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By definition, an antibiotic is either a natural product of a micro-organism, an identical synthetic product or a similar semi synthetic product, that inhibits the growth of other microorganisms (bacteriostatic effect) or destroys other microorganisms (bactericide effect).

In the field of food hygiene and food control we deal with analysis of antibiotic residues in food of animal origin due to the potential of unwanted consequences . Among them are sensitivity to antibiotics, allergic reactions and imbalance of intestinal microflora in people, spread of resistance to antibiotics in microorganisms and losses in the food industry where antibiotics can influence starter cultures used in the production of meat and milk products.

Microbial methods were the first choice of systematic detection of antibiotic residues in food in the past and are still mainstream screening methods. They allow determination of the presence of antibiotics in the sample and identification of specific antibiotic groups.

The most frequently used microbial method is based on the principle of inhibition of growth of testing strains which is known as the STAR five-plate method.It is used for detection of antibiotics from the macrolide, aminoglicoside, tetracycline and cephalosporine families.

Material and methods
Microbiological methods are based on the measurement and evaluation of zones of inhibited bacterial growth on media. Two test strains are used to assess the presence of each antibiotic – one maximally sensitive and the other resistant to the tested substance. With the combination of different sensitive and resistant bacterial strains, specific antibiotic groups present in the sample can be identified. In our research we used the following strains with previously established sensitivity and resistancy profiles: Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 2341, Escherichia coli ATCC 10536, Staphylococcus epidermidis ATCC 12228 and E. coli ATCC 10536 (manufactured by OXOIDTM).

Preparation of bacterial cultures and media
Bacterial strains stored as cultures in original bacterial loops (Culti loop) were applied to a test tube containing 1ml Trypton soya broth (TSB) medium and incubated at 37 oC for one hour. The culture was then inoculated on blood agar and incubated for further 16 hours at the same temperature. Afterwards the purity of bacterial colonies was assessed with a light microscope and pure colonies were stored in a fridge at temperatures between 2 oC and 8 oC for up to one month. To compose test plates, bacterial culture was diluted with normal saline containing peptone water to produce a suspension which was then incubated at 37 oC for one hour and afterwards added to the agar medium specified below. The suspension density was standardised with the Mc Farland method.

Basic media for preparation of test plates were antibiotic agar No. 1 (MerckTM) and antibiotic agar No. 2 (MerckTM). Antibiotic agar No. 1 was prepared as follows: 1000 ml of distilled water was added to 30, 5 g of the medium, left for 15 min and then heated to boiling point so that the medium was completely dissolved. The medium was then autoclaved at 121 oC for 15 min. For antibiotic agar No. 2 1000 ml of distilled water was added to 15, 5 g of medium and then the same procedure was followed. After autoclaving, the pH of the media was set to desired values: pH 8 for Er, I BGA, Kin and AC plates and pH6 for E plates.

Preparation of test plates
Test plates were marked according to the bacterial strain added to the medium: AC plate – Micrococcus luteus ATCC 2341, ER plate - Staphylococcus epidermidis ATCC 12228, I-BGA plate - Bacillus subtilis BGA, Kin plate - E. coli ATCC 10536 and E plate – Bacillus cereus ATCC 11778. The pH of the medium was maintained at 8.0 for AC, E and ER plates and at 6.0for I-BGA and Kin plates. We defined the tolerance for the width of inhibition zone at (as) 8.5 mm – 0.5 mm wider than the width of the metal cylinder containing the sample. Inhibition zones between 8 mm and 8.5 mm wide were considered a non-specific reaction. To prepare a test plate 0.45 ml of suspension of bacterial culture was added to 40 ml of basic medium and heated to 40 0C. Kin plate was an exception where 0.2 ml of suspension was added to 50 ml of medium. The mixture of medium and bacterial culture was poured into a petri dish (5 ml of mixture into each petri dish). At room temperature the petri dishes with silified medium were enveloped in a parafilm and stored in a fridge. The storage period of test plates was one week. Before application of samples to test plates, plates were warmed at room temperature for 20 to 30 min.

Preparation of milk samples
To test the sensitivity of our method, milk samples containing known concentrations of standard antibiotics were inoculated on test plates. Prior to the addition of antibiotics, milk was always tested for the presence of inhibitory substances.

As the initial step, standard antibiotic solutions were prepared using reference chemical composition and purity. Standard antibiotics in powder were dissolved in appropriate solvents: tetracyclines in phosphate buffer with pH value 4.5, cephalosporines in phosphate buffer with pH value 6.0, aminoglicosides in phosphate buffer with Ph value 8.0, and macrolides in methanol. Standard solutions were diluted to desired concentrations with UHT milk containing 1.6% fat (Ljubljanske mlekarne). These samples of milk with known concentrations of antibiotics were then poured into 10-ml test tubes and heated to 80 oC for 5 min to avoid later non-specific reaction on test plates. After heating, the samples were cooled and transferred to plates in 8 mm wide cylinders. Test plates were incubated at 37 oC (I-BGA, AC, Er, Kin) or at 30 oC (E plate) for 18-24 hours. For each antibiotic we used milk samples containing antibiotic concentrations equal to MRL and half the MRL for that substance. If at half the MRL the result was still positive, lower concentrations of antibiotic were applied until the minimal level of detection was reached.

Confirmation solutions
To confirm the presence of antibiotic groups or their individual representatives we used confirmation solutions. These solutions inhibit the action of certain antibiotics and can help to distinguish between antibiotic groups which cause inhibition zones on the same test plates. Magnesium sulphate (MgSO4) was used to neutralise the aminoglicosides and cephalosporinase enzyme to neutralise the cephalosporines.

25 μl of 20% MgSO4 solution in water was added to the sample on E, AC and I-BGA plates where inhibition zones are produced by aminoglicosides, macrolides or tetracyclines. 25 μl of cephalsporinase was added to samples on AC and I-BGA plates to identify cephalosporines.

Evaluation of results
Results of microbial methods can be evaluated both qualitatively and quantitatively. Qualitative results are obtained by analysing the effect of antibiotics on a combination of sensitive and resistant bacterial strains. When required, neutralizing substances (confirmation solutions) can help to differentiate between antibiotics with similar action on test bacterial strains.

Quantitatively the concentration of antibiotic can be assessed with microbial methods if the sample contains a known antibiotic or an antibiotic that has previously been identified qualitatively. In each case a calibration curve is required.

We have confirmed sensitive and resistant bacterial strains for all antibiotic groups tested in our study. Based on our results we chose to use Bacillus cereus ATCC 11778 (E plate) as the sensitive and Micrococcus luteus ATCC 9341 (AC plate) as the resistant strain for tetracycline and chlortetracycline from the tetracyclines group. For tylosine and erythromycine from the macrolides group Micrococcus luteus ATCC 9341 (AC plate) was chosen as the sensitive and Escherichia coli ATCC10536 (Kin plate) as the resistant strain. For gentamycine, sterptomycine and neomycine from the aminoglicosides group Bacillus subtilis BGA (I-BGA plate) was chosen as the susceptible and Staphylococcus epidermidis ATCC 12228 (ER plate) the resistant strain. For cephalexine, cephoperasone and cephasoline from the sensitive group Micrococcus luteus ATCC 9341 (AC plate) was chosen as the susceptible and Staphylococcus epidermidis ATCC 12228 (ER plate) as the resistant strain.

We differentiated between antibiotic groups using a combination of five test plates (Table 3). To discriminate between aminoglicosides and macrolides we had to utilise used magnesium sulphate which inactivates the aminoglicosides. To discriminate between cephalosporines and macrolides we used the cephalosporinase enzyme.

Table 4 shows the limit of detection for milk samples containing standardised antibiotic solutions on selected test plates. The level of detection was at or below the MRL in all tested antibiotics.

Microbial methods for detection of antibiotic residues in food of animal origin are used as a screening method in the majority of laboratories in Europe that deal with analyses of drug residues in food.They are always the method of choice for screening purposes as they allow qualitative detection of antibiotics in the sample and identification of antibiotic groups. This facilitates subsequent confirmation of specific antibiotic residues with chemical methods. Microbial methods are relatively inexpensive, easy to use, do not require expensive equipment and can be efficiently adopted by laboratory staff. Although minimal expenditure is a significant factor of analyses, no test is valuable if it does not give reliable results. We succeeded in developing a microbial method which is sensitive and meets the legislative requirements – to detect concentrations of antibiotics below the MRL. For some antibiotics the level of detection was at half the MRL or lower.Microbial methods are semi quantitative, therefore any positive or suspicious result should be confirmed by chemical methods. In accordance with the EC 2002/657/EC regulation results of microbial methods are not reported as negative and positive, but as satisfactory or suspect when the MRL is exceeded.

Although the STAR five-plate test is the official method approved by the Community Reference Laboratory, many variations of microbial methods are used across the world and most laboratories apply a specific approach with a different number and types of bacterial strains and therefore a different number of test  plates.

1. Indian Pharmacopoeia 2007, Published By The Indian Pharmacopoeia Commission,Government Of India Ministry Of Health & Family Welfare,Central Indian Pharmacopoeial Laboratory,Printed By National Institute Of Science Communication And Information Resources (NISCAIR) Council Of Scientific & Industrial Research, Page No.45
2. The United State Pharmacopoeia,The National Formulary,Published By United States Pharmacopoeial Convention,INC.12601,Twinbrook Parkwag,Rockville,MD 20852,Asian Edition 2005,Page No.2256
3. slovetres.si/files/pdf/volume2006/vol43_4/SlovVetRes_43_4_pp161-168.pdf


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