Esterases in Serum-Containing Growth Media Counteract Chloramphenicol Acetyltransferase Activity In Vitro

Abstract

The spirochete Borrelia burgdorferi was unexpectedly found to be as susceptible to diacetyl chloramphenicol, the product of the enzyme chloramphenicol acetyltransferase, as it was to chloramphenicol itself. The susceptibilities of Escherichia coli and Bacillus subtilis, as well as that of B. burgdorferi, to diacetyl chloramphenicol were then assayed in different media. All three species were susceptible to diacetyl chloramphenicol when growth media were supplemented with rabbit serum or, to a lesser extent, human serum. Susceptibility of E. coli and B. subtilis to diacetyl chloramphenicol was not observed in the absence of serum, when horse serum was used, or when the rabbit or human serum was heated first. In the presence of 10% rabbit serum, a strain of E. coli bearing the chloramphenicol acetyltransferase (cat) gene had a fourfold-lower resistance to chloramphenicol than in the absence of serum. A plate bioassay for chloramphenicol activity showed the conversion by rabbit, mouse, and human sera but not bacterial cell extracts or heated serum of diacetyl chloramphenicol to an inhibitory compound. Deacetylation of acetyl chloramphenicol by serum components was demonstrated by using fluorescent substrates and thin-layer chromatography. These studies indicate that esterases of serum can convert diacetyl chloramphenicol back to an active antibiotic, and thus, in vitro findings may not accurately reflect the level of chloramphenicol resistance by cat-bearing bacteria in vivo.

Chloramphenicol, one of the first broad-spectrum antibiotics discovered, inhibits protein synthesis by interacting with the peptidyl transferase center of ribosomes (24). The cat gene coding for the enzyme chloramphenicol acetyltransferase (CAT) provides resistance to chloramphenicol. This enzyme acetylates the antibiotic at the C-3 hydroxyl, which can then undergo a nonenzymatic rearrangement of the acyl group to the C-1 hydroxyl. This frees the C-3 for a further round of acetylation, producing 1,3-diacetyl chloramphenicol (reviewed in reference 31). Neither the mono- nor the diacetylated form of chloramphenicol has been reported to inhibit the growth of prokaryotes.

Many different cat genes have been used as selectable markers in gram-positive and -negative bacteria (2, 5, 30), as well as the eukaryote Saccharomyces cerevisiae (11). The cat gene also provided resistance to chloramphenicol in two spirochetes: Serpulina hyodysenteriae and Treponema denticola (16, 28). Based on CAT’s ability to provide resistance in a wide range of organisms, the cat gene was a promising candidate for the same purpose in the spirochete Borrelia burgdorferi, a Lyme disease agent.

B. burgdorferi is susceptible to chloramphenicol (8). Recently, the cat gene of the Staphylococcus aureus plasmid pC194 was used as a reporter gene in transiently transfected B. burgdorferi cells where functional CAT was produced (33). However, to date, no chloramphenicol-resistant mutants have been reported for any Borrelia species (32). At a minimum, for CAT to provide resistance the host should not be susceptible to the acetylated form of chloramphenicol. This appears to be universal for prokaryotes, with no known exceptions.

We attempted to resolve the paradox of the apparent inability of cat to produce chloramphenicol resistance in B. burgdorferi although producing functional CAT. For this, the interaction of this bacterium with chloramphenicol products was undertaken. We found that sera, to varying degrees, have esterase activity that converts acetylated chloramphenicol to chloramphenicol that is sufficient to alter the susceptibilities to chloramphenicol of different classes of bacteria. This phenomenon may influence the results of chloramphenicol MIC tests of cat-containing bacteria if serum is present and may prevent the use of cat as a genetic selection marker in some bacterial species.

MATERIALS AND METHODS

Media, strains, and culture conditions.

Escherichia coli XL1-Blue MRF′ (Δ[mcrA]183 Δ[mcrCB-hsdSMR-mrr]173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac[F′ proAB lacI^q ZΔM15 Tn10]) (Stratagene, La Jolla, Calif.) was the chloramphenicol-susceptible E. coli strain. E. coli XL1-Blue MRF′ with pGOΔ1, a high-copy plasmid containing the S. aureus pC194 cat gene (33), was the chloramphenicol-resistant strain. Bacillus subtilis EUR9030 (aroI916 purB33 trpC2 spoIIAC::erm) (6) and the spirochetes Spirochaeta aurantia M1 (4) and B. burgdorferi B31 (ATCC 35210) were used.

E. coli and B. subtilis were grown in Luria-Bertani (LB) broth (Difco Laboratories, Detroit, Mich.) at 37°C with shaking. S. aurantia was grown in MPY (0.2% [wt/vol] maltose–0.2% peptone–0.1% yeast extract–10 mM potassium phosphate buffer, pH 7.5) at 22°C. B. burgdorferi was grown at 34°C. Barbour-Stoenner-Kelly medium (BSK) is used as the general term for Borrelia medium, while in specific cases either BSK II (3) or BSK H (23) is indicated. BSK H was supplied by Sigma (St. Louis, Mo.). BSK II was made with the following components: CMRL 1066 (Gibco BRL, Grand Island, N.Y.), bovine serum albumin fraction V (Intergen, Purchase, N.Y.), neopeptone (Difco), HEPES (Sigma), sodium citrate (Sigma), glucose (Sigma), yeastolate (Difco), sodium bicarbonate (EM Industries, Inc., Gibbstown, N.J.), sodium pyruvate (Sigma), N-acetylglucosamine (Sigma), and gelatin (Difco).

Serum contained no preservatives and had not been heat treated. When used, it was filter sterilized through a 0.22-μm-pore-size filter (Corning Glass Works, Corning, N.Y.) and added to media at 10% (vol/vol). To isolate serum, collected blood was allowed to clot overnight at 4°C and the serum was then collected and frozen at −20°C. Rabbit serum (trace hemolyzed, delipidized) was obtained from Pel-Freez Inc. (Rogers, Ark.) or collected from one female New Zealand rabbit by ear bleed. Horse serum was from Omega Inc. (Tarzana, Calif.). Human serum was obtained from Irvine Scientific, Inc. (Irvine, Calif.), or drawn from a volunteer. Mouse (female C3H/HeN mice [Harlan Laboratories, Indianapolis, Ind.) blood was collected from the tail.

Chemicals.

For stock solutions, chloramphenicol (Sigma) and chloramphenicol diacetate (Sigma) were dissolved in dimethyl sulfoxide (American Type Culture Collection, Manassas, Va.). Spectrophotometric-grade ethyl acetate (Sigma) was used for all extractions.

MIC.

MICs were determined in duplicate. Approximately 10⁷ B. burgdorferi cells from a logarithmic-phase culture were pelleted by centrifugation for 5 min at 10,000 × g and then resuspended in phosphate-buffered saline solution to a concentration of 10⁸ cells/ml. A 50-μl volume of 5 × 10⁶ washed cells was added to 5 ml of BSK medium (final concentration; 10⁶ cells/ml). Samples were incubated at 34°C, and the cell count was determined with a Petroff-Hausser counting chamber (Hausser Scientific Partnership, Horsham, Pa.). The MIC was the lowest concentration that inhibited growth (<10⁷ cells/ml). In tubes without antibiotic, the cell count was 10⁸ cells/ml or greater after 72 h. For E. coli and B. subtilis, 10⁶ cells in a 10-μl volume from an 18-h culture were added to 5 ml of LB medium and grown for 18 h with shaking at 37°C. The MIC was the lowest concentration that inhibited growth defined as an optical density at 600 nm of greater than 0.05. A MIC of chloramphenicol of greater than 25 μg/ml was regarded as resistance.

Bioassay for chloramphenicol.

Plate bioassays to detect chloramphenicol were done by mixing medium with or without 10⁷ cells and with or without 20 μg of diacetyl chloramphenicol or chloramphenicol per ml. After the indicated interval at 30°C, the medium was extracted twice with an equal volume of ethyl acetate. The organic phase was evaporated in a Speed Vac evaporator (Savant, Farmingdale, N.Y.) with heat until dry. Each sample was resuspended in 20 μl of ethyl acetate and applied to a blank paper disc (BBL; 1/4-in. diameter). These were allowed to air dry completely before placement on a lawn of indicator bacteria of either B. subtilis or E. coli pGOΔ1. Lawns were made by swabbing an 18-h culture onto a fresh Mueller-Hinton (Difco) plate. The plates were then incubated at 37°C overnight. To estimate the amount of chloramphenicol present, a standard curve with known amounts of chloramphenicol was prepared.

Acetyl chloramphenicol esterase assay.

Reagents for the fluorescent chloramphenicol acetate esterase (CAE) assay were from the FASTCAT assay kit from Molecular Probes, Inc. (Eugene, Oreg.). The indicator reagent, AcBCAM (boron dipyromethane difluoride 1-deoxychloramphenicol-3-acetate), was used for acetyl chloramphenicol, and reagent A, BCAM (boron dipyromethane difluoride 1-deoxychloramphenicol), was used for chloramphenicol. This assay was performed without the addition of acetyl coenzyme A. Cell extracts were prepared from 5 × 10⁸ cells by first pelleting them at 10,000 × g for 5 min and washing them twice in phosphate-buffered saline. Cells were resuspended in 400 μl of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and lysed by the addition of 40 μl of 50 mM Tris-HCl (pH 8.0)–100 mM EDTA–100 mM dithiothreitol and a drop of toluene. The suspension was briefly vortexed and incubated for 30 min at 30°C (33). Cell extracts (10 μl) or sera (100 μl) were added to 10 μl of substrate and incubated at 34°C for 2 h unless otherwise indicated. To determine the background level of conversion, the identical reaction was performed with TE in place of the cell extract. The reaction was stopped by the addition of 1 ml of ice-cold ethyl acetate and extracted once with ethyl acetate. The organic phase was dried in a Speed Vac evaporator with heat until dry. The pellet was resuspended in 20 μl of ethyl acetate, spotted onto a thin-layer chromatography (TLC) plate (Whatman, Clinton, N.J.), and separated in chloroform-methanol (85:15). Densitometric quantitation was performed as previously described (33). The conversion activity was measured as the percent conversion of AcBCAM to BCAM after subtraction of the background.

RESULTS

Susceptibilities of different bacteria to diacetyl chloramphenicol.

The MICs of chloramphenicol and diacetyl chloramphenicol were determined for E. coli, B. subtilis, and B. burgdorferi, in a medium commonly used for each (Table 1). E. coli in LB medium was susceptible to 5 μg of chloramphenicol per ml while the strain with the cat-containing plasmid pGOΔ1 required a MIC of 100 μg/ml. B. subtilis was twice as susceptible as E. coli, and B. burgdorferi was twice as susceptible as B. subtilis. E. coli, with and without pGOΔ1, and B. subtilis were not susceptible to a minimum of 1,000 μg of the diacetylated form of chloramphenicol per ml. In contrast, B. burgdorferi was susceptible to 1.25 μg/ml.

TABLE 1.

MICs of chloramphenicol and diacetyl chloramphenicol for bacteria in different media

Bacterium	Medium	Serum	Heat	MIC (μg/ml)
				Chloram-phenicol	Diacetyl chlor-amphenicol
E. coli	LB	None	−	5.0	>1,000
		Rabbit	−	5.0	5.0
		Rabbit	+	10.0	>1,000
		Horse	−	5.0	400
		Human	−	2.5	200
		Human	+	5.0	>1,000
	BSK	None	−	5.0	400
		Rabbit	−	5.0	5.0
		Rabbit	+	10.0	400
E. coli pGOΔ1	LB	None	−	100	>1,000
		Rabbit	−	25	100
		Rabbit	+	200	>1,000
		Horse	−	100	>1,000
		Human	−	25	>1,000
		Human	+	50	>1,000
	BSK	None	−	100	>1,000
		Rabbit	−	100	400
		Rabbit	+	200	>1,000
B. subtilis	LB	None	−	2.5	>1,000
		Rabbit	−	2.5	2.5
		Rabbit	+	5.0	>1,000
		Human	−	1.3	400
		Human	+	2.5	>1,000
	BSK	None	−	2.5	200
		Rabbit	−	2.5	2.5
		Rabbit	+	5.0	200
B. burgdorferi	BSK	None	−	1.25	20
		Rabbit	−	1.25	1.25
		Rabbit	+	1.25	5.0
		Horse	−	1.25	5.0

Most prokaryotes are susceptible to chloramphenicol, and thus it was conceivable that the inhibition of B. burgdorferi by diacetyl chloramphenicol was due to the presence of chloramphenicol, rather than to a direct inhibitory effect of the diacetyl form. To determine if B. burgdorferi cell extracts could modify either chloramphenicol or the diacetylated form, the CAE assay was performed (Fig. 1). Neither E. coli nor B. burgdorferi cell extracts appeared to modify chloramphenicol or acetyl chloramphenicol. S. aurantia, a spirochete that converts diacetyl chloramphenicol to chloramphenicol (34), was included as a positive control. Under these conditions, in medium without serum, S. aurantia extracts converted the AcBCAM to BCAM. Thus, the susceptibility of B. burgdorferi to diacetyl chloramphenicol could not be attributed to a bacterial esterase.

FIG. 1.

CAE assay of esterase activity in cell extracts of different bacteria. Cell extracts from the indicated bacteria were prepared and incubated with either AcBCAM or BCAM for 2 h at 34°C. The reaction was then stopped with the addition of ice-cold ethyl acetate and extracted once. After drying, the reaction products were resuspended in ethyl acetate and separated by TLC.

Medium effects on diacetyl chloramphenicol.

To determine if the medium was responsible for the apparent sensitivity to diacetyl chloramphenicol, the MICs for E. coli and B. subtilis were determined in BSK. Each bacterium was susceptible to concentrations of diacetyl chloramphenicol as low as 2.5 μg/ml (Table 1). The chloramphenicol-resistant E. coli with plasmid pGOΔ1 was also more susceptible to diacetyl chloramphenicol in BSK. This suggested that the medium was converting diacetyl chloramphenicol to chloramphenicol, which in turn inhibited growth of the bacteria.

Serum contains nonspecific carboxylesterases that could be responsible for converting diacetyl chloramphenicol to chloramphenicol (15, 19, 29). In agreement, E. coli inoculated into LB medium made with the addition of each component of BSK II (3) and 20 μg of diacetyl chloramphenicol per ml grew with each component with the exception of rabbit serum. The chloramphenicol-resistant E. coli pGOΔ1 grew in all samples (data not shown). The MICs for E. coli and B. subtilis of both forms of chloramphenicol were determined in LB medium with 10% rabbit serum (Table 1). E. coli and B. subtilis were susceptible to low levels of diacetyl chloramphenicol in LB medium supplemented with rabbit serum. The MIC of chloramphenicol for E. coli pGOΔ1 decreased to a level considered to indicate susceptibility. B. burgdorferi does not grow in LB medium and could not be directly tested.

To verify the role of rabbit serum, the CAE assay was performed. The acetylated form of chloramphenicol was added to BSK with and without serum, as well as to serum alone, and the reaction was allowed to continue for 2 h (Fig. 2). Only the reactions containing serum converted the fluorescent acetyl chloramphenicol to chloramphenicol while the reaction containing BSK without serum did not. For further proof of the role of serum, a microbiological plate assay for chloramphenicol in the medium was used. Diacetyl chloramphenicol was incubated with BSK, with or without serum, for 18 h. The medium was extracted, dried, and applied to a paper disc. This was placed on a lawn of chloramphenicol-susceptible B. subtilis, and the culture was incubated for 18 h. A zone of inhibition of growth of the B. subtilis lawn around a disc indicated the production of chloramphenicol. A zone of inhibition was observed only with serum and diacetyl chloramphenicol together and not with either alone (Table 2). Based on the size of the zone, an estimated 3.5 μg of chloramphenicol was produced from 20 μg of diacetyl chloramphenicol in 18 h. There was no change in the zone with the addition of live B. burgdorferi cells. In all cases, there was no zone of inhibition if E. coli pGOΔ1 was used for the lawn.

FIG. 2.

CAE assay of the conversion of acetyl chloramphenicol to chloramphenicol. The medium or components indicated were incubated with the AcBCAM substrate for 2 h at 34°C. The reaction was then stopped with the addition of ice-cold ethyl acetate and extracted once. After drying, the reaction products were resuspended in ethyl acetate and separated by TLC.

TABLE 2.

Growth inhibition after incubation of different media and sera with diacetyl chloramphenicol^a

Medium	Serum	Bacterium	Diacetyl chloram-phenicol	Zone of inhibition (mm)	Chloramphen-icol equiva-lent (μg)
BSK	Rabbit	None	−	<6	<0.4
BSK	Rabbit	B. burgdorferi	−	<6	<0.4
BSK	Rabbit	None	+	21	3.5
BSK	Rabbit	B. burgdorferi	+	21	3.5
BSK	None	None	+	<6	<0.4
BSK	None	B. burgdorferi	+	<6	<0.4
BSK	Rabbit (heat)	None	+	<6	<0.4
LB	None	None	+	<6	<0.4
BSK	Rabbit	E. coli	+	21	3.5
MPY	None	None	+	<6	<0.4
MPY	None	S. aurantia	+	12	1.0
None	Rabbit	None	+	23	5.0
None	Rabbit (heat)	None	+	<6	<0.4
None	Mouse	None	+	23	5.0
None	Horse	None	+	7	0.4
None	Human	None	+	21	3.5

^aThe indicated components were mixed and incubated for 18 h at 30°C. The reaction mixture was then extracted twice with ethyl acetate. After drying, the products were resuspended in 20 μl of ethyl acetate and applied to a paper disc. This was placed on a plate seeded with chloramphenicol-susceptible B. subtilis and incubated for 18 h. 

From these tests, we concluded that rabbit serum contained diacetyl chloramphenicol esterase activity while B. burgdorferi cells did not. The inhibition produced on plates may be due to inhibitory compounds other than chloramphenicol. However, the growth of the chloramphenicol-resistant E. coli with pGOΔ1 was not inhibited. In addition, the fluorescent compound produced in the CAE assay appeared identical to BCAM.

Rabbit serum has conversion activity.

Diacetyl chloramphenicol deacetylase activity has been detected in many eukaryotic cell lines and can be an interfering factor in CAT assays (7). This interference was eliminated by heating the cell extract for 15 min at 70°C (21). To assess the heat susceptibility of the diacetyl chloramphenicol-converting activity in rabbit serum, the serum was heated and the MICs of the compounds were determined for E. coli and B. subtilis (Table 1). The susceptibility to diacetyl chloramphenicol in LB medium with heated serum was identical to that in LB medium without serum. A CAE assay with heated serum showed no conversion to chloramphenicol (Fig. 3). In the plate bioassay, no zone of inhibition was detected after diacetyl chloramphenicol was added to heated serum and incubated at 30°C for 18 h (Table 2). Therefore, the diacetyl chloramphenicol-converting activity of serum had been completely inactivated by the heat treatment.

FIG. 3.

CAE assay of the conversion of acetyl chloramphenicol to chloramphenicol by serum. The serum indicated was incubated with the AcBCAM substrate for 2 h at 34°C. The reaction was then stopped with the addition of ice-cold ethyl acetate and extracted once. After drying, the reaction products were resuspended in ethyl acetate and separated by TLC.

BSK was also made with heated rabbit serum, and the MICs of diacetyl chloramphenicol for E. coli, B. subtilis, and B. burgdorferi were determined (Table 1). The MICs for all three of the bacteria increased with the use of heat-treated serum in comparison to BSK with normal serum. The susceptibility of B. burgdorferi to diacetyl chloramphenicol decreased fourfold with heating of the serum, but B. burgdorferi was susceptible in comparison to E. coli and B. subtilis.

Conversion activity with other sera.

The CAE assay was performed with serum from several different sources (Fig. 3). The conversion activity of mouse serum was similar to that of rabbit serum. Human and horse sera converted less diacetyl chloramphenicol than did either rabbit or mouse serum. These sera were also tested by the plate bioassay (Table 2). By this method, human serum appeared to have activity approximately equal to that of rabbit serum. The MICs of chloramphenicol and diacetyl chloramphenicol for E. coli were determined in LB medium with 10% horse serum (Table 1). B. subtilis did not grow in the presence of horse serum, and neither it nor E. coli grew with mouse serum. This was probably due to complement, since these bacteria could grow in the presence of heat-treated serum. This heating also destroyed esterase activity (32). The addition of horse serum to LB medium had no effect on the susceptibility of E. coli to chloramphenicol. There was also no change in the susceptibility to diacetyl chloramphenicol of E. coli pGOΔ1, but that of E. coli without pGOΔ1 was increased.

The addition of 10% human serum to LB medium increased the susceptibility of E. coli, E. coli pGOΔ1, and B. subtilis to chloramphenicol (Table 1). Addition of human serum to LB medium also increased the susceptibilities of E. coli and B. subtilis to diacetyl chloramphenicol. The converting activity of human serum was undetectable after heating (Table 1). Both horse and human serum contained diacetyl chloramphenicol esterase activity. The results of the MIC experiments and the CAE assays suggested that the chloramphenicol-converting activity of human serum was not as great as that of rabbit serum, but greater than that of horse serum.

Conversion by BSK.

The experiments with sera explained the susceptibilities of E. coli and B. subtilis to diacetyl chloramphenicol, but it was still possible that there were other factors besides serum responsible for the susceptibility of B. burgdorferi to diacetyl chloramphenicol. To determine the susceptibilities of these bacteria in the absence of serum, the MICs for E. coli and B. subtilis in BSK without serum were determined (Table 1). Although both bacteria were not susceptible to at least 1,000 μg of diacetyl chloramphenicol per ml in LB medium, they were significantly more susceptible in BSK. BSK H, but not BSK II, supported the growth of B. burgdorferi without additional serum; therefore, the MIC was determined in this medium without serum. The susceptibility of B. burgdorferi again decreased, 16-fold from that in BSK H with rabbit serum and 4-fold from that in BSK H with heat-inactivated serum.

The susceptibility to diacetyl chloramphenicol in BSK without serum but not in LB medium raises the possibility that there was still weak conversion activity in BSK that was not detected in the original assay because of the strength of the activity present in serum. The protocol for the plate bioassay provided for only an overnight incubation of 18 h and may not have allowed detection of lower levels of diacetyl chloramphenicol hydrolysis. To determine if conversion was occurring, diacetyl chloramphenicol was added to BSK without serum and then extracted at 24-h intervals for a plate bioassay. No conversion could be detected after 24 h, but slight conversion, producing a zone of inhibition of 10 mm (approximately 0.6 μg of chloramphenicol), could be detected at 48 h. After 3 days, the zone size was 13 mm (1.2 μg), and after 4 days, it was 16 mm (1.8 μg). Thus, even in the absence of serum there was conversion of diacetyl chloramphenicol into chloramphenicol by BSK.

To compare the conversion in BSK with serum to that in BSK without serum, the CAE assay was used. Acetyl chloramphenicol was added to LB medium, or BSK, with and without serum, and samples were taken at timed intervals (Fig. 4). All of the acetyl chloramphenicol was converted to chloramphenicol by 2 h in samples with serum, but there was only slight conversion by 12 h in BSK without serum. A longer incubation revealed the slow conversion to chloramphenicol by BSK, with less than 50% conversion by 96 h. No conversion to BCAM was seen in LB medium even at 96 h.

FIG. 4.

CAE assay of the conversion of acetyl chloramphenicol to chloramphenicol by LB medium or BSK with or without serum. The medium was incubated with the AcBCAM substrate for the time indicated, and then the reaction was terminated with the addition of ice-cold ethyl acetate. After extraction, the reaction products were separated by TLC. Open circles, BSK with 10% rabbit serum; filled circles, BSK without serum; open inverted triangles, LB with 10% rabbit serum; filled inverted triangles, LB without serum.

DISCUSSION

Both mammalian and nonmammalian species have carboxylesterases. These enzymes often have wide substrate specificity, especially towards lipophilic substrates. They hydrolyze a variety of foreign compounds, including drugs and pesticides, but their physiological function is not completely understood (15, 17). In mammals, the carboxyesterases are concentrated in the liver, but are also found in serum and intestinal mucosa, among other locations (15, 19, 29). The hydrolysis, by nonspecific carboxylesterases, of esters of chloramphenicol is thought to occur mainly in the liver, as only low levels of this activity have been detected in the blood (9, 12, 35). These enzymes are probably responsible for the conversion of diacetyl chloramphenicol to chloramphenicol reported. This suggests that even low levels of activity may have a profound effect.

Some actinomycete species and the spirochete S. aurantia have esterase activities capable of converting diacetyl chloramphenicol to chloramphenicol (20, 34), but B. burgdorferi apparently does not, as determined by the CAE assay and plate bioassay. Therefore, a bacterial esterase was unlikely to be the explanation for the susceptibility of B. burgdorferi to diacetyl chloramphenicol, and we focused on medium components.

Components of growth medium can interfere with MIC determinations for different antibiotics. Low levels of thymidine affect the activity of diaminopyrimidines (10), while high levels of cations such as magnesium and calcium interfere with the action of tetracycline and kanamycin (1). B. burgdorferi was originally reported to be resistant to trimethoprim (18, 25). The activity of this antibiotic was inhibited in the presence of several components of BSK (26, 27). When the medium was modified, it was subsequently shown that B. burgdorferi was susceptible to this drug (27).

There were several effects attributed to the serum component of medium that were detected here. First, when heated rabbit serum was added to either BSK or LB medium, the MICs of chloramphenicol for B. subtilis and E. coli slightly increased, probably due to binding of chloramphenicol to serum proteins (22). A more substantial effect was the conversion by rabbit serum of acetylated chloramphenicol to chloramphenicol, thus counteracting the reaction catalyzed by CAT. The resistance or susceptibility of a bacterium to chloramphenicol in the presence of serum is due to two opposing reactions. One is the inactivation by acetylation of chloramphenicol by the activity of CAT. On the opposite side of the reaction, the esterase activity of the serum converts the diacetyl chloramphenicol back to chloramphenicol. The balance of these two reactions would determine the resistance or susceptibility phenotype. In the present study, the MIC of chloramphenicol for E. coli pGOΔ1 decreased in the presence of rabbit serum to a level that would be considered to indicate susceptibility (Table 1). Thus, a bacterium that appeared resistant in vitro might be susceptible in vivo.

There are also important considerations for the further development of genetic tools for Borrelia. Chloramphenicol resistance is used widely as a selection marker for prokaryotes, where cat appears to provide almost universal resistance. Chloramphenicol resistance from cat genes is a selection marker that has been used for a variety of bacteria. But this resistance has not been obtained in B. burgdorferi after several attempts in this (32) and other laboratories. Perhaps this failure to obtain transformants of B. burgdorferi with the chloramphenicol resistance phenotype is due in part to the presence of esterases in the growth medium or to hydrolysis by BSK.

There are many bacteria that are cultured in the presence of either serum or blood and could be affected by this interfering activity of esterases. Often, serum that contains lower levels of esterase activity is used in media. However, in most cases heated serum is used to avoid the effects of complement, and this heating is sufficient to inactivate the esterase responsible for the conversion to chloramphenicol. B. burgdorferi is resistant to complement in many kinds of serum and is often grown in serum that has not been heat inactivated (13, 14).

Both E. coli and B. subtilis were more susceptible to diacetyl chloramphenicol when grown in BSK. This is due to the low-level conversion to chloramphenicol caused by the complex medium even in the absence of serum. B. burgdorferi cannot be cultured in a medium such as LB that lacks this low level of activity. Therefore, an inherent susceptibility of B. burgdorferi to diacetyl chloramphenicol cannot be ruled out. But it is likely that the susceptibility of this bacterium to diacetyl chloramphenicol is due in part to the low level of conversion activity of the medium in the absence of serum. Two additional factors may also be important. B. burgdorferi has a longer dividing time than either E. coli or B. subtilis, thus providing additional time for the conversion to chloramphenicol (3, 23). And B. burgdorferi is more susceptible to chloramphenicol than is either E. coli or B. subtilis. The low level of hydrolysis may prove an important consideration for other slow-growing bacteria that require complex media.

This report has identified an activity in some sera used in bacterial media that converted mono- and diacetyl chloramphenicol to chloramphenicol. This activity interfered with the MIC determination of different classes of bacteria and could result in the inaccurate determination of chloramphenicol resistance.