Detection, Identification and Characterization of Staphylococci in Street Vend Foods—Characterization of Staphylococcus Isolates

Rohinishree Yadahalli Shrihari, Pradeep Singh Negi

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DOI: 10.4236/fns.2011.24044                

Abstract

In the present investigation the diversity of the Staphylococcus species in different street vend food samples was studied. A total of 35 staphylococcal food isolates comprising of various species from 14 different street vend food samples were identified and characterized phenotypically. Staphylococcus aureus was found to be the most prevalent species in these foods. A PCR-RFLP analysis based on 16S rRNA gene was used for identification of Staphylococcus species. Isolates showing distinct RFLP pattern for AluI restriction digestion were selected for nucleotide sequence analysis. Phylogenetic tree constructed using the multiple alignments of 16S rRNA gene sequences of isolates showed a hotspot region of 169 bp and the relationship among species was evaluated by bootstrap values generated in phylogenetic analysis. 16S rRNA gene sequences allowed bacterial identification that was reproducible and more accurate than that obtained by phenotypic testing. 16S rRNA gene sequence analysis would be helpful in timely and correct identification of pathogens.

Keywords

Staphylococcus, 16S rRNA Gene Sequence, RFLP, Phylogenetic Tree

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	W. E. Kloos and K. H. Schleifer, “Genus IV Staphylococcus,” In: P. H. A Sneath, et al., Eds., Bergey’s Manual of Systematic Bacteriology, Vol. 2, Williams & Wilkins, Baltimore, 1986, pp. 1013-1035.
[2]	H. Asperger and P. Zangerl, “Staphylococcus aureus,” In: H. Roginski, J. W. Fuquay and P. F. Fox, Eds., Encyclopedia of Dairy Sciences, Vol. 4, Academic Press and Elsevier Science, Amsterdam, 2003, pp. 2563-2569.
[3]	J. A. Boerema, R. Clemens and G. Brightwell, “Evaluation of Molecular Methods to Determine Enterotoxigenic Status and Molecular Genotype of Bovine, Ovine, Human and Food Isolates of Staphylococcus aureus,” International Journal of Food Microbiology, Vol. 107, No. 2, 2006, pp. 192-201. doi:10.1016/j.ijfoodmicro.2005.07.008
[4]	G. Normanno, A. Firinu, S. Virgilio, G. Mula, A. Dambrosio, A. Poggiu, et al, “Coagulase–Positive Staphylococci and Staphylococcus aureus in Food Products Marketed in Italy,” International Journal of Food Microbiology, Vol. 98, No. 1, 2005, pp. 73-79. doi:10.1016/j.ijfoodmicro.2004.05.008
[5]	J. M. Jay, “Staphylococcal Gastroenteritis. Modern Food Microbiology,” 4th Edtion, Van Norstrand Reinhold, New York, 1992, pp. 455–478.
[6]	A. A. Adesyn, S. R. Tatini and D. Hoover, “Production of Enterotoxin by Staphylococcus hyicus,” Veterenary Microbiology, Vol. 9, No. 5, 1984, pp. 487-495. doi:10.1016/0378–1135(84)90069–5
[7]	K. Becker, B. Keller, C. Von Eiff, M. Brück, G. Lubritz, J. Etienne and G. Peters, “Enterotoxigenic Potential of Staphylococcus intermedius,” Applied and Environmental Microbiology, Vol. 67, No. 12, 2001, pp. 5551-5557. doi:10.1128/AEM.67.12.5551–5557.2001
[8]	E. Y. Hirooka, E. E. Muller, J. C. Freitas, E. Vicente, Y. Yoshimot and M. S. Bergdoll, “Enterotoxigenicity of Staphylococcus intermedius of Canine Origin,” International Journal of Food Microbiology, Vol. 7, No. 3, 1988, pp. 185-191. doi:10.1016/0168–1605(88)90036–0
[9]	F. M. Khambaty, R. W. Bennet and D. B. Shah, “Application of Pulse–Field Gel Electrophoresis to the Epidemiological Characterization of Staphylococcus intermedius Implicated in a Food–Related Outbreak,” Epidemiology and Infection, Vol. 113, No. 1, 1994, pp. 75-80. doi:10.1017/S0950268800051487
[10]	L. Bautista, et al., “A Quantitative Study of Enterotoxin Production by Sheep Milk Staphylococci,” Applied and Environmental Microbiology, Vol. 54, No. 2, 1988, pp. 566-569.
[11]	M. A. Al–Bustan, E. E. Udo and T. D. Chugh, “Enterotoxin Production by Coagulase–Negative Staphylococci in Restaurant Workers from Kuwait City May be a Potential Cause of Food Poisoning,” Journal of Medical Microbiology, Vol. 48, No. 9, 1999, pp. 819-823. doi:10.1099/00222615–48–9–819
[12]	M. E. Marin, M. C. de la Rosa and I. Cornejo, “Enterotoxigenicity of Staphylococcus Strains Isolated from Spanish Dry–Cured Hams,” Applied and Environmental Microbiology, Vol. 58, No. 3, 1992, pp. 1067-1069.
[13]	J. Valle, L. E. Gomez, S. Piriz, J. Goyache, J. A. Orden and S. Vadilo, “Enterotoxins Production by Staphylococcal Isolated from Healthy Goats,” Applied and Environmental Microbiology, Vol. 56, No. 5, 1990, pp. 1323-1326.
[14]	F. Vandenesch, S. J. Projan, B. Kreiswirth, J. Etienne and R. P. Novick, “Agr-Related Sequences in Staphylococcus lugdunensis,” FEMS Microbiology Letter, Vol. 111, No. 1, 1993, pp. 115-122. doi:10.1111/j.1574–6968.1993.tb06370.x
[15]	T. L. Bannerman, “Staphylococcus, Micrococcus, and Other Catalase–Positive Cocci That Grow Aerobically,” In: P. R. Murray, et al., Eds., Manual of Clinical MicroBiology, ASM Press, Washington DC, 2003. pp. 384-404.
[16]	D. W. Russell and J. Sambrook, “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, 2001.
[17]	L. H. Lambert, T. Cox, K. Mitchell, R. A. Rossello–Mora, C. D. Cueto, D. E. Dodge, P. Orkand and R. J. Cano, “Staphylococcus succinus sp. Nov, Isolated from Dominican Amber,” International Journal of Systematic Bacteriology, Vol. 48, No. 2, 1998, pp. 511-518.
[18]	M. Kuroda, A. Yamashita, H. Hirakawa, M. Kumano, K. Morikawa, M. Higashide, A. Maruyama, Y. Inose, et al., “Whole Genome Sequence of Staphylococcus saprophyticus Reveals the Pathogenesis of Uncomplicated Urinary tract Infection,” Proceedings of the National Academy of Science, Vol. 102, No. 37, 2005, pp. 13272-13277. doi:10.1073/pnas.0502950102
[19]	I. Tiago, A. P. Chung and A. Verissimo, “Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic aerobic Populations,” Applied and Environmental Microbiology, Vol. 70, No. 12, 2004, pp. 7378-7387. doi:10.1128/AEM.70.12.7378–7387.2004
[20]	M. T. Holden, E. J. Feil, J. A. Lindsay, S. J. Peacock, N. P. Day, M. C. Enright, T. J. Foster, C. E. Moore, et al. “Complete Genomes of Two Clinical Staphylococcus aureus Strains: Evidence for the Rapid Evolution of Virulence and Drug Resistance,” Proceedings of the National Academy of Science, Vol. 101, No. 26, 2004, pp. 9786-9791. doi:10.1073/pnas.0402521101
[21]	J. G. Neigel and A. C. Avise, “Phylogenetic Relationship of Mitochondrial DNA under Various Models of Speciation,” In: S. Karlin and E. Nevo, Eds., Evalution Process and Theory, Academic Press, New York, 1986, pp. 515-534.
[22]	N. Takahata and M. Nei, “Gene Genealogy and Variance of Interpopulational Nucleotides Difference,” Genetics, Vol. 110, No. 2, 1985, pp. 325-344.
[23]	K. Becker, D. Harmsen, A. Mellmann, M. Christian, P. Schumann, G. Peters and C. von Eiff, “Development and Evaluation of a Quality–Controlled Ribosomal Sequence Database for 16S Ribosomal DNA–Based Identification of Staphylococcus Species,” Journal of clinical Microbiology, Vol. 42, No. 11, 2004, pp. 4988-4995. doi:10.1128/JCM.42.11.4988–4995.2004
[24]	A. Y. C. Kwok, et al., “Species Identification and Phylogenetic Relationships Based on Partial HSP60 Gene Sequences within the Genus Staphylococcus,” International Journal of Systematic Bacteriology, Vol. 49, No. 3, 1999, pp. 1181-1192. doi:10.1099/00207713–49–3–1181
[25]	M. Palka-Santini, S. Puzfeld, B. E. E. Cleven, M. Kronke and O. Krut, “Rapid Identification, Virulence Analysis and Resistance Profiling of Staphylococcus aureus by Gene Segment–Based DNA Microarrays: Application to Blood Culture Postprocessing,” Journal of Microbiological Methods, Vol. 44, No. 7, 2006, pp. 2389-2397.
[26]	J. E. Clarridge III, “Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases,” Clinical Microbiology Review, Vol. 17, No. 4, 2004, pp. 840-862. doi:10.1128/CMR.17.4.840–862.2004