Environ. Sci. Technol. 2005, 39, 2301-2306
Accelerated Transformation and Deactivation of Erythromycin in Superheated Water. 2. Transformation Reactions and Bioassays MICHELLE N. BUTLER AND WALTER J. WEBER JR.* Department of Chemical Engineering, Energy and Environment Program, University of Michigan, Ann Arbor, Michigan 48109-2099
The work presented in this second part of a two-part series on the accelerated transformation of erythromycin in superheated water focuses on the chemical nature and resultant antimicrobial implications of the overall reaction observed. Analyses of reactor effluents employing infrared spectroscopy and liquid chromatography/mass spectrometry indicated that the initial step in the decomposition pathway is one of dehydration. Subsequent hydrolysis in the presence of an acetate buffer appeared to result in the loss of the sugar cladinose. Chemical transformation under superheated conditions was tied to the loss of antibiotic function by an agar dilution test. The sensitivity of this test was verified by determination of the minimum inhibitory concentration (MIC) of erythromycin corresponding to each of two test microorganisms. MIC values for the selected strains of Escherichia coli and Bacillus subtilis were 35 and 0.5 mg/L, respectively. To relate the loss of antimicrobial activity to increased reaction temperature and thus to increased extent of parent compound transformation to microbially benign intermediates, bioassays using E. coli as test microorganism were performed on flow reactor effluents resulting from reaction of initial erythromycin concentrations of 75-150 mg/L.
Introduction and Background This paper examines pathways by which destabilization of erythromycin occurs in superheated water. The work makes an important connection between the accelerated chemical transformation reactions observed in this study and reports in the literature describing much slower decomposition reactions under ambient temperature conditions. Agar dilution tests, commonly used to assess antibiotic activity, were employed as a complement to the study of chemical transformation reactions. These microbiological evaluations link the work described herein and the reactor study presented in Part 1 of the series (1) to the overarching treatment objective of curbing the development of resistance among microbial species indigenous to environmental systems by reducing their exposure to active substances. Erythromycin was selected as the model compound for the study in part because mechanisms of degradation of the * Corresponding author telephone: (734)763-2274; fax: (734)9364391; e-mail:
[email protected]. 10.1021/es049397n CCC: $30.25 Published on Web 02/09/2005
2005 American Chemical Society
FIGURE 1. Mechanism of erythromycin decomposition under acidic conditions. (Des ) Desosamine, Clad ) Cladinose). compound in aqueous solutions under ambient conditions have been well-characterized. Studies conducted by Atkins et al. (2), Connors (3), Cachet et al. (4), Kibwage et al. (5), and Vinckier et al. (6) have all shown that under acidic conditions and ambient temperatures decomposition of erythromycin results in anhydroerythromycin formation. Figure 1 presents a schematic illustration of this reaction. Anhydroerythromycin is produced directly from erythromycin, while the parent compound is in equilibrium with erythromycin enol ether. Further acid hydrolysis of anhydroerythromycin leads to a loss of the cladinose substituent and resulting production of the compound erythralosamine (3, 5, 7). Significantly, the presence of cladinose is essential for retaining the antibiotic activity of the parent compound. Modification of the 3′dimethylamino and 2′-hydroxyl groups on desosamine also results in decreased activity (8). As noted in the first part of this series (1), Connors (3) determined that degradation of erythromycin in aqueous solutions can occur by either acid- or base-catalyzed reactions within a pH range of 6-8 and that within this range the parent compound is most stable at a pH of ∼7-7.5. Several mechanisms of decomposition in basic media have been described. Kibwage et al. (5) found that psuedoerythromycin enol ether, a therapeutically insignificant metabolite, is formed by translactonization in neutral media (pH 6-8). Waddell and Blizzard (9) determined that erythromycin’s lactone ring is hydrolyzed upon reaction with a strong base and more polar carboxylate products are formed. Paesen et al. (10) investigated the stability of the pharmaceutical over a pH range of 7.5-11. Their experiments, performed in 1:1 mixtures of methanol/phosphate buffer, resulted in the formation of psuedoerythromycin A hemiketal, psuedoerythromycin enol ether, and a hydrolysis product having an open lactone ring. In contrast, Volmer and Hui (11) more recently found, using a combination of solid-phase microextraction and liquid chromatography/electrospray ionization tandem mass spectroscopy, that anhydroerythromycin is the major product of erythromycin degradation in aqueous solutions, not only at pH values of less than 4 but also under basic conditions; i.e., a pH range of 10-13. VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Links between the degradation pathways of erythromycin and reactions observed in superheated water are supported by Katritzky and co-workers (12, 13), who successfully used this medium to target the behavior of many of the functional groups carried by the antibiotic. They reported (i) that ketals are highly reactive in superheated water, undergoing complete hydrolysis within 30 min at 205-250 °C, (ii) that the hydrolysis of esters results in the formation of carboxylic acids that can catalyze further reactions, and (iii) that ether and ester bond formation can also occur. In short, their work suggests that, under superheated conditions, acid-basecatalyzed reactions involving polar residues of erythromycin that are linked to binding, such as its carbonyl and sugar moieties, can result in modifications to the compound that will impair its antimicrobial functions.
Experimental Methods Preparation of Solutions. Stock and dilute solutions of erythromycin A (Sigma-Aldrich Co.) were prepared as described in Part 1 of this two-part series (1). Acetate buffer solutions (0.1 M) were used to adjust the pH of select test systems operated under superheated conditions. An acetateamended system was chosen because it did not interfere with the liquid chromatographic method developed for detection of the parent compound and its reaction products. The conjugate acid/base pair, glacial acetic acid and ammonium acetate (HPLC grade), were obtained from Fisher Scientific. Buffer solutions were prepared in distilled deionized water (Millipore). The pKa of the conjugate acid (4.75) limited the test range for experiments utilizing these solutions to pH values between 4 and 6. Batch and Flow Reactor Operation. The batch and flow reactor configurations were operated as described in Part 1 of this two-part series (1). High-Pressure Liquid Chromatography. Quantitative measurements of parent compound concentrations in reactor effluents were made using high-pressure liquid chromatography (HPLC) with evaporative light scattering detection. The complete analytical method is outlined in Part 1 of this two-part series (1). Infrared Spectroscopy. Reactions of erythromycin in superheated water were qualitatively monitored using attenuated total reflectance (ATR) infrared spectroscopy. The instrument employed was a ThermoNicolet Nexus 670 spectrometer with Smart Ark Accessory equipped with a DTGS-KBr detector and a KBr beam splitter. Liquid/liquid extraction with ethyl acetate, 2:1 sample\ethyl acetate (Fisher Scientific) by volume, was used to concentrate reaction effluents for analysis. Extracted liquid samples were applied in three aliquots to the ZnSe crystal mounted in the instrument’s ATR unit. The organic solvent was allowed to evaporate away after each application. Comparisons between spectra collected from erythromycin standards and from residual effluent samples were used to draw conclusions about the structural changes being observed after reaction. Liquid Chromatography/Mass Spectrometry. The complete liquid chromatography/mass spectrometry (LC/MS) system consisted of a Hewlett-Packard 1090 series liquid chromatograph and a Hewlett-Packard 5989B mass spectrometer. An atmospheric pressure ionization (API) electrospray chamber operated at the interface between the HPLC and the MS. The HPLC conditions used in this system were designed to duplicate those used in earlier analyses. Solutes were carried through a Jones Apex silica column and a precolumn Alltech Econosil guard cartridge at a rate of 1.3 mL/min for a total run time of 9 min. The solvent media used was a 90% methanol (HPLC grade, Fisher Scientific) and 10% 0.2 M ammonium acetate (HPLC grade, Fisher Scientific) solution. Analyses were performed at an ambient 2302
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column temperature of approximately 25 °C. The sample injection volume was 25 µL. Preparation of Microbial Cultures. Microorganisms selected for use in this study included Bacillus subtilis (ATCC 6633), purchased directly from the American Type Culture Collection, and Escherichia coli (264F-2), provided by Dr. Betsy Foxman at the University of Michigan. The Gramnegative Bacillus strain was selected because its growth is inhibited at relatively low concentrations of erythromycin. B. subtilis is also commonly used as a reference strain in standard assay methods published by the National Committee for Clinical and Laboratory Standards (NCCLS) (14). E. coli was selected because it is present in most natural systems. The minimum inhibitory concentration of erythromycin corresponding to this microorganism also allowed the effects of transformations of erythromycin in superheated water, at the test concentrations chosen for this study, to be observed. Both bacteria were cultivated on plates containing Mueller-Hinton agar (Difco) and then transferred to MuellerHinton broth (Sigma-Aldrich Co.). Cultures were stored in liquid media at 4 °C and used to inoculate fresh broth once a week. Cell cultures were also periodically streaked onto agar plates to examine whether contamination of the culture had occurred. Growth Inhibition Test. Agar dilution is a documented methodology for the determination of minimum inhibitory concentrations and, therefore, of the susceptibility of microorganisms to known antibiotics (14-16). In this study, the method was extended to the evaluation of treated samples. Effluent samples were filter-sterilized using 0.22µm PVDF disposable syringe filters (Millipore). Filtered samples were then incorporated, within a range of volume percentages, into Mueller-Hinton agar. Plates were prepared using these mixtures. Microbial cultures were streaked onto prepared plates using a standardized inoculating loop. The plates were incubated overnight at a temperature of 37 °C for E. coli and 30 °C for B. subtilis. After visual inspection of the plates, growth was recorded. Growth was recorded as positive if any colonies were observed on the plate. A failure to grow, and, therefore, inhibition by erythromycin was denoted with a negative sign. To standardize the assay, the agar concentration was fixed at 38 g/L in all prepared solutions. Also, all culture broths used for streaking were diluted with sterile broth until a UV absorbance of 1.35 was reached. UV absorbance was measured using a spectrophotometer. All measurements were made at a wavelength of 625 nm. The minimum inhibitory concentration (MIC), the concentration of antibiotic at which microbial growth is first inhibited, was also determined for each of the test microorganisms using a comparable methodology. A range of known concentrations of erythromycin was incorporated into poured plates by serially diluting a stock mixture of erythromycin and agar with sterile Mueller-Hinton media. Microbial cultures were streaked onto these plates, the plates were incubated, and growth was recorded as described above.
Results and Discussion The difficulty of distinguishing between potential reaction reversibility and the effects of reactor dynamics discussed in Part 1 of this series (1) allowed only a qualitative examination of HPLC results for reaction products. Figure 2 is a graphical representation of product formation as afunction of reaction time in the superheated water during batch-mode reactor experiments at 150 °C. Peak areas rather than masses are used to represent the species identified on this graph because, with the exception of erythromycin, a calibrated relationship between detector response and concentration was not available.
TABLE 1. Characteristic Absorption Bands Used for Spectral Analysis (17)
1 2 3 4 5 6
FIGURE 2. Unidentified reaction product formation at 150 °C.
FIGURE 3. Spectral comparison of erythromycin and sample effluent. Two major products, arbitrarily designated here as A and B, were observed at HPLC retention times of tR ) 3.4 and 5.0 min. Figures 2 emphasizes the fact that the observed disappearance of erythromycin can be tied directly to the formation of quantifiable products. The ability to chromatographically separate products in reactor effluents suggests that a mechanism of decomposition may be determined for this system. The inability to explicitly quantify resulting products, as noted above, also indicates that further mass balance analysis, for example, an evaluation of total organic carbon (TOC) in reactor influent and effluent streams, is necessary to confirm that erythromycin was completely converted to the products observed. Structural Analysis and Potential Product Identification. Reactor effluents were collected and examined further using infrared spectroscopy. Clear structural changes were evident after reaction of erythromycin in superheated water. The significance of these changes is made apparent by comparison of a spectrum for an effluent from the batch reactor operated at 150 °C with that for the parent compound, as illustrated in Figure 3. Effluent spectra were also compared to that of ethyl acetate to ensure that residual solvent did not bias the analytical results.
spectral range (wavenumbers)
functional group
3450-3300, 3650-3590 3000-2840 (often multiplet) 1725 1300-1200 1150-1050 815
N-H, O-H C-H CdO (lactone) C-O-C (ether) C-O, CH2-O-CH2 cyclic ether
Table 1 lists relevant absorption bands for this analysis. The loss of peaks characteristic of ketones (peak 3) and the appearance of those associated with ether bonds (peaks 4-6) suggest that dehydration is an important step in parent compound transformation. This conclusion is also supported by the loss of the peak associated with the hydroxide functionality. Although the data are not presented here, the intensity of peaks corresponding to observed structural changes increased as reaction temperature increased. This was an expected outcome of the increased degree of conversion observed in the batch studies as temperature was raised (1). While the infrared spectra provide important qualitative information about the reactions occurring in superheated water, definitive product structures cannot be inferred from them because they were generated using parent/product mixtures. Liquid chromatography/mass spectrometry (LC/ MS) was therefore utilized to gain more insight into the structures of the transformation products. A retention time shift, as compared with HPLC separations presented in the first part of this series (1), of approximately 1 min for both erythromycin and its reaction products was observed using LC/MS. Erythromycin eluted at tR ) 6.3 min. The chromatogram shown in Figure 4 confirms the appearance of a product peak at a retention time of approximately 6.0 min. This peak corresponds to the compound designated as product A (HPLC tR ) 5.0 min). As previously mentioned, an API electrospray chamber operated at the interface between the HPLC and the MS in the analytical system employed in this study. Fragmentation of species does not occur in an API electrospray chamber. Because erythromycin is slightly basic, positively charged ions were formed. As shown in Figure 4, the mass-to-charge (m/z) ratio of the protonated form of unidentified product A is 717, and thus the compound has a molecular weight (MW) of 716 g/mol. The 18 g/mol difference between the molecular weight of product A and that of erythromycin, 734 g/mol, suggests that a water molecule was lost during the first stage of erythromycin decomposition. This finding is in agreement with conclusions drawn from the infrared analyses of these effluents. This transformation also mimics those found in the erythromycin stability literature under a range of acid/base conditions. Hence, it appears that water is autocatalyzing the decomposition reactions being observed, even at the moderate temperatures and pressures used in these experiments. Relevant work in the literature on superheated water chemistry further supports this hypothesis. Katritzky and Allin (12) found that carbonyl condensation reactions occur readily in superheated water and result in formation of ether bonds. Katritzky and Allin (12) also determined that this reaction is reversible in nature. Given the known pH sensitivity of erythromycin, adjustment of system pH was viewed as an important investigative tool. In an effort to decrease system pH under superheated conditions and to examine the resulting effect on reaction progression, batch reactions at temperatures of 125 and 150 °C and pH values ranging from 4 to 6 were carried out in 0.1 VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. E. coli Agar Dilution Test Results for Flow Reactor Effluentsa reaction conditions vol % of sample in agar solution
100 °C, 100 mg/L
100 °C, 125 mg/L
100 °C, 150 mg/L
125 °C, 75 mg/L
125 °C, 100 mg/L
125 °C, 125 mg/L
125 °C, 150 mg/L
70 60 50 40
+ (s) + +
+ +
+ (s)
+ + + +
+ + +
+ (s) + +
+ +
a
+, growth observed; -, no growth observed; s, sparse, colony density very light.
FIGURE 5. LC/MS chromatogram for unidentified product in pHadjusted reaction.
FIGURE 4. Chromatographic identification of product A using LC/ MS. M acetate buffer solutions. The disappearance/ reappearance trend noted in Part 1 of this series (1) at reaction temperatures greater than 150 °C, which was assumed to be the result of inadequate mixing at early reaction times, was again observed during this set of experiments. Given the temperatures at which these experiments were conducted, additional questions were raised about the possibility of reaction reversibility. This issue was explored using LC/MS. A representative chromatogram associated with this analysis is shown in Figure 5. It should be noted that the pH values included in the legend of this figure and throughout the text of this paper are experimentally determined values for the 50 mg/L erythromycin/ 0.1 M acetate inlet solution at room temperature. Significantly, instead of decreases in conversion, as calculated using batch reactor concentration-time data for the pH-adjusted system, corresponding to the reappearance of erythromycin (m/z ratio ) 735), a third product having a molecular weight of 557 g/mol was identified that eluted at approximately the same time as the parent compound had. The difference of 159 between the m/z ratios of this third compound, denoted product C, and product A suggests that 2304
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the sugar cladinose was subsequently being lost during treatment in the amended reaction medium. This finding suggests that during this set of experiments the acid-catalyzed mechanism advanced by Connors (3), Kibwage et al. (5), and Flynn et al. (7) was occurring. The results of experiments carried out in pH-adjusted reaction media also indicate the importance of water chemistry under superheated conditions, particularly changes in the self-ionization of water, on the extent of reaction observed in this system. Given the results of the LC/MS analysis, the corresponding HPLC data was revisited. The formation of product C was marked by a shift in retention time on the HPLC from 5.2 min, the elution time of erythromycin, to 5.4 min. Apparent decreases in conversion observed in acetate-amended systems were therefore caused by an inability to resolve product C and erythromycin. Microbial Assays. Agar dilution tests were employed as a means of evaluating the impact of observed transformations on selected target microorganisms. The initial step in the application of this microbiological method involved determination of a MIC for each target microorganism. The MIC of erythromycin was determined to be 35 mg/L for E. coli strain 264F-2. For the B. subtilis strain 6633 a MIC of 0.5 mg/L was obtained. The experimentally determined MIC values compared well with literature values, thus confirming the accuracy of the methodology as developed for this application. For example, the MIC for B. subtilis is reported as 0.1-0.4 mg/L (18, 19) and as 25 mg/L for several strains of E. coli at a medium pH of 7.0 (20). Once the sensitivity of the test was established using the MIC determinations, flow reactor effluents resulting from reactions at temperatures of 100-175 °C and an initial erythromycin concentration of 50 mg/L were tested using this functional assay. For the E. coli system, growth was observed on all sample plates. The erythromycin concentra-
tion in the agar on these plates never exceeded the erythromycin MIC of 35 mg/L for this microorganism. Under these test conditions, however, the MIC for the B. subtilis strain was almost always exceeded. No growth was observed except at the highest reaction temperature and lowest sample volume percentages. Although these data validate the sensitivity of the test, they did not explicitly indicate that the reactions in superheated water caused a loss in antimicrobial activity, the result anticipated for this suite of experiments. Test conditions were altered in an attempt to more clearly define the effects of reactions in superheated water on the antibiotic activity of erythromycin. Table 2 shows the results of a set of experiments involving sample effluents that were generated by reaction of higher erythromycin concentrations than previously considered. The concentrations listed in Table 2 are initial concentrations of erythromycin introduced into the flow reactor. The results of these tests should be evaluated within the framework of the conversion data for the flow reactor. At 125 °C and a reactor residence time of 30 min, erythromycin concentrations in flow reactor effluents of 19.5 ( 0.5, 29.9 ( 0.9, 36.5 ( 2.6, and 46.2 ( 2.4 mg/L corresponded to inlet concentrations of 75, 100, 125, and 150 mg/L, respectively. If 70 vol % of the reactor sample effluent was incorporated into the agar solution, as indicated in Table 2, and the initial erythromycin concentration was 75 mg/L, then the actual concentration of erythromycin present in the plate is calculated to be approximately 13.7 mg/L. At sample volume percentages higher than 70%, the agar solution could not be mixed homogeneously and failed to solidify properly. To eliminate the impacts of density variations created by nonhomogeneity on antibiotic diffusion and resulting inconsistencies in the data, a 70 vol % upper limit was set for the experimental test range. The data presented in Table 2 show that as erythromycin conversion increases with increased reaction temperature, the incidence of microbial growth observed also increases. This set of experiments makes the anticipated connection between transformations that occur under superheated conditions and the loss of antibiotic activity. Because growth trends are linked to both inlet erythromycin concentration and reaction conversion, these data also seem to suggest that any residual antibiotic activity can be attributed to the action of the parent compound and not to the products. It should be noted, however, that growth is not observed at erythromycin concentrations greater than 25 mg/L. This finding contradicts the MIC determination for E. coli of 35 mg/L. Since the procedure used to obtain the results listed in Table 2 mimicked that used to determine the MIC for this microorganism, this disparity may be the result of the increased activity of erythromycin at higher aqueous concentrations and, therefore, at higher pH values or the presence of a reaction intermediate(s) that is still biologically active. The results of both the analytical work and bioassays presented in this paper underscore water’s ability to autocatalyze and accelerate the aqueous degradation reactions of erythromycin commonly observed at ambient temperatures. Identifying the functional groups involved in these transformations will allow extension of the spectrum of compounds to which this treatment technology can be applied, such that it includes not only compounds from the macrolide class of antibiotics but also other environmental contaminants of concern. In this paper, compound degradation in superheated water was viewed as a targeted attack on specific functionalities and utilized to eliminate the erythromycin’s biological activity. A significant amount of work remains to explore both the selectivity and broad spectrum applicability of using this medium as a means for treating waste streams.
Acknowledgments The authors acknowledge the invaluable laboratory support provided by University of Michigan undergraduate students Tikoshia Davis, Douglas Chenoweth, and Kendra Carter. Special thanks go to Laboratory Director Tom Yavaraski for his assistance in development of the analytical components of this study. Development of the superheated water reactor system and protocols was supported in part by companion Grants DMI-9985423 and R828246 from the National Science Foundation and the U.S. Environmental Protection Agency, respectively. Fellowship support to M.N.B., a graduate student in the Departments of Chemical Engineering and Civil and Environmental Engineering, was provided by the University of Michigan’s Horace H. Rackham Graduate School and the National Science Foundation.
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(19) Novikova, S. I.; Bushueva, A. M.; Trachuk, L. A.; Konstantinova, G. E.; Serkina, A. V.; Hoischen, C.; Gumpert, J.; Chestukhina, G. G.; Mankin, A.; Shevelev, A. B. Introduction of mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis. FEMS Microbiol. Lett. 2000, 182, 213-218. (20) Sabath, L.; Lorian, V.; Gerstein, D.; Loder, P.; Finland, M. Enhancing effect on alkalinization of the medium on the activity
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of erythromycin against Gram-negative bacteria. Appl. Microbiol. 1968, 16: 1288-1292.
Received for review April 21, 2004. Revised manuscript received December 21, 2004. Accepted December 22, 2004. ES049397N
