8 - Negative control

Click here to view



   Discussion Top


The present study substantiates previous studies that actinomycetoma in Sudan can be caused by at least by two types of actinomycetes: Streptomyces spp. and Nocardia spp. The prevalence of these organisms was found to be 12%. The phenotypic and genotypic data show that 75% of these organisms were Streptomyces spp., whereas, 25% were Nocardia spp. Occurrence of Streptomyces among actinomycetoma patients in Sudan has long being recognized. [2],[3],[4],[8] But to our knowledge this is the first report to isolate Nocardia spp. from actinomycetoma patients in Sudan.

Three strains isolated in the present study showed distinct phenotypic profiles in accordance with those of the genus Nocardia. Further taxonomic studies are needed to clarify the taxonomic position and fully identify these isolates. The results of the present study underline the importance of isolating and characterizing the causal agents of actinomycetoma in an appropriate clinical setting as it is evident that the color and morphology of grains isolated from biopsy material is not sufficient for the accurate identification of actinomycetes that cause mycetoma. In view of the present findings, we advocate the use of different phenotypic, chemotaxonomic, and genotypic properties to recognize the causative agents of mycetoma. Similar recommendations have been given in the past. [8],[9],[17]


   Conclusions Top


In many developing countries no simple test is currently available for the diagnosis of mycetoma other than clinical assessment and the invasive procedure of surgical biopsy to take grain from non-open sinuses. When appropriate diagnostic tests are done the identity of the causative agents could be revealed. It could be concluded that actinomycetoma exists with significant prevalence (12%) among patients investigated in the present study. Streptomyces is the most important etiological agent of actinomycetoma compared with Nocardia. PCR could be a quick alternative given its high sensitivity and specificity, which may encourage its use rather than the tedious and time consuming conventional methods in diagnosis of mycetoma in the Sudan.


   Acknowledgments Top


The authors acknowledge Prof. A.H. Fahal (Mycetoma Research Center, University of Khartoum) who facilitated samples collection and Prof. M. Goodfellow (School of Biology, University of Newcastle) for the supply of reference strains.

 
   References Top

1.Lichon V, Khachemoune A. Mycetoma: A review. Am J Clin Dermatol 2006;7:315-21.  Back to cited text no. 1
[PUBMED]    
2.Mahgoub ES. Mycetoma. Int J Dermatol 1985;24:230-9.  Back to cited text no. 2
    
3.Fahal AH, Hassan MA. Mycetoma. Br J Surg 1992;79:1138-41.  Back to cited text no. 3
[PUBMED]    
4.Fahal AH. Mycetoma: A thorn in the flesh. Trans R Soc Trop Med Hyg 2004;98:3-11.  Back to cited text no. 4
[PUBMED]    
5.Mariat F. On the geographic distribution and incidence of mycetoma agents. Bull Soc Pathol Exot Filiales 1963;56:35-45.  Back to cited text no. 5
[PUBMED]    
6.Gumaa SA. The aetiology and epidemiology of mycetoma. Sudan Med J 1994;32:14-22.  Back to cited text no. 6
    
7.Welsh O, Vera-Cabrera L, Salinas-Carmona MC. Mycetoma. Clin Dermatol 2007;25:195-202.  Back to cited text no. 7
[PUBMED]    
8.Quintana ET, Wierzbicka K, Mackiewicz P, Osman A, Fahal AH, Hamid ME, et al. Streptomyces sudanensis sp. nov., a new pathogen isolated from patients with actinomycetoma. Antonie Van Leeuwenhoek 2008;93:305-13.  Back to cited text no. 8
    
9.Hamid ME. Variable antibiotic susceptibility patterns among Streptomyces species causing actinomycetoma in man and animals. Ann Clin Microbiol Antimicrob 2011;10:24-28   Back to cited text no. 9
    
10.Mahgoub ES, Gumaa SA, El Hassan AM. Immunological status of mycetoma patients. Bull Soc Pathol Exot Filiales 1977;70:48-54.  Back to cited text no. 10
[PUBMED]    
11.Fahal AH. Mycetoma Management Guideline. Khartoum, Sudan: Mycetoma Research Centre; 2011. p.5-8. Available from: http://www.mycetoma.uofk.edu/academic/updates_MMG.htm. [Last accessed 2011 Nov 22].  Back to cited text no. 11
    
12.El Hassan AM, Fahal A, EI Hag IA, Khalil EA. The pathology of mycetoma. Sudan Med J 1994;32:23-45.  Back to cited text no. 12
    
13.Kämpfer P. The family streptomycetceae. In: Dworkin M, Rosenberg E, Schleifer KH, Stackebrandt E, editors. The Prokaryotes. 3 rd ed., Vol. 3. NY: Springer; 2007. p. 538-604.  Back to cited text no. 13
    
14.Hamid ME, Minnikin DE, Goodfellow M, Ridell M. Thin-layer chromatographic analysis of glycolipids and mycolic acids from Mycobacterium farcinogenes, Mycobacterium senegalense and related taxa. Zentralbl Bakteriol 1993;279:354-67.  Back to cited text no. 14
[PUBMED]    
15.Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9.  Back to cited text no. 15
[PUBMED]    
16.Huddleston AS, Cresswell N, Neves MC, Beringer JE, Baumberg S, Thomas DI, et al. Molecular detection of streptomycin-producing streptomycetes in Brazilian soils. Appl Environ Microbiol 1997;63:1288-97.  Back to cited text no. 16
[PUBMED]    
17.Wallace RJ Jr, Steele LC. Susceptibility testing of Nocardia species for the clinical laboratory. Diagn Microbiol Infect Dis 1988;9:155-66.  Back to cited text no. 17
[PUBMED]    

Top
Correspondence Address:
Mogahid M Elhassan
Department of Microbiology, College of Medical Laboratory Science, Sudan University of Science and Technology, P.O. Box 407, Khartoum
Sudan
Login to access the Email id

Source of Support: This work have been partially funded by Sudan University of Science and Technology.,, Conflict of Interest: None


DOI: 10.4103/1755-6783.116523

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *


    Abstract
   Introduction
   Materials & Methods
   Results
   Discussion
   Conclusions
   Acknowledgments
    References
    Article Figures

 Article Access Statistics
    Viewed5038    
    Printed62    
    Emailed0    
    PDF Downloaded24    
    Comments [Add]    

Recommend this journal