ES2263361A1 - Method of detecting antibiotic residues and other antimicrobial compounds - Google Patents

Abstract

The invention relates to a novel microbiological assay method for the rapid detection of the presence or absence of antibiotic residues and other antimicrobial compounds, such as sulfamides, in biological samples, such as milk, eggs, meat, honey, serum and urine. Said novel method offers many notable advantages over current rapid microbiological assay methods that are marketed with the same or a similar objective. More specifically, the invention relates to a novel method which is based on inhibiting the growth of Geobacillus kaustophilus in the presence of samples contaminated with residues of antimicrobial compounds. Within a period of between 1 1/2 and 4 1/2 hours, the novel assay method can be used to detect a large number of antibiotics in concentrations close to the maximum residue levels (MRLs) permitted in food intended for human consumption (EEC-directive 96/23).

Description

Antibiotic residue detection method and other antimicrobial compounds.

Technical sector

Food industry.

Object of the invention

This invention aims at a new method Microbiological test for rapid presence detection or absence of residues of antibiotics and other compounds antimicrobials such as sulfa drugs in biological samples such as milk, egg, meat, honey, whey and urine. This new method it presents remarkable advantages over the current rapid methods of microbiological assay marketed with the same or similar objective.

More specifically, a new method based on the inhibition of the growth of Geobacillus kaustophilus in the presence of samples contaminated with residues of antimicrobial compounds has been devised in the invention. This new test method allows a large number of antibiotics to be detected in concentrations close to the Maximum Residue Limits (MRLs) allowed in foods intended for human consumption (Directive 96/23 EEC) within a period of 1½ to 4½ hours.

State of the art

Drug residue detection and, especially antibiotics and sulfa drugs in food, it has of extraordinary importance given its repercussions on Health Public and its effects on the technological processes of Food processing

The problem is of such magnitude that the Union European has established that they cannot be used for human consumption those foods whose substance residue content Pharmacological agents exceed a Maximum Residue Limits (MRLs), established according to Directive 96/23 EEC, which mark the current minimum requirements for trade international.

At present, it is possible to determine specifically the presence of various compounds antimicrobials, in both solid and liquid matrices, by immunochemical, enzymatic, HPLC techniques, etc., but for their simplicity, low cost and broad spectrum, based on the microbial growth inhibition are the most widely used Within this group, techniques based on placement of a disk of an absorbent material, soaked in the shows problem, on an agar plate seeded with a Certain microbial species are the oldest. These methods allow to confirm the presence of inhibitory substances by appearance, after the corresponding incubation, of a "halo of inhibition "around the disc.

There are several variants of the "plaque disc" technique that differ in the use of various species / microbial strains ( Geobacillus stearothermohilus, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Streptococcus thermophilus, Sarcina lutea , etc.), of various media and cultivation conditions, etc., which in general are useful in specific situations. However, all of them present the common inconvenience of their industriousness that prevents their systematic use for performing routine tests both in official control centers and in the laboratories of the agri-food industries.

To overcome this limitation, various ready-to-use methods based on the detection of changes induced by the metabolic activity of Geobacillus stearothermophilus var. C953 calidolactis in various media, in the presence of the problem sample. Examples of such tests have been described in EP 0005891, EP 0611001, US 4946777, US 3941658, ES 2176220 or ES 2153562. Generally, these methods provide a result within 1½ to 4½ hours by changing color of an acid-base or redox indicator added to the test system. They allow to process large number of samples so they are the most used in our days.

Geobacillus stearothermophilus var. Calidolactis C953 has rapid growth and the additional advantage that it is thermophilic, that is, its optimum growth temperature is between 50 and 70 ° C. These extreme temperatures prevent the growth of possible contaminating microorganisms present in the test medium that could affect the test result. In addition, its minimum growth temperature is greater than 45 ° C, which allows the test method to be stable at storage temperatures or room temperature.

While the use of these test methods offers numerous advantages, they also show some shortcomings in relation to the lack of sensitivity to some groups of antibiotics commonly used in therapeutic / prophylactic treatments of animals intended for human consumption. Assay methods based on the growth inhibition of Geobacillus stearothermophilus var. Calidolactis are especially sensitive to beta-lactam antibiotics, and thanks to the addition of adjuvant substances and the modification of the test conditions they are also sufficiently sensitive to tetracyclines and sulfa drugs. However, the detection limits that they present against a large majority of aminoglycoside and macrolide antibiotics sometimes differ in several logarithmic orders from established MRLs.

This specification demonstrates that the use of Geobacillus kaustophilus as a test organism makes it possible to expand the range of antimicrobial compounds that can be detected by a rapid test method ready for use in a period of time less than 4½ (see Figures 1 and 2 ).

Explanation of the invention.

The test method of detecting antibiotics and other antimicrobial compounds, object of this report, claims the use of Geobacillus kaustophilus as a test microorganism of a new rapid test method that allows one or more antimicrobial compounds to be detected in a biological sample, such as example milk, egg, meat, honey, whey or urine. This new test method comprises contacting the test sample with a test microorganism selected from the thermophilic strains of Geobacillus kaustophilus , and the provision of the conditions for carrying out the test in which: in the absence of antimicrobial compounds the activity metabolic of said test microorganism happen and can be detected; and in the presence of antimicrobial compounds said metabolic activity is inhibited and therefore cannot be detected.

This new test method allows to extend the range of antimicrobial compounds that can be detected by a quick method ready for use over a period of time less than 4½ hours.

The test medium may optionally be a solid medium, such as an agar medium, or liquid, such as for example a nutritious broth, to which the microorganism of test, the nutrients needed to support the growth of said microorganism, an indicator of metabolic activity microbial, such as a pH indicator or one or more indicators redox, and optionally, substances that allow modifying the sensitivity to certain antimicrobial compounds. All these components or part of them can optionally be added to the test medium separately using a tablet or a disc of paper.

Geobacillus kaustophilus has rapid growth and the additional advantage that it is thermophilic, so it can grow at extreme temperatures (50-70 ° C) preventing interference from possible contaminating microorganisms that could affect the result of the test method. In addition, it cannot grow at room temperature which allows the test method to be stable at storage temperatures until use.

The microorganism is preferably added to the test medium in the form of a spore suspension. Function of the time of carrying out the test or the degree of sensitivity to the different antimicrobial compounds that are intended to be achieved said microorganism will be present in a concentration of 10 4 to 10 9 CFU / ml.

The microorganism can either be preserved dispersed in a solid medium, for example an agar medium solidified, liquid, for example in a nutrient broth, or It can be preserved in a dehydrated or lyophilized form.

The test microorganism is activated when placed in contact with the problem sample and when incubated in the medium test at temperatures between 40 and 75 ° C.

Suitable nutrients include assimilable carbon sources, assimilable nitrogen sources and sources of growth factors and minerals.

The growth of the test microorganism can be detected either visually, or by changing the color of the test medium in which an indicator of the metabolic activity This may be a pH indicator, preferably bromo-cresol purple, or one or several redox indicators, preferably gloss black.

The sensitivity of the test method can be modified by various means: for example by changing the test conditions, such as temperature and time of incubation, or the pH of the test medium, varying the ratio of volumes of the detection medium and the substance to be analyzed, or by adding substances that allow modifying the sensitivity to certain antimicrobial compounds. As an example, it suggests the addition of trimethoprim to increase sensitivity of the sulfamide test method.

In relation to the test units, these They can be of different sizes, understanding that the relationship between amount of detection medium and sample problem determines The sensitivity of the test method. Thus, they can be used optionally tubes between 3-30 mm high and 1-20 mm cross section, plates, plates microtiter or blocks with tubular grooves that house the test medium The volume of the test medium and the sample Problem may vary depending on the test unit. By For example, when the test unit is a tube the volume of the test medium may range between 10 µl and 3 ml and the volume of the test sample between 10 µl and 1 ml.

In relation to the conditions of realization of the test, the test temperature may be between 40 and 75 ° C, more preferably between 60-65 ° C; the pH of the medium test between 5-8; and the incubation time can be between 1½ and 4½ hours.

Incubation can optionally be performed in a water bath, in a stove, or in any other type of conventional incubator

Reading the result of the test method can be obtained visually or optionally by measurement spectrophotometric of the absorbance changes experienced by the test medium.

Description of the drawings

Figure 1.- Comparison of the sensitivity of Geobacillus kaustophilus and Geobacillus stearothermophilus var. C953 calidolactis to various antibiotics and sulfonamides, determined according to the plate disc method. The figure represents the diameter of the inhibition halo of petri dishes inoculated with Geobacillus kaustophilus or Geobacillus stearothermophilus var. calidolactis, caused by the presence of a paper disk containing known concentrations of penicillin, ampicillin, cefazolin, tetracycline, oxytetracycline, chloramphenicol, sulfanilamide, norfloxacin, gentamicin, neomycin, polymyxin or erythromycin.

As seen in the figure, Geobacillus kaustophilus showed greater sensitivity than Geobacillus stearothermophilus var. calidolactis to all tested antimicrobial compounds, although in different amounts. While the sensitivity to beta-lactams was similar in both species, Geobacillus kaustophilus was between 30 and 45% more sensitive than Geobacillus stearothermopnilus var calidolactis against tetracycline, sulfanilamide, gentamicin, chloramphenicol, and neomycin.

Figure 2.- Establishment of the detection limit for streptomycin of the test method based on the inhibition of the growth of Geobacillus kaustophilus and performed according to the description of a preferred test described in the following section. The figure represents in abscissa the incubation time in hours and in ordinates the absorbance changes that occur as a result of the metabolic activity of the test organism.

To carry out this test, it was used a microtiter plate manufactured as described in the next section (Description of a preferred test). How control solution was inoculated 0.1 ml of distilled water, and as problem samples were inoculated 0.1 ml of distilled water contaminated with 50, 100, 200, 300, 400, 600 and 1000 ppbs of streptomycin. All samples were inoculated by quadruplicate.

As seen in the figure, the method of trial detected the presence of streptomycin in concentrations equal to or greater than 200 ppb in a time less than 4½ hours.

Description of a preferred trial

To complement the description, although of Illustrative and non-limiting, this report is attached of the description of a preferred test carried out in the following conditions:

Preparation of test plates

Test unit: as test plates are they used 96-well microtiter plates which we allowed 96 independent test units.

Test medium: a solution of 15 g was prepared agar, 5 g peptone, 3 g meat extract, and 1.2 g of dextrose in 1000 ml of distilled water. The final solution is sterilized at 121 ° C for 20 min and cooled to approximately 45-50 ° C.

Test microorganism: a volume of a suspension of Geobacillus kaustophilus spores in distilled water was added to the test medium to obtain a final concentration of 108 spores /
ml.

Indicator: a certain amount of one was added Brilliant black solution to obtain a final concentration of 200 mg per 1000 ml.

Once the components have been mixed, 150 µl of the solution was dispensed in each of the 96 microtiter plate wells under aseptic conditions. TO then the plate was closed with an aluminum foil and it was stored under refrigeration (<7 ° C).

Carrying out the test: detection of milk samples of contaminated cow with different concentrations of streptomycin

Once the test plate is tempered to At room temperature the aluminum foil was removed. TO then 0.1 ml of a sample was added in quadruplicate of distilled water, and 0.1 ml of different water solutions distilled contaminated with 50, 100, 200, 400, 600 and 1000 ppbs of streptomycin. Test performance conditions: a then the test plate was incubated for 4 hours and 30 minutes in an oven at 65 ° C.

Reading the results: while the test units inoculated with the control samples or with the samples contaminated with 50 or 100 ppbs of streptomycin viraban from the original black to acquire an orange color, test units inoculated with fresh milk samples of cow contaminated with 200 ppbs or more of streptomycin they remained dark obtaining readings spectrophotometers greater than the detection limit set at 0.3 absorbance

Claims (8)

1. Method for detecting one or more antibiotics or other antimicrobial compounds present in a biological sample based on the inhibition of the metabolic activity of a test microorganism selected from the thermophilic strains of Geobacillus kaustophilus , and comprising: a) the contact of the problem sample with the test microorganism and, b) the provision of the conditions of test performance in which: in the absence of compounds antimicrobials the metabolic activity of said organism of test happen and can be detected; and in the presence of compounds antimicrobials said metabolic activity is inhibited and by Therefore, it cannot be detected. 2. Method according to claim 1, characterized in that the test medium can optionally be a solid medium, such as an agar medium, or liquid, such as a nutrient broth, to which the test microorganism, nutrients, is added necessary to support the growth of said microorganism, and an indicator of metabolic activity. 3. Method according to claim 2, characterized in that all or part of said components may optionally be added to the test medium separately by means of a tablet or paper disk. 4. Method according to claim 2, characterized in that the test microorganism is preferably added to the test medium in the form of spores in a concentration of between 104 and 109 CFU / ml, which allows modifying the time of the test and its sensitivity to the different antimicrobial compounds. 5. Method according to claim 2, characterized in that the metabolic activity indicator can optionally be an acid-base indicator or one or more redox indicators. 6. Method according to claim 2, characterized in that it allows modifying the sensitivity of the method according to the conditions for carrying out the test such as the temperature and incubation time, or the pH of the test medium, varying the volume ratio of the medium of detection and the substance to be analyzed, or by adding substances that modify the sensitivity to certain antimicrobial compounds, such as the addition of trimethoprim to increase the sensitivity to sulfa drugs. 7. Test unit to carry out a method according to claim 2, wherein said unit can be optionally a tube or a plate, a microtiter plate or a block with tubular grooves that house the test medium as described in previous claims. Method according to previous claims, characterized in that the reading of the results can be obtained visually or optionally by spectrophotometric measurement of the absorbance changes experienced by the test medium.