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Microbiological bioassay using Bacillus pumilus to detect tetracycline in milk

Published online by Cambridge University Press:  27 February 2015

Melisa Tumini ,
Orlando Guillermo Nagel  and
Rafael Lisandro Althaus
Melisa Tumini
Affiliation:
Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral-R.P.L., Kreder 2805, 3080 Esperanza, Argentina
Orlando Guillermo Nagel
Affiliation:
Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral-R.P.L., Kreder 2805, 3080 Esperanza, Argentina
Rafael Lisandro Althaus*
Affiliation:
Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral-R.P.L., Kreder 2805, 3080 Esperanza, Argentina
*
*For correspondence; e-mail: ralthaus@fcv.unl.edu.ar
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Abstract

The tetracyclines (TCs) are widely used in the treatment of several diseases of cattle and their residues may be present in milk. To control these residues it is necessary to have available inexpensive screening methods, user-friendly and capable of analysing a high number of samples. The purpose of this study was to design a bioassay of microbiological inhibition in microtiter plates with spores of Bacillus pumilus to detect TCs at concentrations corresponding to the Maximum Residue Limits (MRLs). Several complementary experiments were performed to design the bioassay. In the first study, we determined the concentration of spores that produce a change in the bioassay's relative absorbance in a short time period. Subsequently, we assessed the concentration of chloramphenicol required to decrease the detection limit (DL) of TCs at MRLs levels. Thereafter, specificity, DL and cross-specificity of the bioassay were estimated. The most appropriate microbiological inhibition assay had a B. pumilus concentration of 1·6 × 109 spores/ml, fortified with 2500 μg chloramphenicol/l (CAP) in Mueller Hinton culture medium using brilliant black and toluidine blue as redox indicator. This bioassay detected 117 μg chlortetracycline/l, 142 μg oxytetracycline/l and 105 μg tetracycline/l by means of a change in the indicator's colour in a period of 5 h. The method showed good specificity (97·9%) which decreased slightly (93·3%) in milk samples with high somatic cell counts (>250 000 cells/ml). Furthermore, other antimicrobials studied (except neomycin) must be present in milk at high concentrations (from >5 to >100 MRLs) to produce positive results in this assay, indicating a low cross specificity.

Keywords

Bacillus pumilusbioassaymicrobial inhibition methodmilktetracycline
Type
Research Article
Information
Journal of Dairy Research , Volume 82 , Issue 2 , May 2015 , pp. 248 - 255
Copyright
Copyright © Proprietors of Journal of Dairy Research 2015 

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References

Abee, T, Groot, MN, Tempelaars, M, Zwietering, M, Moezelaar, R & van der Voort, M 2011 Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors. Food Microbiologycal 28 199–208CrossRefGoogle ScholarPubMed
Aga, DS, Goldfish, R & Kulshrestha, P 2003 Application of ELISA in determining the fate of the tetracyclines in land-applied livestock wastes. Analyst 128 658–662Google Scholar
Bogialli, S, Curini, R, Corcia, AD, Lagana, A & Rizzuti, GA 2006 Rapid confirmatory method for analyzing tetracycline antibiotics in bovine, swine, and poultry muscle tissues: matrix solidphase dispersion with heated water as extractant followed by liquid chromatography-tandem mass spectrometry. Journal Agric Food Chemistry 54 1561–1570Google Scholar
Charm, S & Zomer, E 1995 The evolution and direction of rapid detection/identification of antimicrobial drug residues. In Residues of Antimicrobial Drugs and Other Inhibitors in Milk. FIL-IDF Special Issue No. 9505, 224–233. Brussels, Belgium: International Dairy FederationGoogle Scholar
Chopra, IP, Hawkey, M & Hinton, M 1992 Tetracyclines, molecular and clinical aspects. Journal of Antimicrobial Chemotherapy 29 245–277Google Scholar
Codex Alimentarius 2010 Codex committee on residues of veterinary drugs in foods. 9th session of the 30 August – 3 September 2010. Discussion paper on methods of analysis for residues of veterinary drugs in foods (CX/RVDF 10/19/6). Vermont, USA. Avaliable on line: ftp://ftp.fao.org/codex/ccrvdf19/rv19_06e.pdfGoogle Scholar
Council Regulation 2009 Council Directive n° 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Official Journal of the European Union 15 1–72Google Scholar
Demoly, P & Romano, A 2005 Update on Beta-lactam allergy diagnosis. Current Allergy and Asthma Reports 1 9–14CrossRefGoogle Scholar
Dewdney, J, Maes, L, Raynaud, J, Blanc, F, Scheid, J, Jackson, T, Lens, S & Verschueren, C 1991 Risk assessment of antibiotic residues of beta-lactams and macrolides in food-products with regard to their immunoallergic potential. Food Chemistry and Toxicology 29 477–483Google Scholar
Diserens, J, Beck Henzelin, A, Le Breton, M & Savoy Perroud, M 2005 Antibiotics in Milk: Actual Situation & Compilation of Commercial Available Screening Methods for the Detection of Inhibitors/Antibiotics Residues in Milk. Lausanne, Switzerland: Quality & Safety Deparment, Nestlé Research CenterGoogle Scholar
Hlavka, JJ & Boothe, JH 1985 The Tetracyclines. In Handbook of Experimental Pharmacology, Vol. 78. (Eds. Hlavka, JJ & Boothe, JH), 451. Heidelberg, New York, USA: SpringerGoogle Scholar
IDF (International Dairy Federation) 2010 Current Situation & Compilation of Commercially Available Screening Methods for the Detection of Inhibitors/Antibiotics Residues in Milk. Brussels, Belgium: FIL-IDF Standard No. 442Google Scholar
Logan, NA & de Vos, P 2009 Bacillus. In The Firmicutes in Bergey's Manual of Systematic Bacteriology, 2nd edition, Vol. III. 1450 (Eds De Vos, P, Garrity, G, Jones, D, Krieg, NR, Ludwig, W, Rainey, FA, Schleifer, K-H & Whitman, WB). Nueva York, USA: SpringerGoogle Scholar
Luitz, M & Suhren, G 1995 Advantages of photometric evaluation of microbial inhibitor test. In Residues of Antimicrobial Drugs and Other Inhibitors in Milk. FIL-IDF Special Issue No. 9505, pp. 177–181. Brussels, Belgium: International Dairy FederationGoogle Scholar
Nagel, OG, Zapata, ML, Basílico, JC, Bertero, J, Molina, MP & Althaus, RL 2009 Effect of chloramphenicol on a bioassay response for the detection of tetracycline residues in milk. Journal of Food and Drug Analysis 17 36–42Google Scholar
Nagel, OG, Molina, MP & Althaus, RL 2011 Optimization of bioassay for tetracycline detection in milk by means of chemometric techniques. Letters in Applied Microbiology 52 245–252Google Scholar
Nagel, OG, Molina, MP & Althaus, RL 2013 Microbiological system in microtiter plates (MSmp) for detection and classification of antibiotic residues in milk. International Dairy Journal 32 150–155Google Scholar
Navrátilová, P 2008 Screening methods used for the detection of veterinary drug residues in raw cow milk- A review. Czech Journal of Food Sciences 26 393–401Google Scholar
Packham, W, Broome, MC, Limsowtin, GKY & Roginski, H 2001 Limitations of standard antibiotic screening assays when applied to milk for cheesemaking. Australian Journal Dairy Technology 56 15–18Google Scholar
Pikkemaat, M 2009 Microbial screening methods for detection of antibiotic residues in slaughter animals. Analitical and Bioanalitical Chemistry 395 893–905Google Scholar
Pikkemaat, M, Ostra-van Dijk, S, Schouten, J, Rapallini, M & van Egmond, H 2008 A new microbial screening method for the detection of antimicrobial residues in slaughter animals: the Nouws antibiotic test (NAT-screening). Food Control 19 781–789Google Scholar
Rasmussen, B, Noller, HF, Daubresse, G, Oliva, B, Misulovin, Z, Rothstein, DM, Ellestad, GA, Gluzman, Y, Tally, FP & Chopra, I 1991 Molecular basis of tetracycline action: identification of analogs whose primary target is not the bacterial ribosome. Antimicrobial Agents and Chemotherapy 35 2306–2311CrossRefGoogle Scholar
Roberts, MC 1996 Tetracycline resistance determinants: mechanisms of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiology Reviews 19 1–24Google Scholar
Sischo, W & Burns, C 1993 Field trial of four cowside antibiotic residue screening test. Journal of the American Veterinary Medical Association 202 1249–1254Google Scholar
Suhren, G, Reichmuth, J & Walte, H 1996 Detection of b-lactam antibiotics in milk by the penzym-test. Milchwissenschaft-Milk Science International 51 269–273Google Scholar
Toldra, F & Reig, M 2006 Methods for rapid detection of chemical and veterinary drug residues in animal foods. Trends in Food Science and Technology 17 482–489Google Scholar
Wilke, MS, Lovering, AL & Strynadka, NCJ 2005 β-Lactam antibiotic resistance: a current structural perspective. Current Opinion in Microbiology 8 525–533Google Scholar