Since use of antibiotics and to monitor the antibiotic

the discovery of penicillin G (the world’s first antibiotic; a ?-lactam
antibiotic synonymously known as benzylpenicillin) in 1928 by Sir Alexander
Fleming 1, antibiotics have revolutionized
the world of modern medicine. The treatment of wounds and infections with
penicillin has been saving millions of people around the world. Moreover,
antibiotics are being used to a great extent in livestock production. Due to the
extensive use in animal husbandry and inappropriate prescribing of antibiotics
in human medicine, many types of bacteria have developed resistance; some have even
become resistant to more than one type of antibiotic (reviewed e.g. in 2, 3). The antibiotic resistance of disease-causing
bacteria is of particular concern since they can be transmitted to humans both
by animal products and by daily contact with other humans, or upon medical
therapy. The incidence and spread of different resistance mechanisms is
threatening our ability to treat even common infections, which can result in
prolonged illness, epidemic/pandemic spread of infections, use of more toxic
drugs and increasing mortality rates of infectious diseases 4, 5. Therefore, it is important to
support efforts to minimize the inappropriate use of antibiotics and to monitor
the antibiotic residues in wastewater, offal and animal products, especially when
entering the human food chain via meat, milk and eggs. In order to control this
threat, the European Union (EU) has issued strict regulations including
specific maximum residue limits (MRLs) for each veterinary drug in animal
husbandry (Council Regulation 2377/90/EEC).
For instance, the MRL of benzylpenicillin (penicillin G) in milk is
4 µg/kg (? 12 nM) 6, 7. A multitude of analytical
methodologies has been developed (as e.g. comprehensively reviewed in 8-13). Some
methods were designed to easily yield clinically relevant information on
whether or not bacteria from hospitals can degrade antibacterial drugs and thus
are resistant to those. Important detection methods are based on the cleavage
of the ?-lactam ring of antibiotics such as penicillin and cephalosporin by the
bacterial enzyme ?-lactamase (Figure 1A). The activity of ?-lactamases, like
penicillinase, has been assayed by a variety of chemical methods such as
manometric techniques (detection of CO2 evolution in bicarbonate
buffers) 14, 15, growth inhibition, and a diversity
of chromogenic methods, including acidometric methods with pH indicators (halochromic
dyes 16, 17; Figure 1C), or directly with chromogenic
substrates (e.g. chromogenic cephalosporin 18; Figure 1D), or iodometric
assays (compared e.g. in 18-20; Figure 1B) in combination
with spectrophotometry. Acidometric methods, as
used in this work, are based on the formation of penicilloic acid with a
pH change, monitored by a pH indicator in a low-buffered system
(Figure 1C). The halochromic indicators mostly applied are phenol red
(e.g. 16, 21-23, 20) and bromcresol purple (19, 24, 25, for review 12).