Antibiotics, detection 3-lactams

One approach to combating antibiotic resistance caused by P-lactamase is to inhibit the enzyme (see Enzyme inhibition). Effective combinations of enzyme inhibitors with P-lactam antibiotics such as penicillins or cephalosporins, result in a synergistic response, lowering the minimal inhibitory concentration (MIC) by a factor of four or more for each component. However, inhibition of P-lactamases alone is not sufficient. Pharmacokinetics, stability, ability to penetrate bacteria, cost, and other factors are also important in determining whether an inhibitor is suitable for therapeutic use. Almost any class of P-lactam is capable of producing P-lactamase inhibitors. Several reviews have been pubUshed on P-lactamase inhibitors, detection, and properties (8—15). 

Sanz ML, Gamboa PM, Antepara I, Uasuf C, Vila L, Garda-Aviles C, Chazot M, De Week L Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate-type reactions to (J-lactam antibiotics. Clin Exp Allergy 2002 32 277-286. 

Penicillins are one of the most important families of antibiotics used in veterinary and human medicine. But due to their rapid transformation in environmental media (easy hydrolysis of the /1-lactam ring), their persistence in environmental samples should be low. Thus, some works aimed at detecting antibiotic residues in water samples point out the absence of penicillin residues in spite of this drug being widely used [24,25]. 

Disc Assay - This is the simplist of the procedures and involves the placing of a standard 1/2 disc saturated with milk onto the surface of B. stearothermophilus seeded agar plate and co-incubating with suitable control discs at 55 or 64 C until well-defined zones of inhibition are obtained, usually 3-4 h. Confirmation using penicillinase-treated milk is required. Zones 14.0 mm are positive. The lower limit of detection is 0.008 units penicillin/mL. This type of assay is simple, reasonably rapid and reasonably sensitive. Quantitation is possible by using graded concentrations of penicillin in the control milk. The technique is limited, however, to 3-lactam antibiotics, primarily penicillin ( ). 

Other miscellaneous assays for penicillin or other 3-lactams in milk is the Penzyme Test which uses cell wall enzjrmes inhibited by 3-lactam drugs in a kinetic assay. This test system is purported to be able to detect 0.005 units penicillin/mL and requires approximately 30 min to complete. It, like many other assays, detects 3-lactam antibiotics only. 

Table II. Chromatographic Methods for Detection of B-lactam Antibiotic Residues in Foods...

Penicillins form several major metabolites which are excreted in the urine (83,84). These metabolites are usually inactive microbio-logically and they would not be detected by the usual microbiological tests. There are no analytical methods for these metabolites in tissues and, therefore, little is known as to their occurrence and persistence in tissues. There are no methods available for identifying residues of some commonly used B-lactam antibiotics including carbenicillin and ticarcillin. For cephapirin and ampicillin, except for one HPLC method for ampicillin in milk (79) only TLC procedures (72-74,76) with detection by bioautography are reported. 

Although some European countries still accept the results of the four plate test as confirming the presence of antibiotic residues in samples ( ), other work indicates that FPT test is not necessarily reliable. The occurrence of natural microbial inhibitors in tissues has frequently been noted (4,9,49,82), It has also been frequently observed that the results obtained by microbial and physicochemical procedures sometimes differ considerably (9,10,45,82,86), Results obtained in our laboratory suggest that even inactivation by penicillinase may not be totally specific for B-lactam antibiotics (W), The specificity of immunoassay procedures depends on the specificity of the antibody used in the test (95), Specific antisera are not widely available at present. Physicochemical procedures are therefore essential for identification and confirmation of suspect residues detected by microbiological tests. 

Other sources of PPCP contamination to groundwater can originate from farms, leaking septic tanks, and lagoons. For instance, Campagnolo et al. (2002) detected several types of antibiotics including macrolides, tetracycline, sulfonamides, and (3-lactams in groundwater samples collected from sites that were in proximity of a swine farm. 

Results showed a total of 2.8% of the samples (n 2972) to be inhibitor positive by the Delvotest SP test further examination identified 1.7% as -lactam antibiotics, and 1.1 % as sulfonamides and dapsone. The percentage of chloramphenicol suspicious samples determined by the Charm II test was amazingly high however, tests for confirmation were not available and contamination of the samples by residues of the chloramphenicol-based preservative azidiol could not be excluded with certainty. Low concentrations of streptomycins were also detected in 5.7% of the samples (n 1221), but the MRL was not exceeded. Macrolide and tetracycline residues were not found in significant levels. Model trials with commercially applied yoghurt cultures confirmed how important the compliance to MRLs can be to dairy industry compared to antibiotic-free milk, a pH of 5.0 was reached with a delay of 15 min in the case of contamination with cloxacillin 30 min in the case of penicillin, spiramycin, and tylosin and 45 min in the case of oxytetracycline contamination. 

Microbial inhibition tests are extremely sensitive for -lactam antibiotics, primarily penicillin, but mostly are more than 100-fold less sensitive for other commonly used antibacterials such as macrolides, sulfonamides, tetracyclines, or chloramphenicol (4, 5). Therefore, inhibition tests usually classify residues as belonging to the -lactam group. Antibiotics other than -lactams and sulfonamides can be detected by use of the enzyme penicillinase and aminobenzoic acid, respectively (1, 6). 

The enzymatic colorimetric format is followed by the Penzyme test. This test is a qualitative enzymatic assay for rapid detection of -lactam residues in milk (28-30). The detection principle of the Penzyme test is based on measurement of the degree of inactivation of the enzyme oo-carboxypeptidase is involved in the synthesis of the bacterial cell wall by -lactam antibiotics. These residues bind specifically with the enzyme and inactivate it, thus interfering with bacterial cell wall formation. 

In the context of residue control, antibiotic residues in milk are the easiest and most practical to accommodate. In the past, inhibitor tests sensitive primarily to -lactam antibiotics were used to control drug residues in milk. More recently, otlier drugs have become the focus of concern, and new concepts for the detection of antibiotics in milk arc repeatedly being reported (31). 

In assays in which bromocresol purple indicator is incorporated into the medium (disc assay for penicillin, disc assay with indicator), a clear bluish zone around the disk appears if samples contain inhibitory substances. When addition of penicillinase to positive milk samples eliminates the zone, the inhibitors can be roughly identified as -lactams. Among these tests, the 3 h Bacillus stearothermophilus disc assay was selected in 1981 as the regulatory test for detecting antibiotic residues in milk. Although particularly sensitive to -lactams, this test is relatively insensitive to a number of commonly used antibiotics (20). 

However, recent investigations on the effect of the tissue matrix on the detection limits attained by this test have indicated that ceftiofur, sulfonamides, streptomycin, and some macrolide antibiotics cannot be detected in intact meat with the plates and the bacterial strains prescribed in the European four-plate test (81, 82). Two plates of this system were not found suitable for screening sulfamethazine or streptomycin at levels far above the MRL the third plate detected tetracyclines and -lactams up to the MRL levels whereas the fourth was sensitive to -lactams and some but not all macrolides. Detection, on the other hand, of the fluoroquinolones enrofloxacin and ciprofloxacin could only be made possible by an additional Escherichia coli plate not included in the four-plate test. 

Penicillin G is by far the most common -lactam antibiotic used in veterinary medicine however, cloxacillin, ampicillin, amoxicillin, cephalexin, and cefuro-xime also share a high proportion of the market. As a result of this rather wide use, a generic -lactam immunochemical assay that can detect several different... 

Antisera to cloxacillin/oxacillin/dicloxacillin and cefuroxime were also produced by similar procedures and successfully utilized in methods for the detection of these antibiotics in milk (34). Unfortunately, a number of other -lactams including aminopenicillins and some cephalosporins were not amenable to this mixed anhydride procedure. Thus, a carrier protein derivatization procedure was used to allow cross-linking of cephalosporins, such as cephataxime that has an acetoxy side chain, to ovalbumin. Because acetoxy groups react readily with the heterocyclic nitrogen atoms, the latter were introduced into ovalbumin through the carbodiimide-mediated derivatization of protein carboxyl groups with amino-methylpyridine (34). 

Following their extraction and cleanup, residues of -lactam antibiotics in sample extracts can be detected by either direct nonchromatographic methods, or thin-layer, gas, or liquid chromatographic methods (Table 29.3). 

Electrochemical detection has also been successively applied for the determination of nonderivatized -lactam antibiotics in edible animal products (70, 99). 

Confirmation of the identity of the -lactam residues detected by liquid chromatography has been attempted through use of photodiode array detectors (73, 75,11-19. This procedure is relatively simple, but does not offer the specificity and the sensitivity required to determine or identify trace levels of residual -lactam antibiotics in edible animal products. Better residue confirmation can be more readily attained by treatment of the suspected samples with -lactamase or penicillinase and their reanalysis (71, 80, 86-89, 105, 106-111). In this instance, absence of a chromatographic peak with the proper retention time provides unequivocal evidence that a given residue is not present above the detection limit of the method. Thus, use of -lactamase provides a simple, inexpensive and... 

The HPLC-receptorgram assay combined the advantages of HPLC separation with the multiresidue detection of the Charm II tests. The procedure was tested for identification and quantitation of the most common veterinary drugs at regulatory levels or lower. It was validated for 40 individual drugs from seven antibiotic families 10 /3-lactams, 13 sulphonamides, 8 tetracyclines, 4 macrolides, 3 amphenicols, and other miscellaneous antimicrobials. This procedure combined a simple aqueous extraction and SPE with HPLC fractionation of individual drugs. Final identification and quantitation was achieved with the Charm II test. A drug contaminant could be identified in less then 3 hours (50). 

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