Identification of Proteus Mirabilis on Banknotes Using 16s rRNA gene in Khartoum State
Background: The presence of pathogenic bacteria in circulated currency was recorded as a public health hazard. In this study, all examined Sudanese banknotes (100%) were found to be contaminated by gram-negative bacteria. Proteus mirabilis were recovered from 10 examined notes (22.2%, f = 10), E. coli (13.3%, f = 6) and Klebsiella spp. (8.9%, f = 4) were also identified. Only the most resistant P. mirabilis isolate was identified using culture-based and 16S rRNA gene sequencing techniques.
Methods: Proteus isolates were identified phenotypically and tested for their susceptibility to 16 of commonly used antibiotics, then most resistant isolate was confirmed genotypically via 16S rRNA gene amplification and sequencing. Bioinformatics analysis using BLAST for sequence similarity search, Clustal W program for multiple sequence alignment, MEGA7 software for phylogenetic analysis. Tree was constructed to show the evolutionary relationships of the obtained sequence
with similar sequences in the databases using.
Results: The obtained sequence was found to be 100% identical to P. mirabilis 16S rRNA gene using BLAST. The phylogenetic tree was constructed to show the evolutionary relationships of the obtained sequence with similar sequences in the databases using MEGA7 software, and the closest strain was found to be P. mirabilis strain from India (EU411047)
Conclusion: This study has shown that some currency notes circulated at Khartoum transportation are carriers of antimicrobial-resistant P. mirabilis that could be potential source for their transmission in public.
 Coker, C., Bakare, O. O., and Mobley, H. L. (2000). H-NS is a repressor of the Proteus mirabilis urease transcriptional activator gene ureR. Journal of Bacteriology, vol. 182, no. 9, pp. 2649–2653.
 Bahashwan, S. A. and H.M. El Shafey. (2013). Antimicrobial resistance patterns of Proteus isolates from clinical specimens. European Scientific Journal, vol. 9, no. 27.
 Woo, P. C. Y., Ng, K. H. L., Lau, S. K. P., et al. (2003). Usefulness of the MicroSeq 500 16S ribosomal DNA-based bacterial identification system for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Journal of Clinical Microbiology, vol. 4, no. 5, pp. 1996–2001.
 Tshikhudo, P., Nnzeru, R., Ntushelo, K., et al. (2013). Bacterial species identification getting easier. African Journal of Biotechnology, vol. 12, no. 41, pp. 5975–5982.
 Brisse, S., Stefani, S., Verhoef, J., et al. (2002). Comparative evaluation of the BD Phoenix and VITEK 2 automated instruments for identification of isolates of the Burkholderia cepacia complex. Journal of Clinical Microbiology, vol. 40, no. 5, pp. 1743– 1748.
 Sneath, P. H. A. (1989). Analysis and interpretation of sequence data for bacterial systematics: The view of a numerical taxonomist. Systematic and Applied Microbiology, vol. 12, no. 1, pp. 15–31.
 Logan, N. A. (2009). Bacterial Systematics. John Wiley & Sons.
 Relman, D. A. (1999). The search for unrecognized pathogens. Science, 284, no. 5418,pp. 1308–1310.
 Olsen, G. J. and Woese, C. R. (1993). Ribosomal RNA: A key to phylogeny. FASEB Journal, vol. 7, no. 1, pp. 113–123.
 Van De Peer, Y., Chapelle, S., De Wachter, R., et al. (1996). A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Research, vol. 24, no. 17, pp. 3381–3391.
 Liao, D. (2000). Gene conversion drives within genic sequences: Concerted evolution of ribosomal RNA genes in Bacteria and Archaea. Journal of Molecular Evolution, vol. 51, pp. 305–317.
 Clarridge, J. E. (2004). Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews, vol. 17, no. 4, pp. 840–862.
 Relman, D. A. and Falkow, S. (1992). Identification of uncultured microorganisms: Expanding the spectrum of characterized microbial pathogens. Infectious Agents and Disease, vol. 1, no. 5, pp. 245–253.
 Patel, J. B. (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Molecular Diagnosis, vol. 6, no. 4, pp. 313–321.
 Sogin, M. L., Morrison, H. G., Huber, J. A., et al. (2006). Microbial diversity in the deep sea and the underexplored “rare biosphere,” in Proceedings of the National Academy of Sciences, vol. 103, no. 32, pp. 12115–12120.
 Harker, A. R. and Crandall, K. A. (2014). Life in extreme environments: Microbial diversity in Great Salt. Extremophiles, vol. 1904, pp. 525–535.
 Cheesbrough, M. (2000). District Laboratory Practices in Tropical Countries (second edition). Cambridge University Press.
 Forbes, B. A., Sahm, D. F., and Weissfeld, A. S. (2007). Bailey & Scott’s Diagnostic Microbiology (twelfth edition). Elsevier Inc.
 Giraffa, G., Rossetti, L., and Neviani, E. (2000). An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria. Journal of Microbiological Methods, vol. 42, pp. 175–184.
 Lal, S., Mistry, K. N., and Patel, J. G. (2013). Evaluation of amplified rDNA restriction analysis (ARDRA) for identification and characterization of arsenite resistant. International Science Press, vol. 6, no. 1, pp. 12–20.
 Lee, P. Y., Costumbrado, J., Hsu, C., et al. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments, vol. 62, pp. 1–5.
 Johnson, M., Zaretskaya, I., Raytselis, Y., et al. (2008). NCBI BLAST: A better web interface. Nucleic Acids Research, vol. 36, pp. 5–9.
 Quast, C., Pruesse, E., Yilmaz, P., et al. (2013). The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Research, vol. 41, pp. 590–596.
 Larkin, M. A., Blackshields, G., Brown, N. P., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics Applications Note, vol. 23, no. 21, pp. 2947–2948.
 Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, vol. 41, pp. 95–98.
 Kumar, S., Stecher, G., and Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, vol. 33, no. 7, pp. 1870–1874.
 Thiruvengadam, S., Shreenidhi, K. S., Vidhyalakshmi, H., et al. (2014). A study of bacterial profiling on coins and currencies under circulation and identifying the virulence gene in Chennai (TN). International Journal of ChemTech Research, vol. 6, no. 9, pp. 4108–4114.
 State, O. and State, E. (2014). Antibiotic resistance and public health perspective of bacterial contamination of Nigerian currency. Advances in Life Science and Technology, vol. 24, pp. 4–15.
 Daeschel, M. A. and Fleming, H. P. (1984). Selection of lactic acid bacteria for use in vegetable fermentations. Food Microbiology, vol. 1, no. 4, pp. 303–313.
 Ahmed, S. U., Parveen, S., Nasreen, T., et al. (2010). Evaluation of the microbial contamination of Bangladesh Paper currency notes (Taka) in circulation. Advances in Biological Research, vol. 4, no. 5, pp. 266–271.
 Enemuor, S. C., Victor, P. I., and Oguntibeju, O. O. (2012). Microbial contamination of currency counting machines and counting room environment in selected commercial banks. Scientific Research and Essays, vol. 7, no. 14, pp. 1508–1511.
 New, O. K., Win, P. P., Han, A. M., et al. (1989). Contamination of currency notes with enteric bacterial pathogens. Journal of Diarrheal Diseases Research, vol. 7, pp. 92–94.
 Jiang, X. and Doyle, M. P. (1999). Fate of Escherichia coli O157:H7 and Salmonella Enteritidis on currency. Journal of Food Protection, vol. 62, No. 7, pp. 805–807.
 Macrae, A. (2000). The use of 16S rDNA methods in soil microbial ecology. Brazilian Journal of Microbiology, vol. 31, pp. 77–82.
 Schmidt, T. M., Delong, E. F., and Pace, N. R. (1991). Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. Journal of Bacteriology, vol. 173, no. 14, pp. 4371–4378.
 Surfaces, C., Santo, C. E., Morais, P. V., et al. (2010). Isolation and characterization of bacteria resistant to metallic copper surfaces. Applied and Environmental Microbiology, vol. 76, no. 5, pp. 1341–1348.
 Kalita, M., Szysz, M. P., and Szewczuk, A. T. (2013). Isolation of cultivable microorganisms from Polish notes and coins. Polish Journal of Microbiology, vol. 62, no. 3, pp. 281–286.
 Doi, Y. and Arakawa, Y. (2007). 16S Ribosomal RNA methylation: Emerging resistance mechanism against aminoglycosides. Antimicrobial Resistance, vol. 45, no. 1, pp. 88–94.
 Fournier, P. E. and Raoult, D. (2011). Prospects for the future using genomics and proteomics in clinical microbiology. Annual Review of Microbiology, vol. 65, pp. 169–188.
 Conlan, S., Kong, H. H., and Segre, J. A. (2012). Species-level analysis of DNA sequence data from the NIH Human Microbiome Project. PloS One, vol. 7, no. 10, p. 47075.
 Ellistrem, M. C. and Stout, J. E. (2000). Simplified protocol for pulsed-field gel electrophoresis analysis of Streptococcus pneumoniae. Journal of Clinical Microbiology, vol. 38, no. 1, pp. 351–353.
 Trindade, P. A., McCulloch, J. A., Oliveira, G. A., et al. (2003). Molecular techniques for MRSA typing: Current issues and perspectives. The Brazilian Journal of Infectious Diseases, vol. 7, no. 1, pp. 32–43.
 Babalola, O. O. (2003). Molecular techniques: An overview of methods for the detection of bacteria. African Journal of Biotechnology, vol. 2, pp. 710–713.
 Young, N. D. and Tanksley, S. D. (1989). Restriction fragment length polymorphism maps and the concept of graphical genotypes. TAG Theoretical and Applied Genetics, vol. 77, no. 1, pp. 95–101.
 Wassenaar, T. M. (2000). Genotyping of Campylobacter spp. Applied and Environmental Microbiology, vol. 66, no. 1, pp. 1–9.
 Ahmed, O. B. and Mashat, B. H. (2015). Occurrence of ESBL, MRSA and VRE pathogens in contaminated banknotes in Makkah, Saudi Arabia. Global Advanced Research Journal of Microbiology, vol. 4, no. 9, pp. 27–30.
 Gabriel, E. M., Coffey, A., and O’Mahony, J. M. (2013). Investigation into the prevalence, persistence and antibiotic resistance profiles of Staphylococci isolated from euro currency. Journal of Applied Microbiology, vol. 115, no. 2, pp. 565–571.
 Alemu, A. (2014). Microbial Contamination of Currency Notes and Coins in Circulation: A Potential Public Health Hazard. Biomedicine and Biotechnology, vol. 2, no. 3, pp. 46–53.