6-Lactam antibiotic detection

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. 

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. 

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). 

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... 

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... 

Side chain-specific allergic reactions to beta-lactams are a steadily increasing problem (SEDA-21, 260) (222-224). Apart from epitopes generated by the beta-lactam nucleus, side chains attached to it can serve as additional epitopes recognized by the host immune response. Side chain-specific antibodies can be detected in patients who are allergic to beta-lactam antibiotics, even in the absence of reactivity to the mother compound. The clinical... 

Tyczkowska, K.L. Voyksner, R.D. Straub, R.F. Aronson, A.L. Simultaneous multiresidue analysis of beta-lactam antibiotics in bovine milk by Uquid-chromatography with ultraviolet detection and confirmation by electrospray mass-spectrometry. J. AOAC Int. 1994, 77, 1122-1131. 

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