A Modified Selective Medium Containing Benzalkonium Chloride (BKC) for the Isolation of Pseudomonas aeruginosafrom Raw Milk

Aylin Akoglu, Evrim Gunes Altuntas, Gokce Polat Yemis

Department of Food Engineering, Faculty of Engineering, University of Ankara, Diskapi, Ankara 06110, TURKEY.
Institute of Biotechnology, University of Ankara, Golbasi, Ankara 06830, TURKEY.

DOI: 10.4236/fns.2012.37125                

Abstract

A modified selective medium (modified Cetrimide Agar, mCA) consisting of 200 μg/mL benzalkonium chloride (BKC) was developed for the isolation of Pseudomonas aeruginosa from raw milk. Initially, a total of 55 isolates were obtained from 14 raw milk samples collected from several dairy plants in Ankara, Turkey. Among these isolates, 19 were identified as Pseudomonas aeruginosa, 28 as Pseudomonas fluorescens, 4 as Acinetobacter baumannii, 2 as Enterobacter intermedium, 1 asEnterobacter agglomerans, and 1 as Escherichia coli using Microbact biochemical test kit. BKC was chosen as a selective agent to suppress growth of competitive flora because it is very effective against a wide range of Gram-negative bacteria while P. aeruginosa is resistant. MICs (minimum inhibitory concentration) for BKC were determined by agar dilution method. The concentration of 200 μg/mL BKC inhibited competitive flora, while 90% of P. aeruginosa strains were resistant. When the results of enumeration of P. aeruginosa and other Gram (-) bacteria in Cetrimide Agar (CA) and mCA were compared, it was observed that mCA was more selective than the standard CA in preventing the growth of competitive flora especially of P. fluorescens.

Keywords

P. aeruginosa; Benzalkonium Chloride (BKC); Modified Cetrimide Agar; Raw Milk

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. Flint and N. Hartley, “A Modified Selective Medium for the Detection of Pseudomonas Species that Cause Spoilage of Milk and Dairy Products,” International Dairy Journal, Vol. 6, No. 2, 1996, pp. 223-230. doi:10.1016/0958-6946(95)00007-0
[2]	T. S. Gunasekera, M. R. Dorsch, M. B. Slade and D. A. Veal, “Specific Detection of Pseudomonas spp. in Milk by Fluorescence in Situ Hybridization Using Ribosomal RNA Directed Probes,” Journal of Applied Microbiology, Vol. 94, No. 5, 2003, pp. 936-945. doi:10.1046/j.1365-2672.2003.01930.x
[3]	M. L. Martins, C. L. O. Pinto, R. B. Rocha, E. F. Araújo and M. C. D. Vanetti, “Genetic Diversity of Gram-Negative, Proteolytic, Psychrotrophic Bacteria Isolated from Refrigerated Raw Milk,” International Journal of Food Microbiology, Vol. 111, No. 2, 2006, pp. 144-148. doi:10.1016/j.ijfoodmicro.2006.06.020
[4]	B. Do?an and K. J. Boor, “Genetic Diversity and Spoilage Potentials among Pseudomonas spp. Isolated from Fluid Milk Products and Dairy Processing Plants,” Applied and Environmental Microbiology, Vol. 69, No. 1, 2003, pp. 130-138. doi:10.1128/AEM.69.1.130-138.2003
[5]	W. Dwight, J. R. Lambe and P. Stewart, “Evaluation of Pseudosel Agar as an Aid in the Identification of Pseudomonas aeruginosa,” Applied Microbiology, Vol. 23, No. 2, 1972, pp. 377-381.
[6]	R. Ramalho, J. Cunha, P. Teixeira and P. A. Gibbs, “Modified Pseudomonas Agar: New Differential Medium For the Detection/Enumeration of Pseudomonas aeruginosa in Mineral Water,” Journal of Microbiology Methods, Vol. 49, No. 1, 2002, pp. 69-74. doi:10.1016/S0167-7012(01)00365-7
[7]	L. Laine, J. D. Perry, J. Lee, M. Oliver, A. L. James, C. De La Foata, D. Halimi, S. Orenga, A. Galloway and F. K. Gould, “A Novel Chromogenic Medium for Isolation of Pseudomonas aeruginosa from the Sputa of Cystic Fibrosis Patients,” Journal of Cystic Fibrosis, Vol. 8, No. 2, 2009, pp. 143-149. doi:10.1016/j.jcf.2008.11.003
[8]	H. A. Lilly and E. J. L. Lowbury, “Cetrimide-Nalidixic Acid Agar as a Selective Medium for Pseudomonas aeruginosa,” Journal of Medical Microbiology, Vol. 5, No. 1, 1972, pp. 151-153. doi:10.1099/00222615-5-1-151
[9]	G. Szita, V. Tabajdi, A. Fabian, G. Biro, O. Reichard and P. S. K?rm?czy, “A Novel, Selective Synthetic Acetamide Containing Culture Medium for Isolating Pseudomonas aeruginosa from Milk,” International Journal of Food Microbiology, Vol. 43, No. 1-2, 1998, pp. 123-127. doi:10.1016/S0168-1605(98)00104-4
[10]	P. H. McCay, A. A. Ocampo-Sosa and G. T. A. Fleming, “Effect of Subinhibitory Concentrations of Benzalkonium Chloride on the Competitiveness of Pseudomonas aeruginosa Grown in Continuous Culture,” Microbiology, Vol. 156, No. 1, 2010, pp. 30-38. doi:10.1099/mic.0.029751-0
[11]	K. Nagai, S. Ohta, H. Zenda, H. Matsumoto and M. Makino, “Biochemical Characterization of a Pseudomonas fluorescens Strain Isolated from a Benzalkonium Chloride Solution,” Biological and Pharmaceutical Bulletin, Vol. 19, No. 6, 1996, pp. 873-875. doi:10.1248/bpb.19.873
[12]	S. Langsrud, G. Sundheim and R. Borgmann-Strahsen, “Intrinsic and Acquired Resistance to Quaternary Ammonium Compounds in Food-Related Pdeudomonas spp.,” Journal of Applied Microbiology, Vol. 95, No. 4, 2003, pp. 874-882. doi:10.1046/j.1365-2672.2003.02064.x
[13]	M. E. Campbell, S. W. Farmer and D. P. Speert, “New Selective Medium for Pseudomonas aeruginosa with Phenanthroline and 9-Chloro-9-[4-(diethyamino) phenyl]9,10-dihydro-10-phenylacridine Hydrochloride (C-390),” Journal of Clinical Microbiology, Vol. 26, 1988, pp. 1910-1912.
[14]	A. A. Ternstr?m, M. Lindberg and G. Molin, “Classification of the Spoilage Flora of Raw and Pasteurized Bovine Milk, with Special Reference to Pseudomonas and Bacillus,” Journal of Applied Bacteriology, Vol. 75, No. 1, 1993, pp. 25-34. doi:10.1111/j.1365-2672.1993.tb03403.x
[15]	M. Gennarl and F. Dragotto, “A Study of the Incidence of Different Fluorescent Pseudomonas Species and Biovars in the Microflora of Fresh and Spoiled Meat and Fish, Raw Milk, Cheese, Soil and Water,” Journal of Applied Bacteriology, Vol. 72, No. 4, 1992, pp. 281-288. doi:10.1111/j.1365-2672.1992.tb01836.x
[16]	A. D. Russell and I. Chopra, “Understanding Antibacterial Action and Resistance,” 2nd Edition, Ellis Horwood, Hertfordshire, 1996.
[17]	M. Abraham, P. Venter, J. F. R. Lues, I. Ivanov and O. Smidt, “Influence of Selected Antimicrobials on the Viability, Endotoxicity and Lipopolysaccharide Composition of Pseudomonas aeruginosa in Vitro,” International Journal of Antimicrobial Agents, Vol. 34, No. 5, 2009, pp. 419-423. doi:10.1016/j.ijantimicag.2009.06.017