Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media

FEMS MicrobiologyLetters 96 (1992) 207-212 © 1992 Federation of European MicrobiologicalSocieties 0378-1097/92/$1)5.00 Published by Elsevier

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FEMSLE 05045

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media S t e v e n J. B e a r d a, D i a n a T. C i c c o g n a n i b, M a r t i n N. H u g h e s b a n d R o b e r t K. P o o l e a u Microbiology Group, Division o.f Biosphere Sciences, and b Department of Chemistry, King's College London, London. UK

Received8 June 1992 Accepted 15 June 1992 Key words: AmpiciUin; Hydrolysis of ampiciUin; Metal ion catalysis; Copper; Zinc; Cobalt; Cadmium

1. S U M M A R Y Anodic stripping voltammetry of bacterial growth medium containing copper(ll) and ampicillin shows that Cu(Il) is complexed by the antibiotic and that this complex decomposes to give Cu(ll) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II): the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other /3-1actam antibiotics) to metal ion-catalysed hy-

Correspondence to: M.N. Hughes, Department of Chemistry,

King's College London, Campden Hill Road, London, W8 7AH, UK.

drolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampiciUin and metals such as Cu(ll), Zn(ll), Co(ll) and Cd(lI) is impossible.

2. I N T R O D U C T I O N During studies on genetically modified microbes, it is common practice to exploit antibiotic resistance encoded by a vector or transposon, for example, as a convenient marker for the modified strain and the plasmid and gene; supplementing the growth medium with the antibiotic prevents the loss of the acquired gene. In a recent report on transport of Cu(II) in E s c h e n c h i a coil [1], it was noted that the activity of ampicillin was reduced by the presence of copper(ll) sulphate in the growth medium. The effect of metal ions on the stability of ampicillin [2,3] and penicillins generally [4] is described in the chemical literature but does not appear to be widely recognised by microbiologists. It is noteworthy that at least two /3-1actamases contain zinc (from Bacillus cereus [5] and P s e u d o m o n a s maltophilia [6]), while

208 in model systems Cu(ll) and Zn(II) dramatically catalyse the hydrolysis of a lactam by factors of 106 and 10-'~,respectively, through the attack of a metal-bound hydroxide group on the fl-lactam group [7]. We have observed that Cu(II), as well as Zn(II), Co(II) and Cd(II), all adversely affect the bacteriostatic activity of ampicillin due to their catalytic effect on the hydrolysis of the antibiotic. We report these studies to draw attention to this problem, which has important implications for the use of ~mpicillin, not only in the study of metal resistance in microorganisms, where metal concentrations may be high, but also more generally in view of the sensitivity of ampicillin to low concentrations of strong Lewis acids such as Cu(II) and Zn(II).

3. MATERIALS AND METHODS

3.1. Organisms Two strains of E. coli were used: RG145 [8] and RP68 [(AN2360)aroA,recA)].

3.Z Solutions and growth media Stock solutions of Cu(II) (100 p.g m l - i ) in 5% (v/v) nitric acid and of the sodium salt of ampicillin in double-distilled water were prepared. The ampicillin stock solution was stored at 4°C and diluted in growth medium or buffer to a final concentration of 100/~g ml- ~ immediately before use. The medium used for the growth of E. coli in chemostat cultures [9] contained (g ! -~, mM): K2HPO4-3H20 (43.25, 189.5); KH2PO 4 (8.2, 60.5); NH4C! (1.0, 187); K2SO4 (2.6, 14.9); sodium succinate (9,5, 35.0), CaCI2.2H20 (0.015, 0.1) and trace element solution (10 ml). The medium was adjusted to pH 7.2 and autoclaved. When cool, 1 ml of filter-sterilised 1 M MgCI2"6H20 and 0.5 mg each of thiamine and niacin were added per litre of medium. The trace element solution [10] contained (g l - l raM): NazEDTA. 2H20 (5, 13.43); ZnO (0.05, 0.61); CuCl 2 • 2H20 (0.01, 0.06); Co(NO3) z • 6H20 (0.01, 0.03); HaBO a (0.01, 0.16); (NH4)6Mo~O24.4H20 (0.01, 0.09); FeCI3.6H20 (0.5, 1.82). The CuCl 2 • 2H20 was

omitted in copper-depleted medium. The trace element solution was prepared by dissoMng the Na2EDTA. 2H20 in about 900 ml distilled water, adjusting the pH to 7.2 with NaOH solution, and then dissolving each of the other components separately, ensuring that the pH remained between 7.2 and 7.4 after each addition. The final volume was made up to 1 I and the solution was stored at 0°C. Copper-depleted medium was prepared by the method of Hubbard et al. [11] in which copper was extracted by use of Chelex 100 resin (Biorad Laboratories). E. coli RP68 was grown on Luria-Bertani medium [12].

3.3. Anodic stripping voltammetry Cu(II) catalysis of the hydrolysis of ampicillin was studied directly in the polarographic ceil. Citric acid buffers were used as the supporting electrolyte in initial studies, the pH of 0.1 M citric acid being adjusted to the required pH (initially 3) with ammonium hydroxide solution. Subsequently, standard buffers were used to provide a range of pH values, without adverse effects on the determination of Cu(II), although the potentials varied with pH. At each pH value, the voltammogram was obtained for the aquacopper (II) ion. All experiments were carried out on an E G & G Princeton Applied Research polarograph (model 264A) with a model 303A dropping mercury electrode and a Houston Instruments Omnigraphic 2000 recorder. A known volume of electrolyte (generally 9.60 ml) was placed in the acidwashed polarographic cells and scanned to obtain a base line. The volume was made up to 10.00 ml with the ampiciUin solution and scanned again. A small volume (usually 20/zl) of the Cu(II) solution was added at time zero and scans were made at known time intervals.

4. RESULTS

4.1. Effects of Cu(ll) on the stability of ampicillin in aqueous solution In an attempt to isolate extracellular ligands associated with the uptake of copper by E. coli, growth studies were carried out in chemostats under copper-limited conditions [9] using strain

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Fig. 1. A. Voltammograms obtained after standard additions of Cu(ll) to ~ ~ 0.1 M ammonium citrate electrolyte, pii 3. Figures show the concentrations of Cu(II) (p.g ml- t) after each addition: (a) is the base line. B. Voltammograms obtained after standard additions of Cu(ll) to 400/zl of copper-extracted medium plus 9.6 ml of an 0.1 M ammonium citrate electrolyte, pH 3. Figures show the concentration of Cu(II) (p,g m l - t ) after each addition: (b) is the base line, The additional features in the voltammograms result from the complexation of Cu(ll) by ampicillin,

RG145. This strain lacks the cytochrome bd-type quinol oxidase but over-expresses [13] the cytochrome o-type quinol oxidase from the cyo gene cloned into cosmid vector pcG1 [8] containing the genes for ampicillin resistance. The medium contained 100 p.g m l - i ampicillin. Attempts to analyse the copper concentration of the medium flowing into the chemostat by anodic stripping voltammetry by the method of standard additions were frustrated as the voltammogram showed the presence of two Cu(ll) species, one of which was characteristic of Cu(II) in the citrate electrolyte (Fig. 1). Systematic testing of the medium components was undertaken to identify the second Cu(ll) species (Fig. lb). E D T A depressed the peak height and shifted the potential of the Cu(II), as expected, but it was clear that the second Cu(II) complex involved ampicillin, which has suitable donor atoms for complexing Cu(ll). Repetitive scanning of medium containing Cu(II) and ampicillin showed that the Cu(II)ampicillin complex was unstable (Fig. 2). The peak at -0.005 V due to free Cu(II) increased in

height with time, while the peak at about -0.075 V decreased with time, showing a well-defined cross-over point of fixed height. Changes may also be seen in the +0.095 V peak over a time

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Fig. 2. Voltammograms of 0.2 t~g ml- i Cu(II) in 400 wl of copper-extracted medium plus 9.6 ml of an 0.1 M ammonium citrate electrolyte, pH 3. Voltammogramswere started at the followingtimes (rain) after addition of Cu(ll): (a), 2.4; (b), 6.3; (c) 12.2;(d), 20.8; (e), 31.6; (f), 41.6. The voltammogramof the medium sample is (g), while(h) is the baseline.

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stability of ampicillin or ampicillin fragments at the lower concentrations of Cu(II). The stability of ampicillin (10.8 p.M) in the presence of Cu(II) (3.15 p.M) was measured over the pH range 3.0-7.0. The rates and products of decomposition are pH-dependent (results not shown), with a different pattern of behaviour at pH > 5. This may reflect changes in protonation of ampicillin or the speciation of Cu(II). At pH 7, complete loss of certain peaks was observed over 20 rain, while at lower pH values smaller changes in these features took 8-20 h.

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The figuresshow the concentrationof Cu(II)(/zg ml-l)after successiveadditions.

period of about 12 min. These results suggest that a well-defined decomposition is taking place over a short time period to give at least two products. Addition of Cu(II) to ampiciUin-containingsolutions provides a convenient method for determining the presence of ampicillin or ampicillin fragments. Figures lb and 3 show the voltammograms obtained after standard additions of Cu(II) to growth medium containing ampicillin and either normal trace levels of Cu(II) (0.1/~M, Fig. 3) or Cu-extracted medium (concentration too low to be detected by atomic absorption spectroscopy, Fig. lb). Decomposition of the ampicillin has occurred in both cases. In a control experiment, it was shown that a solution of ampicillin in distilled water was stable for some days. It should be noted that the results obtained using ampicillin in Cu-extracted medium appeared to be more complex, possibly due to the greater

4.2. Growth o f an ampicillin-sensitive Escherichia coli strain on plates containing ampicillin and added Zn(II), Co(lI) or Cd(II) An E. coil genomic DNA library was constructed in the cosmid vector pHC79 [14] and transduced into a recA strain of E. coil, RP68. Transductants (Ap r) were screened for the ability to grow on Luria-Bertani plates (with ampicillin at 50 /zg ml -I) containing concentrations of ZnSO4, CoCl 2 or CdCI 2 inhibitory to growth of E. coil RP68 transformed with pHC79. Growth of putative metal-resistant clones was compared with growth of E. coil RP68/pHC79 by streaking across Luria-Bertani plates containing ampiciilin and a concentration gradient [15] or ZnSO4 (5 mM maximum), CoCl 2 (3 mM maximum) or CdCI 2 (1.5 mM maximum). Strain RP68 (Ap s) was included as a negative control for the presence of ampicillin. Unexpectedly, strain RP68 showed growth part way across all three plates. In all cases the growth increased with increasing metal concentration until the minimum inhibitory concentration of the metal was reached. At Zn(II) concentrations close to zero, strain RP68 grew only as single colonies as the ampicillin was still active. Increase in metal concentration results in greater hydrolysis of ampicillin and enhanced bacterial growth, until the metal concentration is high enough to inhibit growth.

5. DISCUSSION This polarographic study of the Cu(II)-ampicillin system shows conclusively that the ampicillin

molecule coordinates to Cu(ll) and that the ampicillin then undergoes metai-catalysed decomposition. No attempt has been made in this work to identify the detailed pathway of ampicillin decomposition, although it probably involves initial cleavage of the C - N / ] - I a c t a m group to give penicilloic acid, with alleviation of the strain associated with four-membered ring. A n u m b e r of o t h e r r e a r r a n g e m e n t s may then take place. Similar reactions will occur for fl-lactam antibiotics in general. It is also clear that the Cu(II)-catalysed decomposition of ampicillin is rapid at p H 7, taking minutes r a t h e r than the few days over which ampicillin decomposes in the absence of Cu(ll). Hydrolysis of ampicillin was observed even in media from which Cu(ll) had been extracted, showing the efficiency of Cu(II) as a catalyst, possibly via a C u - O H pathway in view of the increased rate of reaction at pH 7 compared to p H 5. T h e significance of these observations to microbial studies has been amply demonstrated by the observation that an ampicillin-sensitive strain of E. coli grew on plates containing ampicillin and Zn(II), Cd(II) or Co(II) at millimolar concentrations. A n important consequence of this observation, for example, is that double selection for resistance to ampicillin and metals such as Cu(ll), Zn(II), Co(ll) and Cd(II) is impossible. From a practical view-point, it should be noted that the efficiency of the transition metal ions as catalysts of ampicillin decomposition should follow the Irving.Williams series, with the sequence M n ( l l ) < Fe(II) < Co(ll) < Ni(lI) < Cu(lI) > Zn(II). T h e reactivity of metal ions would, however, be affected by their speciation in the medium [16]: a complex medium might well provide ligands to bind strongly to all the coordination positions on the metal centre and so reduce the possibility of coordination of the ampicillin.

ACKNOWLEDGEMENTS We t h a n k the S E R C and Castrol plc for support for D.T.C. and the S E R C for support for S.B. We are grateful to Professor R.B. G e n n i s for the gift of strain RGI45.

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