AJMR- 20 September, 2012 Issue

African Journal of

Microbiology Research Volume 6 Number 36 ISSN 1996-0808

20 September, 2012

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Donovan Anthony McGrowder

Dr. Jianfeng Wu Dept. of Environmental Health Sciences, School of Public Health, University of Michigan USA Dr. Ahmet Yilmaz Coban OMU Medical School, Department of Medical Microbiology, Samsun, Turkey.

Editorial Board Dr. Kwang Young Song Department of Biological Engineering, School of Biological and Chemical Engineering, Yanbian Universityof Science and Technology, Yanji, China. Dr. Kamel Belhamel Faculty of Technology, University of Bejaia Algeria. Dr. Sladjana Jevremovic Institute for Biological Research Sinisa Stankovic, Belgrade, Serbia. Dr. Tamer Edirne Dept. of Family Medicine, Univ. of Pamukkale Turkey. Dr. R. Balaji Raja M.Tech (Ph.D) Assistant Professor, Department of Biotechnology, School of Bioengineering, SRM University, Chennai. India Dr. Mohd Fuat ABD Razak Institute for Medical Research Malaysia. Dr. Minglei Wang University of Illinois at Urbana-Champaign USA. Dr. Davide Pacifico Istituto di Virologia Vegetale – CNR Italy. Prof. Branislava Kocic Specaialist of Microbiology and Parasitology University of Nis, School of Medicine Institute for Public Health Nis, Bul. Z. Djindjica 50, 18000 Nis Serbia.

Dr. Ntobeko A. B. Ntusi Cardiac Clinic, Department of Medicine, University of Cape Town and Department of Cardiovascular Medicine, University of Oxford South Africa and United Kingdom. Prof. N. S. Alzoreky Food Science & Nutrition Department, College of Agricultural Sciences & Food, King Faisal University, Saudi Arabia. Dr. Sivakumar Swaminathan Department of Agronomy, College of Agriculture and Life Sciences, Iowa State University, Ames, Iowa 50011 USA. Dr. Alfredo J. Anceno. School of Environment, Resources and Development (SERD), Asian Institute of Technology, Thailand. Dr. Okonko, Iheanyi Omezuruike Department of Virology, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, University College Hospital, Ibadan, Nigeria. Dr. S. Meena Kumari Department of Biosciences Faculty of Science University of Mauritius Reduit Mauritius. Luki Subehi Parasitology & Mycology Dept, Baghaeei Lab., Shams Abadi St. Isfahan Iran.

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African Journal of Microbiology Research International Journal of Medicine and Medical Sciences Table of Contents:

Volume 6

Number 36 20 September, 2012

nces ARTICLES Research Articles Conventional and molecular characterization of Trichophyton rubrum Farzad Aala, Rosimah Nulit, Umi Kalsom Yusuf and Sassan Rezaie

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Inhibitory effect of some plant extracts on clinical isolates of Staphylococcus aureus Rajaa Milyani and Nahed Ashy

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Role of CSE1034 in bacterial lipids and polysaccharides involved in biofilm formation: a comparison with other drugs Chaudhary Manu and Anurag Payasi

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Analytical specificity and sensitivity determination of 16SrRNA gene based diagnostic polymerase chain reaction (PCR) for molecular detection of Coxiella burnetii Mohammad Soleimani, Keivan Majidzadeh A., Amirhossein Mohseni and Mohammad Khalili

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Effect of Bacillus cereus Br on bacterial community and gossypol content during fermentation in cottonseed meal Xin Wang, Jiang-wu Tang, Xiao-hong Yao, Yi-fei Wu, Hong Sun and Yao-xing Xu

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Identification and characterization of a fungal strain with lignin and cellulose hydrolysis activities Ran Jin, Hongdong Liao, Xuanming Liu, Mang Zheng, Xianqiu Xiong, Xinwu Liu, Liyong Zhang and Yonghua Zhu

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Table of Content: Volume 6 Number 23 21 June, 2012 Table of Contents: Volume 6 Number 36 20 September, 2012

nces ARTICLES ARTICLES DNA viral infections and transient bone marrow failure in southern Iranfor Influence of ciprofloxacin on glioma cell line GL26: A new application Kambiz Mohammad Hossein Karimi, Ramin Yaghobi, an oldBagheri, antibiotic Behnam Mohammadi, Mehdi Dehghani and Padideh Ebadi Abdolreza Esmaeilzadeh, Massoumeh Ebtekar, Alireza Biglari and Zuhair Mohammad Hassan

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Survival of microorganisms in high pressure treated minced meat during chilled storage and at pH anddiversity temperature mimicking tract Identification of microbial in caecal contentgastrointestinal of broiler chicken Sami Bulut G. Dhinakar Raj, A. Rajasekar, D. Vijayalakshmi and T. Devasena S. Nathiya,

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Efficacy andquality toxicityofofsome neutralizers against disinfectantsproducts and antiseptics Microbial non-sterile pharmaceutical sourced used insome vaccine production facility from retail pharmacies in Lagos, Nigeria Norhan S. Sheraba, Aymen S. Yassin, Aly Fahmy and Magdy A.A. Amin Adeola Anifowoshe R., Opara Morrison I. and Adeleye Isaac

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Effects of essential oil extracted from Citrullus colocynthis (CCT)in seeds on Molecular detection of adhesins genes and biofilm formation methicillin growth of phytopathogenic bacteria 6572 resistant Staphylococcus aureus Zahra Setayesh Nima Sanadgol and LeylaVafadar Karima BEKIR,Mehr, Omayma HADDAD, Mohammed GRISSA,Ghasemi Kamel CHAIEB, Amina BAKHROUF and Salem IBRAHIM ELGARSSDI 4908 Development and evaluation of a novel TaqMan fluorescence probe-based real-time transcriptase polymerase chainBacillus reaction assayinfor Amylasereverse production by moderately halophilic cereus solid detection and quantification of West Nile virus state fermentation Lijun Shi, HuiqiongD.Yin, Jingang Zhang, P. Vijayabaskar, Jayalakshmi and T.Zhan-zhong Shankar Zhao and Gang Li

Microbial water qualityand in the upper characteristics Olifants River catchment: Networking clusters sequence of clusteredImplications regularly for health interspaced short palindromic repeats (CRISPR) direct repeats and their W.evolutionary J. le Roux, L.comparison M. Schaeferwith and B. Genthe cas1 genes in lactic acid bacteria Kaibo Deng, Fei Liu, Chuntao Gu and Guicheng Huo

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Partial characterization of a bacteriocin produced by Lactobacillus alivarius isolated from oral cavity of desert foxes 6589 Aly E. Abo-Amerscreening and Mohammed Y. Shobrak Antibacterial of the root, stem and leaf extracts of Terminalia albida sc. elliot on selected pathogenic bacteria S. M. Ayodele, G. Alpheus and O. M. Iruaga 1457

African Journal of Microbiology Research Vol. 6(36), pp. 6502-6516, 20 September, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR10.736 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Conventional and molecular characterization of Trichophyton rubrum Farzad Aala1*, Rosimah Nulit2, Umi Kalsom Yusuf2 and Sassan Rezaie3 1

Department of Medical Mycology and Parasitology, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran. 2 Department of Biology, Faculty of Science; Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 3 Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. Accepted 10 September, 2012

Different studies illustrated that Trichophyton rubrum, among all species of Trichophyton, is the most prevalent and consequently the most important genus. T. rubrum as a worldwide filamentous pathogen fungus can infect human keratinized tissue (skin, nails and rarely hair), and causes dermatophytosis. Researchers use two general methods for the identification of dermatophytes namely, conventional methods on the basis of phenotype variations and molecular methods on the basis of molecular differences. Due to some limitations in traditional methods, in the recent years, molecular biological methods are regarded as useful in the exact and rapid recognition of dermatophytes. The present study identified nine clinical isolates and one ATCC as a control strain of T. rubrum by using both conventional and molecular methods. The molecular systematics method was used to elucidate genetic diversity among strains of T. rubrum and within Trichophyton species. Morphological characteristics of all colonies T. rubrum quite varies among each other; we revealed that that conventional methods are generally prolonged and may be indecisive. However, molecular studies based on internal transcribed spacer (ITS) sequencing provides a very accurate result, which is more than 96% the similarity of T. rubrum among all isolates, and more than 90% similarity within Trichophyton spp. Key words: Trichophyton rubrum, conventional method, internal transcribed spacer (ITS) regions, identification, dermatophytes. INTRODUCTION Trichophyton rubrum is one of the most commonly encountered dermatophytes that infect human keratinized tissue such as skin, nails and possibly hair. This pathogen causes well-characterized superficial infections, and also produces skin infections in unusual parts of the body in immunodepressed patients (Cervelatti et al., 2004). Nearly 80% of onychomycosis due to T. rubrum and 90% of the chronic dermatophyte infections are caused mostly by T. rubrum, this pathogen developed mechanisms to avoid or suppress cell- mediated immunity ((Baeza et al., 2006; Baeza et al., 2007).

*Corresponding author. E-mail: [email protected]. Tel: +98-9197544944.

Researchers use two general methods for the laboratory identification of various species of dermatophytes: a) identification on the basis of phenotype differences (conventional methods) and b) Identification on the basis of molecular differences. Faggi et al. (2001) mentioned that identification of dermatophyte species by conventional methods requires the examination of colony, particularly with the method of slide culture and microscopic morphological structures. Morphological and physiological features are dynamic. As a matter of fact, outside factors such as temperature variation, medium and chemotherapy, can greatly influence the phenotypic characteristics and consequently can make the identification more difficult. Molecular biological methods, in the recent years, are regarded as useful in the exact and rapid recognition of

Aala et al.

dermatophytes. Sequencing of the Internal Transcribed Spacer (ITS) region of the ribosomal DNA, Sequencing of protein-encoding genes, Restriction Fragment Lenght Polymorphism (PCR-RFLP) analysis of mitochondrial DNA, Polymerase Chain Reaction (PCR); Random Amplification of Polymorphic DNA (RAPD), Arbitrarily Primed PCR [AP-PCR], and PCR fingerprinting are all instances of molecular techniques which have brought prominent advance in differentiating between species and strains (Faggi et al., 2001; Kanbe et al., 2003; Girgis et al., 2006; Yoshida et al., 2006; Li et al., 2007). In the recent years, quite a few molecular studies have been conducted on the internal transcribed spacer (ITS) region of the rRNA gene. Sequencing analysis of the ITS regions is considered as a useful tool for phylogenetic delineation and the identification of dermatophytes (Yoshida et al., 2006; Li et al., 2007). Even though about 80 to 90% of all isolated are T. rubrum (Brasch and Hipler, 2008), has been isolated to identify the morphological similarity and the variability among this species, but only a few study has been done about the genetic relationship of Trichophyton. The aim of this work is to identify ten clinical isolates of T. rubrum by using both conventional methods and molecular method based on universal fungal primers which are internal transcribed spacer 1 (ITS1). T. rubrum (ATCC-10218) was used as a control strain. The molecular systematics method was used to elucidate genetic diversity among strains of T. rubrum and within Trichophyton species. MATERIALS AND METHODS Isolates Nine isolates of T. rubrum which are T. rubrum (1138), T. rubrum (1059), T. rubrum (1164), T. rubrum (1208), T. rubrum (1160), T. rubrum (1008), T. rubrum (1298), T. rubrum (1044) and T. rubrum (2970), were obtained from the culture collection of clinical isolates preserved at the laboratory of Medical Mycology Department in Tehran University of Medical Sciences, Iran for study; and T. rubrum (ATCC-10218) was used as a control strain. All clinical isolates were kept in sterile saline (0.85%) v/v NaCl at 4°C until required for bioassays.

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(PBS) and finally stored at −70°C.

DNA extraction Fungal genomic DNA from T. rubrum was isolated according to Rezaie et al. (2000) with slight modification. 200 to 300 mg of mycelia was ground with liquid nitrogen to powder form. 500 μl of DNA extraction buffer (50 mM Tris-HCl pH 8.0), 50 mM EDTA, 25 μl 20% SDS, and 10 μl of proteinase-K, was added and mixed gently. Then, incubated at 65°C for 60 min and centrifuged at 3000×g for 15 min. 25 μl Rnase H (10 mg/ml) was added to supernatant and incubated again at 37°C for 30 min. Then mixed with 500 μl of phenol:chloroform:isoamyl alcohol (25:24:1) and and centrifuge at 10000×g for 10 min and the supernatant were collected and transferred to new steril eppendorff tubes. Then mixed again with 500 μl of chloroform:isoamyl alcohol (24:1) and centrifuge at 10000×g for 10 min, and the supernatant were collected and transferred to new steril eppendorff tubes. DNA was precipitated by adding 500 μl isopropanol and 30 μl 3 M sodium acetate followed by centrifugation at 15000×g for 30 min and the supernatant was discarded. DNA pellet was rinsed twice or more with 200 μl of 70% cold ethanol and centrifuged at 10000×g for 10 min. The pellet was air-dry and resuspended DNA pellet in 30 μl of distilled water at 37°C for 60 min and stored at -20°C.

PCR amplification Internal transcribed spacer 1 and 4 (ITS1 and ITS4) (AIT-Biotech, Singapore) were designed as ITS1 forward primer is 5’-TCC GTA GGT GAA CCT GC-3’ and the ITS4 reverse primer 5’-TCC TCC GCT TAT TGA TAT G-3’ (Shehata et al., 2008; Yang et al ., 2008 ). PCR reaction mixtures were prepared in a 25 μl volume containing 2.5 μl of 10× reaction buffer, 1.5 μl of 25 mM MgCl2, 0.5 μl of 10 mM dNTPs, 0.5 μl of 0.2 mM of each ITS 1 primer and ITS 4 primer, 0.5 μl of genomic DNA and 0.5 μl of 1 U Go Taq DNA polymerase (Promega Corporation, USA), and 18.5 μl of distiled water. PCR reactions were carried out on a thermal cycler (MJ Research. Inc. USA) with the following conditions: 1 cycle in an initial step of 94°C for 5 min and then subjected to 30 cycles consisting of denaturation at 94°C for 30 s, annealing at 55°C for 40 s, and extention at 72°C for 40 s. After the last cycle, this was followed by a final extention step at 72°C for 10 min. Then, 5 μl of PCR product was loaded on 1% agarose in 1X Tris–Acetic Acid– EDTA buffer and stained with 0.5 mg/ml ethidium bromide at 80 V for 40 min and visualised with UV transilluminator (Alpha Innotech, USA), compared with a standard DNA size marker; 100 bp DNA ladder (Fermenats, USA), and photographed in UV light.

PCR purification Conventional method All isolates of T. rubrum were cultured on Sabouraud dextrose agar media (Difco Laboratories, Detroit, Michigan) at 28°C for 14 days. Then, slide cultures of isolates were prepared and identified under light microscope (Carl Zeiss, Germany).

DNA PCR products were purified according to the QIAquick PCR Purification Kit (Qiagen, Germany) and send for sequencing (1st Laboratories, Seri Kembangan, Malaysia).

RESULTS AND DISCUSSION

Molecular method

Morphological characteristics of colonies T. rubrum

All isolates of T. rubrum maintained on Sabouraud’s dextrose agar medium and stored at 4°C. Then fungus was cultured in Sabouraud dextrose broth, and incubated at 28°C for 14 days. 200 to 300 mg of mycelia was harvested and centrifuged at 1600×g for 10 min, then washed twice with ice-cold sterile phosphate buffered saline

This study used both conventional and molecular methods to diagnose ten isolates of T. rubrum. Studies revealed that colonies characterization of all isolates quite varies among each other. Of these isolates, isolate

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numbers 1138 and 1059 are white and cottony or fluffy but isolates number 1164, 1160, 1008, and 1298 are cream, flat and downy, but the others are cream with a carmine and woolly or granular type (isolates numbers 1208, 1044, 2970 and 10218) (Figure 1). The microscopic features of the isolates also varies, which are macroconidia and microconidia of isolates numbers, 1138, 1008, 1298, 1044, 2970 and 10218 more abundant than isolates number 1059, 1164, 1208, and 1160. However, the shape of the macroconidia and microconidia of all isolates are almost similar, which is cyclindrical to cigar shaped (Figure 1). Isolation, identification and characterization of ITS1 of T. rubrum

molecular

Figure 2 showed that ITS1 of all isolates T. rubrum had been amplifed and then were isolated and sequenced. The length of nucleotides sequence of all isolates are not similar which is T. rubrum (1138) 658 bp, T. rubrum (1059) 715 bp, T. rubrum (1164) 722 bp, T. rubrum (1208) 713 bp, T. rubrum (1160) 614 bp, T. rubrum (1160) 614 bp, T. rubrum (1008) 719 bp, T. rubrum (1298) 668 bp, T. rubrum (1044) 658 bp, T. rubrum (2970) 660 bp and T. rubrum (ATCC-10218) 633 bp. Nucleotide sequence of all isolates of T. rubrum and ATCC-10218 are shown in Figure 3. Previous studies by Rakeman et al. (2005) and Shehata et al. (2008) also revealed that the universal fungal primers amplified the ITS regions (ITS1-5.8S-ITS2) of the ribosomal DNA nearly 690 bp for T. rubrum isolates. Nucleotide sequence of ten isolates of T. rubrum shown in Figure 3. All nucleotide sequences of T. rubrum isolates were analyzed using online software CLUSTALW (www.Pir.geogetown.edu/pirwww/search/multialn.shtm) to reveal the similarities among isolates. Figure 4 showed that the similarities among nine isolates of T. rubrum are higher, which is more than 96% identities. Nucleotide sequence of isolates T. rubrum were analyzed using online software CLUSTALW (www. Pir.geogetown.edu/pirwww/search/multialn.shtm) to reveal the similarities among isolates T. rubrum and other species of Trichophyton which are Trichophyton raubitschekii strain NOMH 789 (GenBank accession no. AF170469), T. rubrum strain UAMH 8547 (GenBank accession no. AF170471), T. kanei (GenBank accession no. AF170460), T. rubrum strain WM 06.348 (GenBank accession no. EF568093), T. rubrum strain 05-287-3929 (GenBank accession no. EU200395), T. rubrum 5.8S rRNA (GenBank accession no. AJ270808), T. soudanense strain UAMH 8548 (GenBank accession no. AF170474), T. rubrum strain NCPF 295 (GenBank accession no. EU181449), T. megninii strain ATCC 12106 (GenBank accession no. AF170464), and T. rubrum strain ATCC 28188 (GenBank accession no. AF 170472). The similarities of all isolates of T. rubrum and

other species of Trichopthyon is also higher than 90% as shown in Figure 5, CLUSTAL 2.0.12 multiple sequence alignment. DISCUSSION Traditional method such as investigation of macroscopic and microscopic features of cultures of fungi had been applied since early 19th century. However, these methods seem to be difficult to amplify due to the polymorphic feature of these characters, besides increased by differences in media compounds, temperature variations, and other variables of cultivation. Furthermore, in some cases, the dermatophytes fail to make reproductive organization in culture (sterile mycelia) that makes it impossible for final identification (Malinovschi et al., 2009). Besides that, conventional method is often difficult due to abnormal microscopic or macroscopic morphology (Li et al., 2008). Currently, molecular studies become crucial and necessary for identification of pathogenic fungi (Borman et al., 2008; Malinovschi et al., 2009). The internal transcribed spacer (ITS) regions of the fungal ribosomal DNA (rDNA) had been used as one of techniques for species identification becuase it is faster, accurate species determination, specific, and are less feasible to be affected by exterior effects such as temperature changes and chemotherapy (Girgis et al., 2006; Kong et al., 2008). Studies revealed that morphological characteristics of colonies of all isolates T. rubrum are similar to T. rubrum isolated from tinea cruris, tinea pedis, and tinea capitis of human (Graser et al., 2000). Colonies of T. rubrum are fluffy to cotonny and white to cream in colour. Macroconidia are sparse or abundant and microconidia are present in all isolates. In this studies, the length of ITS1 of all isolates is about 690 bp, 10 clinical isolates of T. rubrum were collected from the Clinical Mycology Laboratory at Westmead Hospital, Sydney, and the Women’s and Children’s Hospital, Adelaide, Australia also have almost the same length of ITS1, which is 666 bp (Kong et al., 2008). Consequently, the results of our study are in agreement with these studies and showed that molecular method based on ITS sequencing is a reliable and useful method for the identification of dermatophytes as well as for confirmation of diagnosis of the conventional methods. In this study, the molecular method was also used to clarify genetic diversity among strains of T. rubrum and within Trichophyton species. The results of this study regarding nucleotide sequence of isolates of T. rubrum demonstrated that the similarities among ten isolates of T. rubrum are more than 96% identities. It also showed the similarities among ten isolates of T. rubrum and ten isolates of other genus of Trichophyton are higher than 90%. The results of this study are in agreement with Graser et al. (2000) who showed that the Trichophyton species are supported by high similarities with value of

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Figure 1. The colonies and microscopy of 10 isolates of T. rubrum with (macroconidia and microconidia) × 400.

more than 86% among isolates of T. rubrum and isolates of other genus of Trichophyton. Our results are also in

agreement with Li et al. (2008), who revealed that percentage identity of Trichophyton species with

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Afr. J. Microbiol. Res.

Figure 2. PCR amplification of isolates of T. rubrum on 1% agarose gel electrophoresis. T. rubrum ATCC-10218 as positive control strain also showed DNA amplification at 690 bp.

> T. rubrum (1138) 50 NNNNGGGAGAGCGTAAGTGGGCTGCCACTATAGAGGACCGGACATTCCAT 100 CAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC 150 TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC 200 CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGA 250 CAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGC 300 AAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATC 350 GATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCG 400 TGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGG 450 GCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGAT 500 GGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA 550 GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCTGGCCCCAATCTTTATATATATATATATC 650 TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT 658 ATCAAAAG

> T. rubrum (1059) 50 NCCAGTAACCGTAGGTGACCTGCGCATATCAATAAGCGGAGGACTCCGTG 100 GGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC 150 CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG 200 GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC 250 ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCACAGA 300 CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA 350 AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT 400 CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG 450 CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG 500 ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC 550 CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC 600 TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCA 650 GGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAT 700 AAGCCGGAGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGCTCTCTTTTTG 715 GGGAAGCAAAATGGG > T. rubrum (1164) 50 CNNNNNAGACCGTACGTTGGCTGCGCATATCAGATAACCGGACATGACAT 100 CGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT 150 CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC 200 GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG 600 CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTT 650 TTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAT 700 CAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCCTTTTT 722 TTTTGGGGTAGCGAGAAGGGGG

Figure 3. Nucleotide sequences of 9 isolates of T. rubrum and ATCC10218. Nucleotide sequence numbering is shown on the left.

Aala et al.

> T. rubrum (1208) 50 TNNGCAGACGTACGTGGGCTGCGAATATCAGGAAGCGACATGACTTCGGG 100 GGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC 150 CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG 200 GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGACA 250 CCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCAC 300 AATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAA 350 GAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAATC 400 ATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATGC 450 CTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACGA 500 CCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGCC 550 AGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCCTC 600 AGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCAGG 650 TTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAA 700 GCCGGGAGGAAGGGGGGGCCCCCCAAAATGCCCCCCCCTCTCTTTTTGGG 713 GGGGAGAGCGGGG > T. rubrum (1160) 50 NNNNNAAGAATCGTAAGTGACCTGCGCATATCAATAAGCGGAGGATCCGT 100 AGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTCTACCTCAC 150 CCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGC 200 GGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC 250 ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCA 300 CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA 350 AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT 400 CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG 450 CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG 500 ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC 550 CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC 600 TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTC 650 AGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAA 614 TAAGCGGGGAGGAA

> T. rubrum (1008) 50 NACNAAGAGCCGTAGGTGACCTGCGCATATCAATAAGCGAGAGGACTCCG 100 TAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTCTACCTCA 150 CCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG 200 CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA 250 CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC 300 ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG 350 AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA 400 TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT 450 GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC 500 GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG 550 CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC 600 CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT 650 CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA 700 ATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTCAGGGTGT 719 GTGAAACAAACGGCGGGCC > T. rubrum (1298) 50 NNACNNAGTATCGTAGGTGACCTGCGCATATCAATAAGCGGAGGATTCCG 100 TAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGTTACCTCG 150 CCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG 200 CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA 250 CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC 300 ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG 350 AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA 400 TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT 450 GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC 500 GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG 550 CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC 600 CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT 650 CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA 668 ATAAGCGGAGGAA > T. rubrum (1044) 50 NNNANCGGGACAGCCGTAGTGGGCTGCGCATATCAGATAACGCGGAGATT 100 ACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCT 150 ACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCC 200 GTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGG 250 ACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAG 300 CAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCAT 350 CGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCC 400 GTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGG 450 GGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGA 500 TGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA 550 GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATC 650 TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT 658 ATCAAAAG

Figure 3. Cotnd.

6507

6508

Afr. J. Microbiol. Res.

> T. rubrum (2970) 50 ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACT 100 TCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC 150 TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC 200 CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG 600 CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCT 650 TTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA 660 TCAAAAGCGG > T. rubrum (ATCC-10218) 50 NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACTT 100 CGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT 150 CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC 200 GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA 250 GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA 300 GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA 350 TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG 400 AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC 450 ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG 500 ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT 550 GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG 600 CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATATATCTT 633 GGCAGGTTGACCTCGGATCAGGTAGGGATACGT

Figure 3. Cotnd.

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

--NNNNGGGAGAGCGTAAGTGGGCTGCCA-CTAT-AGAGGAC-CGGACAT ---CNNNNNAGACCGTACGTTGGCTGCGC-ATATCAGATAAC-CGGACAT ----TNNGCAGA-CGTACGTGGGCTGCGA-ATATCAGGAAGC---GACAT NNNANCGGGACAGCCGTAGTGGGCTGCGC-ATATCAGATAACGCGGAGAT -----NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG ----ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG ----NCCAGTAACCGTAGGTGACCTGCGC-ATATCAATAAGC----GGAG --NNNNNAAGAATCGTAAGTGACCTGCGC-ATATCAATAAGC---G-GAG --NNACNNAGTATCGTAGGTGACCTGCGC-ATATCAATAAGC---G-GAG ---NACNAAGAGCCGTAGGTGACCTGCGC-ATATCAATAAGC---GAGAG * ** **** *** * * *

50 50 50 50 50 50 50 50 50 50

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

TCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC TACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC GAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTC GATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGT GACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTC * ***** * ** * * ***

100 100 100 100 100 100 100 100 100 100

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC TACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGC TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC ****** ****************************************** T. rubrum (1138) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1164) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1208) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1044) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (ATCC-10218) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (2970) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1059) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1160) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1298) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA T. rubrum (1008) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA **************************************************

150 150 150 150 150 150 150 150 150 150 200 200 200 200 200 200 200 200 200 200

Figure 4. Comparison of nucleotide sequence between T. rubrum ITS1 orthologues. Nucleotide sequences that are present in all ITS1 are shaded in blue colour. Nucleotide sequence numbering is shown on the right.

Aala et al.

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT ************************************************** T. rubrum (1138) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1164) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1208) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1044) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (ATCC-10218) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (2970) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1059) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1160) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1298) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC T. rubrum (1008) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC **************************************************

250 250 250 250 250 250 250 250 250 250

T. T. T. T. T. T. T. T. T. T.

350 350 350 350 350 350 350 350 350 350

ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT ************************************************** T. rubrum (1138) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1164) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1208) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1044) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (ATCC-10218) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (2970) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1059) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1298) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG T. rubrum (1008) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG **************************************************

T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

400 400 400 400 400 400 400 400 400

GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT ************************************************** T. rubrum (1138) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1164) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1208) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1044) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (ATCC-10218) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (2970) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1059) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1160) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1298) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA T. rubrum (1008) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA **************************************************

450 450 450 450 450 450 450 450 450

T. T. T. T. T. T. T. T. T. T.

GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT ************************************************** T. rubrum (1138) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1164) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1208) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1044) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (ATCC-10218) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATAT T. rubrum (2970) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1059) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1160) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1298) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT T. rubrum (1008) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT ****************************************** ****

550 550 550 550 550 550 550 550 550 550

T. T. T. T. T. T. T. T. T. T.

650 650 650 650 650 650 650 650 650 650

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1008)

300 300 300 300 300 300 300 300 300 300

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

Figure 4. Contd.

ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTGGCAGGTTGACCTCGGATCAGGTAGGGATACGT-----------ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG ***** *****************************

500 500 500 500 500 500 500 500 500 500

600 600 600 600 600 600 600 600 600 600

6509

6510

Afr. J. Microbiol. Res.

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

CATATCAAAAG--------------------------------------CATATCAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCC CATATCAATAAGCCGGGAGGAAGGGGGGGCCCCCCA-AAATGCCCCCCCC CATATCAAAAG---------------------------------------------------------------------------------------CATATCAAAAGCGG-----------------------------------CATATCAATAAGCCGG-AGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGC CATATCAATAAGCGGGGAGGAA---------------------------CATATCAATAAGCGGAGGAA-----------------------------CATATCAATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTC

T. T. T. T. T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum rubrum

(1138) (1164) (1208) (1044) (ATCC-10218) (2970) (1059) (1160) (1298) (1008)

--------------------------TTTTTTTTTGGGGTAGCGAGAAGGGGG TCTCTTTTTGGGGGGGAGAGCGGGG-------------------------------------------------------------------------------TCTCTTTTTGGGGAAGCAAAATGGG-----------------------------------------------------AGGGTGTGTGAAACAAACGGCGGGCC-

700 700 700 700 700 700 700 700 700 700

727 727 727 727 727 727 727 727 727 727

Figure 4. Contd.

T. raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. megninii strain ATCC 12106 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T.rubrum 5.8S rRNA gene T.rubrum strain WM 06.348 T.rubrum strain NCPF 295 T.rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

50 50 50 50 50 50

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

100 100 100 100 100 100

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG --------------------------ACAAGGTTTCCGTAGGTGAACCTG -----------------------------------------------------------------------------------TCCGTAGGTGAACCTG ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

150 150 150 150 150 150 24 16

Figure 5. Comparison sequence between T. rubrum ITS1 T. raubitschekii strain NOMHof789nucleotide CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 T. saudanese UAMH 8548 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G orthologues. Nucleotide sequences that are present in all ITS1 are shaded198in T. megninii strain ATCC 12106 CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 black colour. Nucleotide numbering is shown on the right. T. rubrum strain UAMH 8547sequence CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198 T. T. T. T. T. T. T.

rubrum kanei rubrum rubrum rubrum rubrum rubrum

strain ATCC 28188 5.8S rRNA gene strain WM 06.348 strain NCPF 295 strain 05-287-3929 (1138)

CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG

198 198 72 42 64 42 35

T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum

(1059) (1208) (1264) (2970) (ATCC-10218) (1044)

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 saudanese UAMH 8548 megninii strain ATCC 12106 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G ------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G -------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG -------------NNNNNAAGAATCGTAAGTGACCTGCG-CATATCA-AT -------------NNACNNAGTATCGTAGGTGACCTGCG-CATATCA-AT --------------NACNAAGAGCCGTAGGTGACCTGCG-CATATCA-AT ---------------NCCAGTAACCGTAGGTGACCTGCG-CATATCA-AT ----------------TNNGCAGACGTACGTGGGCTGCG-AATATCA-GG --------------CNNNNNAGACCGTACGTTGGCTGCG-CATATCAGAT --------------ANCGGACAGCCGTA-GTGGCCTGCGACATATCAGAT ---------------NGGGACCGCCGTA-GTGGCCTGCGACATATCAGAT ----------NNNANCGGGACAGCCGTA-GTGGGCTGCG-CATATCAGAT ** * * * *

198 198 198 198 198 198 72 42 64 42 35 35 35 34 33 32 35 35 34 38

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTC-ATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC GA-CCGGACATTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AA-GCG--GAGGAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGG AA-GCG--GAGGATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGG AA-GCGA-GAGGACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCC AA-GCG--GAGGACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCC AA-GCGA-CATGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AA-CCGGACATGACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AACGCGGAGAGGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC AACGCGGAGAGGACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCC AACGCGGAGATTACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC * ** * ***** * ** * *

246 246 246 246 246 246 119 90 112 90 84 81 82 82 80 80 84 85 84 88

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG C-ATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG ATTCCGTTGGTTACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTG ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG *** ****** ****************** *************

296 296 296 296 296 296 168 140 162 140 134 131 132 132 130 130 134 135 134 138

CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG

346 346 346 346 346

Aala et al.

Figure 5. Contd. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188

6511

T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

6512

Afr. J. Microbiol. Res.

CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 135 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 134 CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 138 *** ****** ****************** *************

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1168) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG **** ******************* **** *** ****************

346 346 346 346 346 346 218 190 212 190 184 181 182 182 180 180 184 185 184 188

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG **************************************************

396 396 396 396 396 396 268 240 262 240 234 231 232 232 230 230 234 235 234 238

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH 789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC **************************************************

446 446 446 446 446 446 318 290 312 290 284 281 282 282 280 280 284 285 284 288

TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA

496 496 496 496 496

Figure 5. Contd. T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188

T. T. T. T. T. T.

rubrum rubrum rubrum rubrum rubrum rubrum

(1059) (1208) (1264) (2970) (ATCC-10218) (1044)

TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC **************************************************

280 280 284 285 284 288

T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA **************************************************

496 496 496 496 496 496 368 340 362 340 334 331 332 332 330 330 334 335 334 338

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC **************************************************

546 546 546 546 546 546 418 390 412 390 384 381 382 382 380 380 384 385 384 388

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC **************************************************

596 596 596 596 596 596 468 440 462 440 434 431 432 432 430 430 434 435 434 438

Figure 5. Contd.strain NOMH789 T. raubitschekii T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei

CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC

646 646 646 646 646 646

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T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) Afr. J. Microbiol. Res. T. rubrum (ATCC-10218) T. rubrum (1044)

TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC **************************************************

430 430 434 435 434 438

CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTC--TTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC ********************************** **************

646 646 646 646 646 646 516 490 512 490 484 481 482 482 480 480 484 485 484 488

GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG ***************************************** ******** raubitschekii strain NOMH789 GGCAGCCAATTCAGCGCCCTCAGG-------------------------megninii strain ATCC 12106 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA saudanese UAMH 8548 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain UAMH 8547 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain ATCC 28188 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA kanei GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum 5.8S rRNA gene GGCAGCCAATTCAGCGCCCTCAGGACCGGCNGCCCTGGCCCCAATCTTTA rubrum strain WM 06.348 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain NCPF 295 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum strain 05-287-3929 GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1138) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1164) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1298) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1008) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1059) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1208) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1264) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (2970) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (ATCC-10218) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA rubrum (1044) GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA ********* *************

696 696 696 696 696 696 566 540 562 540 534 531 532 532 530 530 534 535 534 538

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

raubitschekii strain NOMH789 megninii strain ATCC 12106 saudanese UAMH 8548 rubrum strain UAMH 8547 rubrum strain ATCC 28188 kanei rubrum 5.8S rRNA gene rubrum strain WM 06.348 rubrum strain NCPF 295 rubrum strain 05-287-3929 rubrum (1138) rubrum (1164) rubrum (1298) rubrum (1008) rubrum (1059) rubrum (1208) rubrum (1264) rubrum (2970) rubrum (ATCC-10218) rubrum (1044)

T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

Figure 5. Contd.

720 746 746 746 746 746 616 590 612 590 584 581 582 582 580 580 584 585 584 588

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T. raubitschekii strain NOMH789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (1298) T. rubrum (1008) T. rubrum (1059) T. rubrum (1208) T. rubrum (1264) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044) T.raubitschekii strain NOMH 789 T. megninii strain ATCC 12106 T. saudanese UAMH 8548 T. rubrum strain UAMH 8547 T. rubrum strain ATCC 28188 T. kanei T. rubrum 5.8S rRNA gene T. rubrum strain WM 06.348 T. rubrum strain NCPF 295 T. rubrum strain 05-287-3929 T. rubrum (1138) T. rubrum (1164) T. rubrum (2970) T. rubrum (ATCC-10218) T. rubrum (1044)

-------------------------------------------------TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG TATCGCGATATATCTTGGCAGGTTGACCTCGGATCAG------------TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG -------------------------------------------------GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG CTGAACTTAA---------------------------------------CTGAACTTAAGCATATCAAT-----------------------------CTGAACTTAAGCATATCAATAAGCGG-----------------------CTGAACTTAAGCATATCAAAAG---------------------------CTGAACTTAAGCATATCAATAAGCGGGGAGGAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------

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796 796 796 796 796 666 640 662 640 634 631 632 632 630 630 634 635 621 638 846 846 846 846 846 716 650 682 666 656 664

Figure 5. Contd.

reference sequence in GenBank (BLAST search) ranged from 85.9 to 100%. Conclusion By conventional characterization, colonies of all isolates quite varies; however, the shape of macroconidia and microconidia are similar. Beside that, molecular characterization also revealed that all isolates of T. rubrum show high similarity among them and with other Trichophyton species.

ACKNOWLEDGEMENT This study was supported by the Research University Grants Scheme (RUGS) from University Putra Malaysia. REFERENCES Baeza LC, Matsumoto MT, Almeida AMF, Mendes-Giannini MJS (2006). Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer. J. Med. Microbiol. 55:429-436. Baeza LC, Bailao AM, Borges CL, Pereira M, Soares CM, Mendes Gianni MJ (2007). cDNA representational difference analysis used in the identification of genes expressed by Trichophyton rubrum during contact with keratin. Microbes. Infect. 9:1415-1421.

Borman AM, Linton CJ, Miles SJ, Johnson EM (2008). Molecular identification of pathogenic fungi. J. Antimicrob. Chemother. 61: S1, i7-i12, dio: 10.1093/jac/dkm425. Brasch J, Hipler UC (2008). Clinical Aspects of Dermatophyte Infections. Human and Animal Relationships. 2nd Edition. The Mycota. VI. 263-286. Cervelatti EP, Ferreira-Nozawa MS, Aquino-Ferreira R, Fachin AL, Martinez-Rossi NM (2004). Electrophoretic molecular karyotype of the dermatophyte Trichophyton rubrum. Genetics Mol. Biol. 27(1): 99-102. Faggi E, Pini G, Campisi E, Bertellini C, Difonzo E, Mancianti F (2001). Application of PCR to distinguish common species of dermatophytes. J. Clin. Microbiol. 39: 3382-3385. Girgis S, Fakkar N, Badr H, Shaker O, Metwally F, Bassim H (2006). Genotypic identification and antifungal susceptibility pattern of dermatophytes isolated from clinical specimens of dermatophytosis in Egyptian patients. J. Egyptian Dermatol. 2(2): 1-23. Graser Y, Kuijpers AFA, Presber W, De hoog GS (2000). Molecular Taxonomy of the Trichophyton rubrum Complex. J. clin. microbiol. 38(9): 3329-3336. Kanbe T, Suzuki Y, Kamiya A, Mochizuki T, Kawasaki M, Fujihiro M, Kikuchi A (2003). Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCRRFLP targeting of the DNA topoisomerase II genes. J. Dermatol. Sci. 33: 41-54. Kong F, Tong Z, Chen X, Sorrell T, Wang B, Wu Q, Ellis D, Chen S (2008). Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification. J. Clin. Microbiol. 46(4): 1192-1199. Li HC, Bouchara JP, Hsu MML, Barton R, Su S, Chang TS (2008). Identification of dermatophytes by sequence analysis of the rRNA gene internal transcribed spacer regions. J. Med. Microbiol. 57: 592600. Li HC, Bouchara JP, Hsu MML, Barton R, Chang TS (2007).

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Identification of Dermatophytes by an Oligonucleotide Array. J. Clin. Microbiol. 45(10): 3160-3166. Malinovschi G, Kocsube S, Galgoczy L, Somogyvari F, Vagvolgyi C (2009). Rapid PCR based identification of two medically important dermatophyte fungi, Microsporum canis and Trichophyton tonsurans. Acta. Biol. Szegediensis 53(1): 51-54. Rakeman JL, Bui U, LaFe K, Chen YC, Honeycutt RJ, Cookson BT (2005). Multilocus DNA sequence comparisons rapidly identify pathogenic molds. J. Clin. Microbiol. 43(7):3324-3333. Rezaie S, Ban J, Mildner M, Poitschek C, Brna C (2000). Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum. Gene. 241: 27-33. Shehata AS, Mukherjee PK, Aboulatta HN, El Akhras AI, Abbadi SH, Ghannoum MA (2008). Single-Step PCR Using (GACA) 4 Primer: Utility for Rapid Identification of Dermatophyte Species and Strains. J. Clin. Microbiol. 46(8): 2641-2645.

Yang G, Zhang M, Li W, An L (2008). Direct species identification of common pathogenic dermatophyte fungi in clinical specimens by semi-nested PCR and restriction fragment length polymorphism. Mycopathologia. 166: 203-208. Yoshida E, Makimura K, Mirhendi H, Kaneko T, Hiruma M, Kasai T, Uchida K, Yamaguchi H, Tsuboi R (2006). Rapid identification of Trichophyton tonsurans by specific PCR based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) 1 region. J. Dermatolog. Sci. 42: 225-230.

African Journal of Microbiology Research Vol. 6(36), pp. 6517-6524, 20 September, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.119 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Inhibitory effect of some plant extracts on clinical isolates of Staphylococcus aureus Rajaa Milyani* and Nahed Ashy Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Accepted 30 May, 2011

Accumulating data have been dramatically increasing on multiple drug resistant Staphylococcus aureus isolates that are incriminated in nosocomial and community acquired infections causing high mortality and morbidity. Accordingly, 70 resistant isolates were collected from King Fahad General Hospital and National Guard Hospital, Jeddah, Saudi Arabia in an attempt to find alternative antimicrobial substances from various plant extracts. The susceptibility pattern was as follows: 50 isolates were resistant to fusidic acid, 28 isolates were resistant to oxacillin, 20 isolates were resistant to penicillin, 20 isolates were resistant to clindamycin and 21 isolates were resistant to gentamycin. However, all the isolates were sensitive to vancomycin. The antimicrobial activity of the aqueous extracts of five medicinal plants namely Canellia sinensis (green tea), Punnica granatum (pomegranate rind), Psidium guajava Lim (guava leaves), Cinnamomum verum (cinnamon) and Mourus (raspberry) was tested against the 70 resistant isolates of S. aureus using agar well diffusion assay. Significant difference was noted in the inhibitory effect between most of the tested extracts. Pomegranate rind showed the highest activity, followed by green tea, guava leaves, cinnamon bark and raspberry fruits extract respectively, compared to commercial antibiotics. Interestingly, the inhibitory activity of three combined extracts: green tea, pomegranate rind and guava leaves was found to be higher on 20 clindamycin resistant isolates compared to each extract alone, indicating synergistic interaction. These results emphasize the promising role of plant extracts as alternative antibacterial agents against resistant strains of S. aureus if not other pathogenic bacteria. Keywords: Staphylococcus aureus, antimicrobial activity, plant extracts, inhibition, methicillin.

INTRODUCTION Staphylococcus aureus is one of the main causes of human infections. It can cause diseases ranging from minor infections such as pimples and boils to serious systemic fatal infections (Evans and Brachman, 1991). Many neonates, children and adults may be colonized by S. aureus and harbor this organism either in the nasopharynx, skin or any site of their bodies, thus dispersing this hazardous bacterium. In Saudi Arabia, the throat carriage rate of S. aureus has been studied and found to be about 9% among 100 adult and 12% among 150 children (Milyani and Memish, 1987). On the other

*Corresponding author. E-mail: [email protected]

hand, the incidence rate from six sites of the skin in addition to the anterior nares was 30% in 40 examined children and 5% in adults (Milyani, 2001). Moreover, S. aureus predominated among children in both nasopharynx and ear discharge simultaneously recording 26.7 and 20% respectively (Milyani and Mahfouz, 2004). Methicillin resistant S. aureus (MRSA) is a bacterium that has developed resistance to most antibiotics such as methicillin, gentamycin, fucidic acid and clindamycin that are commonly used for Staphylococcus infections, unfortunately, leading to failure of treatment (Shai et al., 2004). The two major strains of MRSA are known to be hospital-acquired (HA) MRSA and community-acquired (CA) MRSA. HA-MRSA includes cases in which the patient has had a current or recent hospitalization,

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receives dialysis, or resides in a long-term care facility. During the period 1970 to 2010, strains of S. aureus resistant to multiple antibiotics including methicillin were increasingly responsible for outbreaks of nosocomial infections in countries around the world, for example, Saudi Arabia (Madani et al., 2001), Argentina (Reyes et al., 2009), South Africa (Shittu et al., 2009), Italy (Soavi et al., 2010) and the United States (Boyce, 1990). In many instances, these outbreaks were associated with individual wards, neonatal, intensive care and burns units (Liu et al., 2011). Furthermore, increasing incidence of CA-MRSA has been a growing public health concern (Mandell et al., 2005; Ma et al., 2007) and has emerged as the predominant cause of skin infections in the USA (Stevens et al., 2010). Only few intervention studies have been published to explore alternative antimicrobial agents to control and prevent diseases due to multidrug resistant S. aureus. Although other practices have been explored such as bacterial interference therapy (Maibach and Aly, 1981) and phage therapy (Jikia et al., 2005), medicinal plants have also been considered by some researchers since they are frequently used in popular medicine as remedies for many infectious diseases (Geyid et al., 2005; Mohana et al., 2008; Rahim et al., 2010). The aim of the present study was to determine the inhibitory effect of different plant extracts on the growth of 70 multiple resistant strains of S. aureus which also includes MRSA obtained from two main hospitals in Jeddah City, Saudi Arabia.

MATERIALS AND METHODS Media and antibiotics used Sheep blood agar, nutrient agar, mannitol salt agar (Hi media, India), Mueller Hinton (Oxoid, England) were used. Commercial antibiotic discs were obtained from Oxoid, England.

Preparation of extracts The plants were extracted by dissolving 50 g of the plant powder in 150 ml of sterile distilled water and boiled for 30 min (aqueous extraction). The extracts were filtered using Whatman filter paper no. 1, and were stored at 4°C until used (Somchit et al., 2003).

Susceptibility test of bacterial isolates to antibacterial agents Susceptibility pattern of the isolates of S. aureus to commercial antibacterial agents was determined by the standard paper disc diffusion method (Baron and Finegold, 1990). According to Koneman et al. (1997), every strain was tested using the following panel of antibiotic discs: clindamycin (2 µg), gentamicin (10 µg), oxacillin (1 µg), penicillin (10 µg), fusidic acid (30 µg), and vancomycin (30 µg). Susceptibility of strains to each antibiotic was determined on the basis of diameter of the inhibition zone. Methicillin-resistant strains were tested by using 1 µg of oxacillin disc. A strain showing inhibition zone of ≤ 10 mm diameter was considered MRSA after 24 h incubation at 35°C (Koneman et al., 1997).

Antibacterial activity of the plant extracts Agar well diffusion assay was used to determine the antibacterial activity of the plant extracts. A standard inoculum size of the S. aureus strains was evenly distributed and streaked onto the surface of a sterile Mueller Hinton Agar plate using sterile cotton swab. The density of the suspension was adjusted to approximately 108 CFU/ml by comparing its turbidity to a McFarland 0.5 BaSO4 standard (Koneman et al., 1997). Agar wells were made by using sterile cork borer (7 mm diameter). Each well was then filled with 200 µl of the tested plant extract after which the plates were incubated at 37°C for 24 h. All tests were performed in duplicate and the antibacterial activity produced by the plant extracts was expressed as the mean diameter of the inhibition zones (mm). In addition, combinations of three plant extracts (green tea, pomegranate and guava) were tested against 20 clindamycin resistant of S. aureus using 200 µl of a mixture of equal volume of each extract.

Statistical analysis Bacterial isolates Seventy S. aureus isolates were obtained from both King Fahad General Hospital and National Guard Hospital in Jeddah City. They were isolated from the following clinical specimens: sputum, wound discharge, blood and cerebrospinal fluid. Identification was according to Winn et al. (2006). The isolates were preserved in cooked meat broth (Oxoid) at 4°C.

Plant materials The medicinal plants were obtained from different markets in Jeddah City, Saudi Arabia. The plants were authenticated at the Herbarium Unit, Department of Biological Sciences, King Abdulaziz University, Jeddah. Five medicinal plants were used namely: Canellia sinensis (green tea leaves), Punnica granatum (pomegranate rind), Psidium guajava Lim (guava leaves), Cinnamomum verum (cinnamon bark) and Mourus (raspberry fruit) was tested. The plant materials were washed and sun dried before being grinded into powder.

The data were analyzed using SPSS version 10. Analysis of variance (ANOVA) and Scheffe tests were used to detect any significant differences among the inhibitory effects of plant extracts on S. aureus isolates. P< 0.05 was considered significant.

RESULTS AND DISCUSSION Methicillin-resistant S. aureus (MRSA) is primarily a nosocomial pathogen that emerged as a major cause of infection and colonization in hospitalized patients, whereas, community-associated MRSA infections are increasing in incidence and said to be severe enough to cause fatality (Mandell et al., 2005). Strains of MRSA that cause infections have also developed resistance to antibiotics commonly used for therapeutic purposes. As shown in Table 1, the majority of the isolates were resistant to five of the six antibiotics tested. However, the

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Table 1. The sensitivity of 70 Staphylococcus aureus isolates represented by diameter of inhibition zone (mm) to different antibiotics and five plant extracts.

Isolate No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Fusidic acid 13.0 00.0 11.0 0.00 13.0 12.0 0.0 13.0 12.0 00.0 14.0 30.0 00.0 00.0 12.0 00.0 00.0 12.0 00.0 14.0 00.0 12.0 13.0 00.0 00.0 12.0 14.0 00.0 00.0 00.0 00.0 00.0 00.0 14.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0

Antibiotics Gentamycin Oxacillin 23 20.0 0 00.0 21 14.0 26 14.0 20 15.0 30 17.0 33 17.0 27 21.0 36 17.0 22.0 20.0 31.0 20.0 20.0 20.0 00.0 00.0 22.0 15.0 19.0 16.0 35.0 20.0 27.0 18.0 32.0 19.0 34.0 18.0 27.0 23.0 25.0 15.0 23.0 17.0 32.0 14.0 33.0 20.0 34.0 17.0 32.0 20.0 23.0 19.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 34.0 00.0 20.0 17.0 28.0 00.0 26.0 00.0 20.0 00.0 21.0 00.0 22.0 19.0

Penicillin 25.0 08.0 11.0 00.0 13.0 14.0 17.0 22.0 10.0 11.0 18.0 19.0 00.0 11.0 12.0 16.0 20.0 18.0 19.0 24.0 9.0 10.0 13.0 17.0 00.0 18.0 20.0 8.0 00.0 00.0 00.0 00.0 10.0 00.0 11.0 08.0 08.0 00.0 00.0 08.0 00.0 25.0 00.0 08.0 09.0 00.0 0.0 12.0

Clindamycin 29.0 00.0 26.0 27.0 24.0 32.0 30.0 35.0 36.0 25.0 33.0 00.0 27.0 00.0 26.0 24.0 32.0 29.0 35.0 30.0 33.0 32.0 25.0 36.0 00.0 38.0 38.0 27.0 00.0 00.0 00.0 00.0 00.0 34.0 00.0 00.0 00.0 00.0 33.0 00.0 00.0 35.0 00.0 00.0 00.0 00.0 00.0 24.0

EXT 1 21.0 22.0 23.0 25.0 30.0 20.0 27.0 20.0 19.0 19.0 20.0 21.0 26.0 22.0 20.0 21.0 23.0 20.0 23.0 21.0 19.0 20.0 20.0 22.0 23.0 20.0 31.0 23.0 26.0 22.0 19.0 16.0 22.0 20.0 20.0 27.0 22.0 26.0 29.0 22.0 21.0 24.0 24.0 27.0 23.0 27.0 28.0 25.0

Plant extracts EXT 2 EXT 3 EXT 4 24.0 11.0 12.0 26.0 14.0 12.0 29.0 17.0 11.0 23.0 16.0 00.0 17.0 15.0 11.0 26.0 13.0 14.0 22.0 15.0 17.0 22.0 20.0 11.0 23.0 13.0 13.0 22.0 18.0 11.0 27.0 17.0 10.0 27.0 14.0 12.0 27.0 17.0 14.0 33.0 16.0 14.0 26.0 14.0 12.0 21.0 00.0 12.0 24.0 11.0 00.0 28.0 08.0 11.0 23.0 08.0 11.0 21.0 10.0 10.0 21.0 15.0 11.0 22.0 19.0 13.0 21.0 15.0 12.0 30.0 16.0 17.0 30.0 16.0 15.0 24.0 19.0 16.0 28.0 15.0 15.0 31.0 30.0 12.0 22.0 13.0 15.0 29.0 13.0 12.0 28.0 16.0 12.0 23.0 00.0 09.0 24.0 19.0 13.0 25.0 12.0 13.0 21.0 11.0 13.0 24.0 16.0 10.0 31.0 13.0 13.0 26.0 16.0 11.0 26.0 20.0 120.0 25.0 18.0 12.0 25.0 19.0 08.0 32.0 00.0 13.0 27.0 16.0 14.0 33.0 16.0 14.0 27.0 19.0 15.0 29.0 00.0 12.0 32.0 00.0 14.0 22.0 13.0 14.0

EXT 5 11.0 14.0 11.0 11.0 13.0 10.0 21.0 10.0 14.0 11.0 10.0 14.0 13.0 14.0 11.0 12.0 00.0 11.0 11.0 09.0 13.0 13.0 10.0 15.0 12.0 15.0 14.0 13.0 14.0 11.0 12.0 09.0 13.0 13.0 11.0 11.0 11.0 09.0 10.0 12.0 16.0 10.0 17.0 15.0 14.0 11.0 11.0 11.0

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Table 1 Contd.

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70

00.0 20.0 29.0 35.0 17.0 28.0 23.0 15.0 21.0 25.0 27.0 25.0 25.0 29.0 24.0 27.0 23.0 32 27 28 33 31

20.0 28.0 26.0 20.0 21.0 20.0 21.0 22.0 21.0 23.0 20.0 18.0 19.0 22.0 19.0 20.0 21.0 21 24 25 24 24

00.0 13.0 22.0 20.0 19.0 21.0 19.0 25.0 22.0 20.0 24.0 19.0 19.0 24.0 21.0 17.0 19.0 22.0 21.0 22.0 27.0 26.0

12.0 27.0 20.0 20.0 17.0 17.0 18.0 21.0 00.0 00.0 22.0 19.0 21.0 22.0 00.0 13.0 18.0 20.0 19.0 17.0 17.0 21.0

33.0 40.0 25.0 23.0 18.0 21.0 20.0 22.0 21.0 23.0 20.0 18.0 19.0 22.0 19 20.0 21.0 21.0 24.0 25.0 24.0 24.0

23.0 22.0 21.0 22.0 20.0 19.0 17.0 20.0 16.0 17.0 19.0 17.0 20.0 21.0 19.0 19.0 22.0 18.0 28.0 22.0 22.0 21.0

22.0 32.0 16.0 17.0 19.0 18.0 16.0 16.0 17.0 16.0 17.0 17.0 18.0 17.0 17.0 17.0 18.0 29.0 25.0 23.0 27.0 26.0

14.0 18.0 12.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 08.0 00.0 08.0 08.0 00.0 09.0 00.0 16.0 21.0 19.0 20.0 16.0

17.0 13.0 12.0 13.0 12.0 10.0 14.0 12.0 13.0 12.0 10.0 14.0 12.0 13.0 12.0 12.0 12.0 12.0 13.0 12.0 13.0 14.0

13.0 14.0 11.0 07.0 12.0 13.0 12.0 11.0 12.0 12.0 11.0 11.0 10.0 13.0 12.0 12.0 13.0 00.0 12.0 13.0 12.0 0.0

EXT 1: Green tea, EXT 2: Pomegranate rind, EXT 3: Guava leaves, EXT 4 cinnamon bark, EXT 5: raspberry.

predominant isolates among the 70 studied isolates were resistant to fusidic acid (50 isolates). 28 isolates were resistant to oxacillin, 20 isolates were resistant to penicillin, 20 isolates were resistant to clindamycin and 21 isolates were resistant to gentamycin. In contrast, all the isolates were sensitive to vancomycin. In accordance to our results, Zaman and Dibb (1994) isolated 7.5% of MRSA from wounds, 83% resistant isolates to gentamycin and 93% resistant isolates to tetracycline in Jeddah City. On the other hand, Udo and Jacob (2000) reported 10% of MRSA that was also resistant to fusidic acid in one of Jeddah hospitals. A total of 587 and 485 isolates of S. aureus were isolated from Abha Maternity Hospital, Saudi Arabia in 1996 and 1998 respectively. In both years, 71.0% were methicillin-resistant S aureus whereas, over 85.0% of all isolates were multi-resistant (Bilal and Gedebou, 2000). Moreover, 34% of fusidic acid resistant isolates among 173 MRSA were isolated in Riyadh City, Saudi Arabia (Al-Digs, 2004). Similarly, high incidence of fusidic acid resistant strains was reported in children complaining of impetigo during five years study at Wales, England (Al-Zimaity et al., 2004). Resistance of S. aureus strains to fusidic acid should be emphasized since fucidin cream and/ or ointment are still in use as topical therapy for boils, sties and superficial skin infections such as impetigo. Obviously, this might lead to failure of treatment, disseminating infection and

increasing resistant strains (Nielsen et al., 2009). Plant products, particularly spices and extracts of various plant parts have been used extensively as natural antimicrobials and antioxidants (Iqbal et al., 1998; Oskay et al., 2009). The sensitivity of 70 resistant isolates of S. aureus to the aqueous extracts of C. sinensis (green tea), P. granatum (pomegranate rind), P. guajava (guava), C. verum (cinnamon) and Mourus (raspberry) has been studied. These plants belong to different families (Table 2) and the active part of each plant was different. Although there are different techniques for plant extraction using organic solvents or water, in the present study, aqueous extraction was used which gave satisfactory and reproducible results. This technique was also utilized by other researchers to investigate the antimicrobial effect of different plant extracts successfully (Tomita et al., 1997; Somchit et al., 2002). Table 1 showed that all the tested S. aureus isolates were sensitive to green tea and pomegranate extracts where the diameters of the inhibition zones ranged from 16 to 30 mm. The inhibitory effect of three plant extracts on S. aureus was shown in Figure 1, where the antimicrobial activity is measured by the mean diameter of the inhibition zones. The antimicrobial activity of green tea may be due to polyphenols and epigallocatechin gallate which are active substances that possess excellent bactericidal activity (Toda et al., 1989, 1991;

Milyani and Ashy

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Table 2. The common and scientific names and used part of five plant extracts.

Common name Green tea Pomegranate Guava Cinnamon Raspberry

Family Theaceae Rosaceae Myrtaceae Lauraceae Rosaceae

Scientific name Canellia sinensis Punnica granatum Psidium guajava Cinnamomum verum Mourus

A

B

Used part of the plant Leaf Rind Leaf Bark Fruit

C

Figure 1. The inhibitory effect of three different plant extracts against resistant Staphylococcus aureus isolate. (A) Green tea extract, (b) Pomegranate extract, (C) Guava extract.

Shiota et al., 1999; Stapleton et al., 2004). Moreover, these substances have at least an indirect influence on biofilm production retarding the formation of dental plaque (Wolinsky et al., 2000). Similar to our studies, pomegranate extract has also been found to exhibit antimicrobial activity against a number of pathogenic bacteria such as Escherichia coli O157: H7 (Voravuthikunchai et al., 2005) which is one of the important emerging virulent strain, Salmonella typhimurium, Salmonella typhi, Shigella dysenteriae and Vibrio cholerae (Pradeep et al., 2008). In respect to guava, raspberry and cinnamon extracts, the numbers of resistant S. aureus isolates were 23, 4 and 7 respectively. Nonetheless, the diameters of the inhibition zones of the sensitive S. aureus isolates inhibited by the three tested extracts ranged from 10 to 20, 10 to 16 and 10 to 17 mm respectively.

Studies to explore the anti-cough activity of guava in rats and guinea pigs have been carried out by Jaiarj et al. (1999), thus encouraged them to suggest the use of guava extracts as anti-tussive product. They also reported the bactericidal effect of guava extract on S. aureus and Streptococcus pyogenes strains. In addition, Vieira et al. (2001) and Anas et al. (2008) have demonstrated that guava extract had a high inhibitory effect against S. aureus strains causing food poisoning in children. Furthermore, using a crude aqueous and water soluble methanol extract from leaf and bark of guava, a strong antibacterial activity against multidrug-resistant Vibrio cholerae O1 was recorded (Rahim et al., 2010), a fascinating finding that should be further studied to be applied clinically especially in controlling epidemics of cholera. On the other hand, cinnamon bark- at the present work- was inhibitory to S. aureus isolates in

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Table 3. Significant differences between the inhibitory activity of plant extracts under study.

Tested plant

Green tea

Pomegranate Guava leaves Cinnamon Raspberry

p value 0.877 0.00* 0.00* 0.00*

Pomegranate

Guava leaves Cinnamon Raspberry

0.00* 0.00* 0.00*

Guava leaves

Cinnamon Raspberry

0.238 0.349

Cinnamon

Raspberry

1.0

Mean diameter of inhibition zone (mm)

*: Significant difference at p