Specificity and Mechanism of Tetracycline

JOURNAL OF BACTERIOLOGY, Feb., 1966 Vol. 91, No. 2 Copyright 1966 American Society for Microbiology Printed In U.S.A. Specificity and Mechanism of Resistance in a Multiple Drug Resistant Strain of Escherichia coli KAZUO IZAKI,I KAN KIUCHI, AND KEI ARIMA Department ofagricultural Chemistry, Faculty of Agriculture, University of Tokyo, Tokyo, Japan Received for publication 16 September 1965 ABSTRACr IZAKI, KAZUO (University of Tokyo, Tokyo, Japan), KAN KIUCHI, AND KEi ARIMA. Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli. J. Bacteriol. 91:628-633. 1966.-A decrease in the uptake of tetracycline occurred concurrently with a rise in the level of resistance of a multiple drug resistant strain of Escherichia coli grown in the presence of tetracycline. Although the strain was also resistant to streptomycin and chloramphenicol, growth in the presence of these two antibiotics did not influence the uptake of tetracycline. The induction of resistance, or decreased uptake of tetracycline, was dependent on growth of the organism in the presence of the drug. Decreased uptake of tetracycline could not be induced in a sensitive strain of the same organism under conditions suitable for induction of the resistant strain. The decrease in accumulating power of the resistant organism cultured in the presence of tetracycline does not appear to be due to selection of a resistant strain from cultures containing both resistant and sensitive strains. Recently, multiple drug resistant bacteria have been found by many Japanese workers. Many biochemical and genetic studies on these bacteria have been carried out. Akiba and Yokota (1) and Okamoto and Mizuno (6, 7) have investigated the mechanism of multiple drug resistance. These workers have concluded that resistance is due to failure of the drug to reach its active site in the resistant bacterium (i. e., the organism is impermeable to the drug). The main evidence leading to this conclusion was the fact that inhibition of amino acid incorporation into polypeptides in cell-free extracts of these organisms was inhibited equally for both sensitive and resistant strains by both chloramphenicol and tetracycline. In a previous report (4), we studied the mechanism of resistance of Escherichia coli to oxytetracycline. We concluded that decreased uptake of this antibiotic by the resistant strain is the probable mechanism of resistance. In this report, we show that decreased uptake of tetracycline by resistant cells occurs simultaneously with an increased level of resistance of this organism to the drug, and that these effects occur only when IPresent address: Department of Pharmacology, University of Wisconsin, Madison. the cells are grown in the presence of antibiotics of the tetracycline group. Other antibiotics, such as chloramphenicol, did not cause such a decrease in the uptake of tetracycline, despite the fact that the organism was also resistant to this drug. Recently, a similar decrease in the uptake of chlortetracycline and tetracycline by resistant E. coli was reported by Franklin and Godfrey (3). MATERIALS AND METHODS Chemicals. and chlortetracycline were the products of Japan Lederle Co. Streptomycin sulfate was supplied by Meiji Seika Co. Ltd. Chloramphenicol and oxytetracycline were supplied by Sankyo Seiyaku Co. Ltd. and Teito Feizer Co. Ltd., respectively. H3-tetracycline was kindly given to us by T. Komai of The National Institute of Health of Japan. Its specific activity was 3.1 mc/mnnie. Bacterial strain. E. coli K-12 sensitive and resistant strains were used. The resistant strain is resistant to tetracycline, chloramphenicol, streptomycin, and sulfonamide. These strains were the kind gift of R. Nakaya, The National Institute of Health of Japan. Culture conditions. Cells of E. coli K-12 cultivated overnight in nutrient broth were inoculated into 10 mi of nutrient broth in a 30-ml Monod's test tube, which was a rectangularly bent test tube specially made for 628

VOL. 91, 1966 TETRACYCLINE RESISTANCE IN E. COLI a shaking culture; cells were grown for 3 hr, with shaking, at 30 C. In some cases, tetracycline, chlortetracycline, oxytetracycline, chloramphenicol, streptomycin, or kanamycin was added to the growth medium. Accumulation conditions. The test for the accumulation of tetracycline was normally carried out at 30 C by use of a Monod's test tube on a shaking machine. Nutrient broth (ph 7.0) was used as the incubation medium except when a nitrogen-free medium composed of glucose (5.5 X 102 M), K2HPO4(10e M, ph 6.5), and MgSO4 (4.0 X 10-4 M) was used. (50 to ug/ml) or H3-tetracycline (20 Ag! ml) was added. Accumulation of tetracycline was usually determined by the increase of turbidity and by the optical density at 360 mmu of boiled extracts of cells which had accumulated tetracycline in the same manner as reported for oxytetracycline (2, 5). In experiments with H3-tetracycline, cells exposed to H3-tetracycine were centrifuged, washed, suspended in 0.5 ml of distilled water, and heated at 100 C for 5 min. Pronase (0.1 ml, 200,g/ml, in 2% K2HPO4 solution, ph 6.5) was added, and the resulting suspension was incubated for several hours at 45 C. After the cells were solubilized, the solution was transferred into a counting vial, and 10 ml of scintillator [7 g of 2, 5-diphenyloxazole, 0.3 g of 2, 2'-p-phenylenebis(5-phenyloxazole), and 100 g of naphthalene in liter of dioxane] was added. The vials were counted in a Packard Tri-Carb liquid scintillation counter. Determination of the level of resistance to tetracycline. E. coli K-12 grown in nutrient broth for 3 hr was centrifuged and washed with distilled water. Two separate tubes containing nutrient broth were inoculated with cells. One tube contained tetracycline (70 lg/mlm), and the other did not. Growth was measured at regular intervals with a Kotaki turbidometer. The inhibition rate was determined from the initial and final values of the turbidity. The same procedures of incubation and measurement of turbidity were used with the resistant cells grown in nutrient broth containing tetracycline (20,g/ml). Inhibition of growth of cells in the presence and absence of tetracycline was compared. REsULTS Accumulation of tetracycline in cells of E. coli K-12. A sensitive strain of E. coli K-12, grown overnight in nutrient broth, was inoculated into fresh nutrient broth. This culture was grown for 3 hr, harvested by centrifugation, washed twice with distilled water, and resuspended in distilled water. Accumulation was measured under various conditions. The results are shown in Table 1. A large amount of tetracycline was accumulated when glucose, K2HPO4, and MgSO4 were present. A low concentration of 2,4-dinitrophenol inhibited the accumulation. These results were similar to results previously reported with oxytetracycline (2, 5). Accumulation of tetracycline by resistant and sensitive E. coli strains. Cells of both sensitive and resistant strains, grown for 3 hr, were centrifuged, washed, and suspended in distilled water. The resulting suspension was inoculated into nutrient broth containing 50 to mg/ml of tetracycline and was incubated for 1 hr at 30 C. The accumulation of the drug was measured. To prevent growth during the incubation, kanamycin (10 Ag/ml) was added in some experiments. As shown in Table 2, a simnilar amount of tetracycline was accumulated in the presence or absence of kanamycin. Sensitive cells accumulated twice as much tetracycline as did resistant cells. TABLE 1. Accumulation of tetracycline in a sensitive straini of Escherichia coli K-12* Tetracy- cline concn Composition of reaction mixture accumulated per mg of cells pag/mi pg Complete 208 Minus MgSO4 40 Minus glucose 60 Complete 7 plus DNP, 10-3 M 100 Complete 37 Minus MgSO4 12 Minus glucose 2 Complete 3 plus DNP, 5 X 10-4 M * Cells were incubated in a medium containing glucose (5.5 X 10-2 M), K2HPO4 (10-2 M, ph 6.5), MgSO4 (4.0 X 10-4 M), and 100 to,ug of tetracycline for 1 hr at 30 C. DNP = 2,4-dinitrophenol. TABLE 2. Accumulation of tetracycline in Escherichia coli K-12 resistant and sensitive strains* Strain Resistant Sensitive concn,g/mi 50 100 200 50 100 200 Kanamycin, 10 pg/ml + + accumulated per mg of cells pg 5 7 60 172 200 11 34 143 300 287 629 * Cells were incubated in nutrient broth (ph 7.0) containing 50 to Mg/ml of tetracycline (with or without kanamycin) for 1 hr at 30 C.

630 IZAKI, KIUCHI, AND ARIMA J. BAC-MRIOL. Decrease of tetracycline accumulation activity in a resistant strain ofe. coli K-12. Resistant and sensitive strains of E. coli K-12 were grown in the presence or absence of 10 to 30 ug/ml of tetracycline for 3 hr. In some experiments, kanamycin was added to inhibit growth of the bacteria. Growth was measured by the turbidimetric method. After 3 hr, the cells were harvested, centrifuged, and suspended in distilled water. The accumulation activity of the cells was measured by incubating the cells in nutrient broth with,g/ml of tetracycline for 1 hr at 30 C. As shown in Table 3, sensitive cells did not grow in the presence of 10 to 30,lg/ml of tetracycline. Only a slight difference in accumulation activity was observed when the cells were incubated for 3 hr with or without tetracycline. Resistant cells grew well in the presence of tetracycline. However, ability to accumulate tetracycline was greatly decreased when the cells were grown in the presence of tetracycline. When growth of these tetracycline-resistant bacteria was inhibited by adding kanamycin, or when the cells were grown in the absence of tetracycline, such a decrease in their ability to accumulate tetracycline was not observed. Therefore, growth in the presence of tetracycline seemed to be essential for occurrence of the decrease in ability to accumulate the drug. When the cells were TABLE 3. Decrease in tetracycline accumulation activity in the cells of resistant Escherichia coli K-12 grown in the presence of tetracycline* Condition Growtht cycline accumulatedt Notetracycline... 488 247, 10l,g/ml. 481 13, l10 g/mi. Kanamycin,... 10,pg/ml 251, 30 Ag/ml. 489 11 No tetracycline 456 285, 10 pug/ml 210, 30 pg/mi 0 191 * Cells were grown in nutrient broth in the presence or absence of tetracycline (10 to 30 pg/ ml) for 3 hr at 30 C; then they were centrifuged, washed, and again incubated in nutrient broth containing pg/ml of tetracycline for 1 hr at 30C. TABLE 4. Decrease in tetracycline accumulation activity in the cells of Escherichia coli K-12 grown in the presence of small amounts of tetracycline* Condition Growtht accumulatedt No tetracycline... 502 240, 0.5 pg/ml... 482 151, 1.0,uglml... 463 64, 4.0,g/ml... 501 26 No tetracycline... 494 273, 0.5,g/ml... 195 171, 1.0,pg/ml... 86 144, 4.0 Ag/ml... 22 166 * Cells were grown in nutrient broth in the presence or absence of tetracycline (0.5 to 4.0,ug/ml) for 3 hr at 30 C; then they were centrifuged, washed, and again incubated in nutrient broth containing ptg/ml of tetracycline for 1 hr at 30 C. grown in the presence of smaller quantities of tetracycline, similar results were obtained (Table 4). One explanation for these observations might be that the population of the resistant strain was heterogenous, being composed of cells from both the sensitive and the resistant strains, and that only the resistant cells could grow in the presence of tetracycline. Therefore, the decrease in uptake of tetracycline by the resistant strain might be explained by selection of resistant cells in the culture, rather than by an actual decrease in the ability of a resistant cell to take up the drug. To check this possibility, the following experiment was performed. The resistant cells were grown in nutrient broth in 10-ml test tubes for 15 to 16 hr. Samples of the culture were diluted with sterilized distilled water. These suspensions were used to inoculate nutrient agar media' which contained tetracycline (30 Ag/ml) or did not contain tetracycline. The number of colonies which appeared on both media were counted after 48 hr at 37 C. As shown in Table 5, the numbe Of colonies was about the same on both media; therefore, the possibility of selection of re lstant cells from a mixed population includ cells is not likely. However, the greater resistance of the cultures resulting from growth of the antibiotic-resistant culture in tetracycline could

VOL. 91, 1966 TETRACYCLINE RESISTANCE IN E. COLI 631 TABLE 5. Effect of tetracycline on the growth of the resistant strain of Escherichia coli K-12* (jg/ml) No. of viable cells/ml Expt 1 Expt 2 None 1.8 X 109 1.8 X 109 30 2.4 X 109 1.6 X 109 * Cells were grown on nutrient agar with or without tetracycline. TABLE 6. Effects of tetracycline group antibiotics on the decrease in tetracycline accumulation activity in cells of Escherichia coli K-12* be the result of a selection of more resistant strains. Effect of tetracycline group antibiotics on the decrease of tetracycline accumulation. Cells of both resistant and sensitive strains were grown in nutrient broth for 3 hr at 30 C in the presence of various antibiotics of the tetracycline group. The growth rate and their ability to accumulate tetracycline were determined. As shown in Table 6, the presence of oxytetracycline or chlortetracycline in the medium reduced the ability of the resistant strain to accumulate tetracycline. The sensitive strain did not grow in the presence of these antibiotics, and no such decrease in accumu- Antibiotic added Concn Growtht accumulatedt cycline Mg/ml None... 465 289... 10 450 33 Oxytetracycline... 10 477 161 Oxytetracycline. 30 447 18 Chlortetracycline. 10 328 130 Chlortetracycline. 30 127 19 None..461 378... 10 0 432 Oxytetracycline. 10 0 385 Oxytetracycline. 30 0 359 Chlortetracycline. 10 0 351 Chlortetracycline... 30 0 204 * Cells were grown in nutrient broth in the presence or absence of antibiotic for 3 hr at 30 C; then they were centrifuged, washed, and again incubated in nutrient broth containing,gg/ml of tetracycline for 1 hr at 30 C. lation activity was observed, except perhaps at the highest dose of chlortetracycine studied (30 Ag/ml). Effects of chloramphenicol and streptomycin on the decrease of tetracycline accumulation ability. The resistant strain of E. coli K-12 used in these studies, as reported previously, is simultaneously resistant to several drugs, such as tetracycline, chloramphenicol, streptomycin, and sulfonamides. It is not yet clear whether resistance to tetracycline, chloramphenicol, and streptomycin may be explained by a common mechanism. To check this point, the effects of both chloramphenicol and streptomycin on tetracycline accumulation were investigated. As shown in Table 7, chloramphenicol had no effect on the tetracycline accumulation activity of the resistant strain. Streptomycin caused an apparent decrease in accumulation activity, but the decrease was only about one-third as great as in the case of tetracycline group antibiotics (at 30,g/ml). Such a decrease in apparent accumulation activity was also observed in the case of the sensitive cells which were incubated with chloramphenicol or streptomycin. Perhaps some other effect, for example, killing action of these antibiotics on the cells, might have caused these decreases in accumulated tetracycline. The resistant strain of E. coli K-12 is also quite sensitive to streptomycin as shown in column 2 of Table 7. Since chloram- TABLE 7. Effects of chloramphenicol anid streptomycin on the decrease in the tetracycline accumulation activity of Escherichia coli K-12* Antibiotic added Concn Growtht cycline accumulatedt Ag/mi None... 503 193 Streptomycin... 10 128 114 Streptomycin... 30 73 133 Chloramphenicol. 10 406 230 Chloramphenicol... 30 504 175 None... 439 237 Streptomycin... 10 19 125 Streptomycin... 30 0 124 Chloramphenicol... 10 33 104 Chloramphenicol... 30 0 137 * Growth and incubation conditions were the same as shown in Table 6. Ag

632 IZAKI, KIUCHI, AND ARIMA ji'l6dti -10,61i, TABLE 8. Increase in the level of resistance to tetracycline by growing the cells of E*hnhrkI coli K-12 resistant strain in the presence of tetracycline* First growth Turbidity at Second growthwth,f 0 min 30 min 60 min 90 min 120 min No antibiotic No tetracycline 50 63 96 140 240 No antibiotic 49 54 61 63 66 No tetracycline 45 67 100 140 240 45 64 93 135 215 Chloramphenicol No tetracycline 53 65 100 140 2 Chloramphenicol 50 54 60 62 64 * First growth: cells were grown in nutrient broth in the presence or absence of 20 Jug/ml of antibiotic for 3 hr at 30 C. Second growth: cells were then grown in nutrient broth in the presence or absence of 70 pg/ml of tetracycline for 2 hr at 30 C. phenicol did not induce decreased tetracycline accumulation, and the apparent decrease with streptomycin may have been due to killing of the cells, the decrease in tetracycline accumulation ability of the resistant strain may be specifically induced by the tetracycline group antibiotics. If this decrease in accumulation is related to the mechanism of multiple drug resistance, the specificity of induction of decreased accumulation is different from drug to drug, although resistance to the drugs occurs simultaneously. The mechanism of resistance to each drug is possibily due to a common factor, for example, impermeability of the bacterium to the drug. However, the specificity of impermeability might differ from drug to drug, and resistance to each specific drug might require the presence of that drug in the medium during growth of the bacterium for the expression of impermeability. Rise in level of resistance to tetracycline by growth of the resistant strain of E. coli K-12 with tetracycline. Experiments were carried out to investigate whether the level of resistance to tetracycline increased simultaneously with the decrease in the tetracycline accumulation activity of the resistant strain E. coli K-12 when cells were grown in the presence of tetracycline. The resistant strain of E. coli K-12 was grown in the absence of drug and in the presence of 20,ug/ml of tetracycline for 3 hr at 30 C. After harvesting and washing, the cells were incubated with or without 70,ug/ml of tetracycline, and the growth rate was measured. Chloramphenicol was also tested in the same way to determine whether it had an inducing effect for raising the tetracycline resistance. As shown in Table 8, only cells grown in the presence of tetracycline increased in resistance; cells grown without tetracycline or in the presence of chloramphenicol did not show such an increase. Therefore, the decrease in tetracycline TABLE 9. Accumulation of H3-tetracycline Escherichia coli K-12 resistant and sensitive strains*.e- Expt Strain KanamycIn, 10 cycline JAg/MI D' accnmu- O Iated$ 1 Resistant + 4 492 _ 66 151 Sensitive + 3 889 _ 0 745 2 Resistant + 0 335 _ 63 133 Sensitive + 1 595 _ 7 622 * Cells were grown in the presence or absence of kanamycin in nutrient broth containing 20 pg/ ml of H3-tetracycline for 1 hr at 30 C. t Expressed as change in turbidity during the 1- I Expressed as counts per minute per milligram of cells. accumulation seemed to be related direcly to an increased level of resistance to tetracycline. Uptake of IP-tetracycline by E. coli X-12 resistant and sensitive strains. Cells we grown for 3 hr at 30 C in nutrient broth. equal amounts of cells were inoctrient broth containing 20 /gml f-#. a- cycline and were incubated for 1 hr a 3 C. The incubation was conducted with -Wu kanamycin. The results are shown inl 9. Cells of the sensitive strintabccdilat amounts of H3-tetracycline than didtie strain. The difference in the amoitt between sensitive and resistant cellswa' when the cells were incubated in the aised in

VOL. 91, 1966 TETRACYCLINE RESISTANCE IN E. COLI 633 kanamycin, that is, when growth of cells occurred in the presence of tetracycline. These results were consistent with experiments already described in which nonlabeled tetracycline was used. DISCUSSION From the experimental results presented in this report, we concluded that the resistant strain of E. coli K-12 accumulated less tetracycline than the sensitive strain. The resistant strain lost tetracycline accumulating activity very rapidly and, simultaneously, its level of tetracycline resistance increased when it was grown in the presence of tetracycline. The sensitive strain did not show such changes. Similar results were obtained in experiments with low concentrations of H3-tetracycline. These results are consistent with the results obtained in experiments with oxytetracycline (4). It was also observed that decreased tetracycline accumulating activity was induced not only by tetracycline itself but also by chlortetracycline and oxytetracycline. However, chloramphenicol and streptomycin seemed to have no such effect, although the resistant strain was resistant to streptomycin and chloramphenicol. Therefore, we can assume that the decrease in tetracycline accumulating activity was specifically induced by tetracycline group antibiotics. If this decrease in uptake is a mechanism of drug resistance, we can assume that multiple drug resistance is not due to a mechanism which is common to several drugs but is due to a mechanism which has specificity corresponding to each drug. This concept may be accepted without difficulty considering that the permeability is specific in its nature. The fact that partial loss of resistance factors occasionally occurs may also support this concept (8). It is possible to consider that the resistance to tetracycline is due to the inactivation of tetracycline in the resistant cells. However, this possibility seems to be unlikely from the following observation. When the cells of a highly resistant strain of E. coli were incubated with tetracycline in a medium containing glucose, K2HPO4, and MgSO4, no appreciable decrease of tetracycline from the medium and no accumulation of tetracycline in the cells was observed. Only one spot identified as tetracycline was detected in the butanol extracts of the cells of both sensitive and resistant strains which were incubated with tetracycline by silica gel thin-layer chromatography [solvent 1, 10% citrate; solvent 2, butanolmethanol-10% citrate (4:2:2)], although much more tetracycline was found in the cells of the sensitive strain. ACKNOWLEDGMENTS We thank J. F. Snell and J. A. Last, Department of Agricultural Biochemistry, Ohio State University, for the correction of this manuscript and for fruitful discussions. We also thank R. Nakaya for cultures of E. coui K-12, and T. Komai for providing us H3- tetracycline. LITERATURE CITED 1. AKIBA, T., AND T. YOKOTA. 1962. Studies on the mechanism of transfer of drug resistance in bacteria. Influences of chloramphenicol and tetracycline on C'4-amino acid incorporation by ribosome isolated from drug sensitive strain and the multiple resistant strain of E. coli. Med. Biol. (Tokyo) 64:34-38. 2. ARIMA, K., AND K. IZAKI. 1963. Accumulation of oxytetracycline in the cells of E. coli relevant to its bactericidal action. Nature 200:192-193. 3. FRANKLIN, T. J., AND A. GODFREY. 1965. Resistance of Escherichia coli to tetracycline. Biochem. J. 94:54-60. 4. IZAKI, K., AND K. ARIMA. 1963. Disappearance of oxytetracycline accumulation in the cells of multiple drug resistant E. coli. Nature 200:384-385. 5. IZAKI, K., AND K. ARIMA. 1965. Effect of various conditions on accumulation of oxytetracycline in Escherichia coli. J. Bacteriol. 89:1335-1339. 5. OKAMOTO, S., AND D. MIzuNo. 1962. Inhibition by chloramphenicol of protein synthesis in the cell-free system of a chloramphenicol resistant strain of E. coli. Nature 195:1022-1023. 7. OKAMOTO, S., AND D. MIzuNo. 1964. Mechanism of chloramphenicol and tetracycline resistance in Escherichia coli. J. Gen. Microbiol. 35:125-133. 8. WATANABE, T. 1963. Infective heredity of multiple drug resistance in bacteria. Bacteriol. Rev. 27:87-109.