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Comparison of Bacterial Communities in the Throat Swabs from Healthy Subjects and Pharyngitis Patients by Terminal Restriction Fragment Length Polymorphism

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Abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis was applied to characterize bacterial flora present in the throats of healthy subjects and pharyngitis patients. The 16S rRNA genes of bacteria present in throat metagenome were amplified by PCR with 6-carboxy-fluorescein (6-FAM)-labeled universal forward primer (27 F) and a universal reverse primer (1513R). The 16S rDNAs were digested with restriction enzymes with 4-bp recognition sites (MspI or RsaI) and analyzed by using an automated DNA sequencer. T-RFLP patterns were numerically analyzed using computer programs. From analysis of the throat bacterial community, patterns derived from MspI and RsaI digested samples of healthy subjects and pharyngitis patients were grouped into different clusters, though RsaI digested samples showed some uncertainty. Pharyngitis throats generated an average species richness of 9 [±2.1 (SD)] and 10 (±2.9) for MspI and RsaI digests, respectively, whereas healthy throats generated 6.3 (±1.2) and 6.1 (±1.5) in MspI and RsaI digests, respectively. These results suggest that samples from pharyngitis patients contain an unexpected diversity of causative bacteria. The pharyngitis throats were colonized with a rich diversity of bacterial species than that of healthy throats. Using T-RFLP, we are able to detect a model bacterium, Streptococcus pyogenes SF370, and T-RF patterns were consistent with the Streptococcal T-RFLP patterns. Our study indicates that T-RFLP analysis is useful for the assessment of diversity of throat bacterial flora and rapid comparison of the community structure between subjects with and without pharyngitis.

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Acknowledgments

The authors gratefully acknowledge the financial assistance rendered by University Grants Commission (UGC), New Delhi [F. No. 34-263/2008(SR)] and the computational and bioinformatics facility provided by the Alagappa University Bioinformatics Infrastructure Facility (funded by Department of Biotechnology, Government of India; Grant No. BT/BI/25/001/2006).

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Authors and Affiliations

  1. Department of Biotechnology, Alagappa University, Karaikudi, 630003, Tamil Nadu, India

    Kannan Balaji, Ramalingam Thenmozhi, Marimuthu Sundaravadivel & Shunmugiah Karutha Pandian

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  1. Kannan Balaji
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  2. Ramalingam Thenmozhi
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  3. Marimuthu Sundaravadivel
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  4. Shunmugiah Karutha Pandian
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Correspondence to Shunmugiah Karutha Pandian.

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Supplementary File 1

Agarose gel showing amplification of 6-FAM labeled 16S rRNA gene of 20 throat metagenome 10 each from healthy subjects (H) and pharyngitis patients (P). 1 kb ladder is loaded in lane M (JPEG 26 kb)

High resolution image (TIFF 565 kb)

Supplementary File 2a

Electropherogram showing T-RFLP patterns of 16S rDNAs from ten throat swab metagenome. Healthy subject sample digested with MspI (Dot represents outlier. Healthy subject sample (H)) (JPEG 64 kb)

High resolution image (TIFF 943 kb)

Supplementary File 2b

Electropherogram showing T-RFLP patterns of 16S rDNAs from ten throat swab metagenome. Pharyngitis sample digested with MspI (Dot represents outlier. Pharyngitis patient sample (P)) (JPEG 61 kb)

High resolution image (TIFF 951 kb)

Supplementary File 2c

Electropherogram showing T-RFLP patterns of 16S rDNAs from ten throat swab metagenome. Healthy subject sample digested with RsaI (Dot represents outlier. Healthy subject sample (H)) (JPEG 65 kb)

High resolution image (TIFF 8509 kb)

Supplementary File 2d

Electropherogram showing T-RFLP patterns of 16S rDNAs from ten throat swab metagenome. Pharyngitis sample digested with RsaI (Dot represents outlier. Pharyngitis patient sample (P)) (JPEG 65 kb)

High resolution image (TIFF 1074 kb)

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Balaji, K., Thenmozhi, R., Sundaravadivel, M. et al. Comparison of Bacterial Communities in the Throat Swabs from Healthy Subjects and Pharyngitis Patients by Terminal Restriction Fragment Length Polymorphism. Appl Biochem Biotechnol 167, 1459–1473 (2012). https://doi.org/10.1007/s12010-011-9508-4

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  • DOI: https://doi.org/10.1007/s12010-011-9508-4

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