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Antimicrobial Agents and Chemotherapy, March 1999, p. 655-660, Vol. 43, No. 3
Departments of Microbiology & Molecular
Genetics and Medicine, University of California Irvine, Irvine,
California 92697-4025
Received 28 May 1998/Returned for modification 18 September
1998/Accepted 13 December 1998
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.
Media, strains, and culture conditions.
Escherichia
coli XL1-Blue MRF' (
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Esterases in Serum-Containing Growth Media
Counteract Chloramphenicol Acetyltransferase Activity In
Vitro
and
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
[mcrA]183
[mcrCB-hsdSMR-mrr]173 endA1 supE44 thi-1 recA1
gyrA96 relA1 lac[F' proAB lacIq ZM15
Tn10]) (Stratagene, La Jolla, Calif.) was the
chloramphenicol-susceptible E. coli strain.
E. coli XL1-Blue MRF' with pGO1, 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.
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 pGO1. 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.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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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 pGO1 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 pGO1, 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.
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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 pGO1 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.
1 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
pGO1 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
pGO1 was used for the lawn.
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1 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.
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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 pGO1, but that
of E. coli without pGO1 was increased.
1, 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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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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 pGO1 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.
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ACKNOWLEDGMENTS |
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We are grateful to William V. Shaw for suggestions and comments. For supplies, we thank Catherine Luke and Debbie Jaworski for providing sera and Howard Peter for B. subtilis.
This research was supported by National Institutes of Health grant AI24424.
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FOOTNOTES |
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* Corresponding author. Mailing address: Departments of Microbiology and Molecular Genetics and Medicine, University of California Irvine, Irvine, CA 92697-4025. Phone: (949) 824-5626. Fax: (949) 824-8598. E-mail: abarbour{at}uci.edu.
Present address: Tuberculosis Research Laboratory (151), Veterans
Administration Medical Center, Long Beach, CA 90822.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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