JOURNAL OF BACTERIOLOGY, Feb. 1968, p. 498-506Copyright @ 1968 American Society for Microbiology

Vol. 95, No. 2Printed in U.S.A.

Accumulation of Tetracyclines by Escherichia coliJOHN R. DE ZEEUW

Medical Research Laboratories, Chas. Pfizer & Co., Inc., Groton, Conniiecticut 06340

Received for publication 27 October 1967

The net accumulation of tetracyclines by Escherichia coli as a function of con-

centration was shown to be biphasic. At concentrations less than the bacteriostaticlevels, the mode of uptake was not azide-sensitive and was considered to be physicaladsorption on the cell surface. At concentrations above the minimal inhibitorylevel, a second, azide-sensitive, uptake component was functional in addition to thesurface adsorption process. This second energy-requiring mode was judged to repre-

sent penetration of the cytoplasmic membrane by tetracycline molecules to theirsites of inhibitory action. Each mode for a given tetracycline and culture is ex-pressed algebraically by a characteristic Freundlich equation. Resistance inE. coli is shown to be a result of diminished transport of antibiotic. However, thisresistance was due not to a reduction or loss of a transport mechanism but ratherto a requirement for higher antibiotic concentrations before the second mode ofuptake could become operative.

The physiological basis for the development ofbacterial resistance to the clinically importanttetracyclines is little understood. It has beenreported that a tetracycline sorption mechanismis somehow diminished in resistant strains (5, 6)Izaki and Arima (6) demonstrated that a resistantEscherichia coli strain accumulated much lessoxytetracycline than did a susceptible strain.Their procedures, however, allowed only forrelative comparisons at antibiotic concentrationswhich were several-fold higher than bacterio-statically effective levels. Franklin and Godfrey(5) worked at lower drug concentrations by theuse of labeled chlortetracycline and tetracycline.They also observed that resistant cells accumu-lated much less of the tetracyclines than didsusceptible cells, but quantitation was difficult.

Indirectly, recent studies, which establish thattherapeutic levels of tetracyclines act by theirinhibition of protein biosynthesis (4), also sug-gest that resistance to these antibiotics is due todecreased penetration of the drugs to the site oftheir inhibitory action. When cell-free extractsfrom susceptible and resistant E. coli cells werecompared, tetracycline inhibition of amino acidincorporation into polypeptides was found to beequivalent in both preparations (5, 10, 13). Thisdemonstration of crypticity indicated to all threegroups that resistance is probably due to aninadequacy in drug permeation.An aberration in the cell-transport process is

one of seven possible biochemical mechanismsproposed for drug resistance by Davis andMaas (3). To detail this mechanism, a program

was initiated to quantitate tetracycline transportby use of both resistant and susceptible strains ofE. coli. Attention was focused particularly onthose concentrations of different tetracyclineswhich are characterized as the minimal inhibitorylevels. In this paper, the methodology used andthe two concepts which were derived from experi-ments aimed at model development are described.Results with resting-cell populations of only twoE. coli strains are reported, and the uptake valuesare those of an equilibrium or a steady state anddo not reflect kinetic phenomena. The conditionsfor the incubation experiments were based on thework reported by Izaki and Arima (7).

MATERIALS AND METHODSE. coli cultures. The tetracycline-susceptible strain

of E. coli used was Crook's culture (ATCC 8739). Theresistant strain was culture 3-85 isolated from aMississippi poultry flock and provided by A. R.English (Chas. Pfizer & Co., Inc.). Both cultures weremaintained by periodic transfer on a nutrient agarmedium.

Preparation of resting-cell population. Portions(25-ml) of an inoculated nutrient broth contained in300-ml Bellco nephaloflasks were incubated at 37 Cwith gyratory shaking. Growth was followed turbidi-metrically in a Klett-Summerson colorimeter (KF-66filter). When the population was in the middle of itsexponential growth period, the flasks were refrigeratedand held at 4 C for 30 rnin. The cells were then re-covered by centrifugation. Typically, the initial cellload was 10 Klett units, while at harvest the readingwas 100 to 105 Klett units. The cells were washed twicewith cold 20 mM 2-(N-morpholino)ethanesulfonic acid(MES) buffer (pH 6.0). The final cell pellet was re-

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constituted to 200 Klett units with the same MESbuffer. This suspension was found to contain 8 X 108

cells per ml by both viable (plate) and total (Petroff-Hausser) counts. The cell mass was 800 ug per ml, asdetermined by the freeze-drying of a 2-ml portion ofcell suspension which was impinged on a MetricelVM-6 filter.

Tetracyclines. All antibiotics were obtained fromPfizer stock as the hydrochloride salts. Aqueous solu-tions were prepared on the day needed by the dissolv-ing of the antibiotics in 800 MM MgSO4-7H20 (withboiled-out water). The pH was adjusted to 6.0 withKOH. Just before use, the solution was passed througha membrane filter (type HA, Millipore Corp., Bed-ford, Mass.). Standard solutions for fluorometry weremade up with barbital-calcium reagent. A 1.0 ,uM solu-tion was prepared daily by dilution of a 200 ,uM stockstandard. The stock standard remained stable forseveral weeks when stcred in an amber bottle at re-frigerator temperature.

Barbital-calcium reagent. The stock solution con-tained 0.25 mole of barbital and 0.015 mole of CaCl2.2H20 per liter of distilled water. Barbital was re-crystallized once from water. This solution was ad-justed to pH 9.0 with KOH. The reagent was preparedby mixing 200 ml of barbital-calcium stock solutionwith 800 ml of aqueous n-propanol azeotrope. Dis-tillation of n-propanol as the azeotrope permits simplepurification to minimize fluorescent contamination.Care was exercised to exclude CO2 from the basicsolutions during preparation and storage.

Assay of tetracyclines. The procedure utilized toassay tetracyclines was an adaptation of the methoddescribed by Kohn (9). In place of a water-immisciblesolvent, n-propanol was used. The aqueous n-propanolin the final reagent (approximately 50%, w/w)was an excellent solvent for the CaCI2 and the barbi-tal, as well as for the uncharged tetracycline complexthat formed. Fluorometric measurement of the tetra-cycline content in barbital-calcium reagent solutionswas performed with an Aminco-Bowman spectro-photofluorometer with an Hanovia 901C-1 Xenonlamp, a RCA 1P21 photomultiplier tube, slit arrange-ment No. 4, and the sensitivity set at 50. The optimalwavelengths for excitation and emission were 390 and520 m,M, respectively. A fluorescence unit (FU) wasdefined as the product of the meter multiplier setting,the recorder deflection, and 1,000. The fluorescentpeak height was a linear function of the tetracyclineconcentration over an instrument range of 20 to20,000 FU. Typical fluorescence values of 1 juM solu-tions of the different tetracyclines are shown in Table1. The usual experiments were conducted to test thequality of the assay. No evidence was found forquenching phenomena or other interferences associ-ated with nontetracycline extractables.

Cellular uptake of tetracyclines. All experiments tomeasure celluar uptake of tetracyclines were carriedout at 32 C in a Dubnoff shaker. Portions (8-ml) of thereaction mixture were added to 25-ml Erlenmeyerflasks fitted with loose glass tops. The reaction mix-ture had an initial pH of 6.0 and was composed of:D-glucose, 50 mM; MES, 20 mnim; MgSO4-7H20, 200,uM; resting E. coli cells, 200 Mug/ml; and a tetracycline,

TABLE 1. Fluorescence of I ,UM standards

Mol wtAntibiotic of HCI FUa

salt

5-Oxytetracycline................ 497 5,9806-Deoxy-5-oxytetracycline........ 481 3,360Tetracycline..................... 481 9,4406-Demethyltetracycline.......... 467 7,6506-Deoxytetracycline.............. 465 5,1006-Demethyl-6-deoxytetracycline. . 451 2,820

a Fluorescence units (FU), corrected for thenonspecific background fluorescence of the bar-bital-calcium reagent (20 to 25 FU).

5 to 2,500 AM. Uptake was initiated by the addition ofthe appropriate antibiotic stock solution and wascontinued for 2 hr. Preliminary time-course studiesshowed little or no antibiotic uptake after 2 hr of in-cubation. To stop the uptake, 1 ml of the reactionmixture was added to 10 ml of a cold special buffer,pH 6 (MES, 20 mM; dithiothreitol, 3 mM; L-cysteine-HCI-H20, 1 mM; and MgSO4-7H20, 1 mM). This wasthen rapidly impinged on a 50% aqueous isopropanol-washed, 25-mm membrane filter (type HA). Thefilter disc and contents were then washed twice withcold 5-ml portions of the special buffer (pH 6). Thefiltrate and washings were collected and freeze-dried ifthe amount of nonsorbed antibiotic was to be de-termined.

Extraction of tetracyclines. At room temperature,10 ml of the barbital-calcium reagent was used to ex-tract the antibiotic from a membrane filter with itsimpinged cells. The aqueous n-propanol solvent hasthe desired lipophilic properties to allow direct ex-traction of the cells without preliminary treatment.Extraction is almost immediate, but the disc was rou-tinely allowed to stay in contact with the reagent for30 min. The extract was clarified by centrifugation ifit was necessary. Nonsorbed antibiotic in the lyophi-lized filtrate was dissolved in 50 ml of barbital-cal-cium reagent.

Antibiotic sensitivity testing. The minimal inhibi-tory concentration (MIC) of each tetracycline for thetwo E. coli strains was determined under conditionswhich paralleled those characteristic of the antibioticuptake experiments. To use the uptake formulationas a growth medium, the MES buffer level was raisedto 200 mM; 20 mM (NH4)2SO4 and 200 ,uM KH2PO4were added along with trace quantities of Fe++, Mn++,and Ca++. Tubes which contained 5 ml of mediumsupplemented with a tetracycline were inoculated with0.05 ml of a 10 Klett unit resting-cell suspension andincubated at 32 C until the control tube read approxi-mately 100 Klett units. To establish the MIC, thegrowth in each tube as a percentage of control wasplotted against the log of the antibiotic concentration.The data were extrapolated to zero growth graphically(Fig. 1). The assigned MIC values are listed in Table 2.

Chromatography. Cell extracts for paper chromato-grams were prepared by a scaling-up of the uptakeand extraction procedures described above. The cells

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- 75-j

0

z0

50 D000T T

DOT

25-

0

0

0.5 1.0 1.5 2.0

LOG,O CONCENTRATION (y M)

FIG. 1. Growth of resistant Escherichiaa functioni of the particular tetracycline (T,DOT, 6-deoxytetracycline; DOOT, 6-tetracycline) and its concentration. Each psingle turbidimetric determination. Extens,sulting lines to zero growth permitted as

minimal inhibitory concentrations.

TABLE 2. Miniimal inhibitory colice

Susceptiblestrain

AntibioticC(oncn Logi l

(pug,mi) puM (

5-Oxytetracycline 5.6 1.056-Deoxy-5-oxytetra-

cycline............. 4.0 0.92Tetracycline.......... 3.8 0.906-Demethyltetra-

cycline............. 1.9 0.606-Deoxytetracycline... 6.0 1. 116-Demethyl-6-deoxy-

tetracycline......... 3.4 0.88

a Average of duplicate determinatscribed in Materials and Methods.

were recovered by centrifugation insteadAfter the cells were washed twice withbuffer (pH 6), they were lyophilized and ea minimal volume of barbital-calcium i

extract was clarified by centrifugation andon Whatman no. 4 paper. The three sywere used in a standard manner at roomwere: no. 33 (ethyl acetate saturated witscending); no. 60B (chloroform, nitromdine, and water, 10:20:3:1, clarifiedthrough glass wool, descending); and thLast and Snell (I l) (isobutyric acid and Inium hydroxide, 5:3, ascending). For sythe paper was treated with Mcllvain's buand dried before it was spotted. For systhe paper was similarly treated but wETetracyclines were located on the parfluorescence under ultraviolet light.

Radioactivity measurementts. Tetracycidrochloride was used (New England N

Boston, Mass.). Cell extracts or digests of 1-ml vol-umes were counted in a Packard Tri-Carb liquidscintillation spectrometer (model 314-DC). All countsobserved were corrected for background. Theefficiency factor for each sample was established bythe internal standard technique (2). Results werecomparedasdisintegrations per minute (DPM). Di-gests of membrane filter-impinged bacteria were pre-pared to measure total radioactivity by an extensionof the formamide digestion procedure described byNeujahr and Ewaldsson (12). The filter suspended in6.5 ml of formamide was held at 60 C for 2 hr under a

2.5 3.0 nitrogen blanket. After this incubation, 3.5 ml of di-methylformamide, which contained 1.5% hydrogenperoxide, was added to dissolve the filter. If allowed

X coli 3-85 as to stand overnight at room temperature, the final di-Itetracycline; gest was a clear, virtually colorless solution.*deoxy-5-oxy-oilnt (0) is a RESULTSlion of the re-Ysigunmenits of Recovery studies. Assessment of the method-

ology was keyed on oxytetracycline becauseKohn (9) reported this to be the one tetracycline

?trationsa in the compound series whose barbital-calciumcomplex was incompletely extracted by his pro-

Resistant cedure. The principal studies were done at anstrain___ initial incubation concenlration of 200 m, which

Concn Logio is higher than the MIC for the susceptible strainpg/ml) '-'1 but less than that for the resistant culture. The

data from recovery experiments are shown in130 2.41 Table 3. Of the 20 individual recovery determina-

49 2.Ot tions, 18 were greater than 95 %. These experi-200 2.62 ments also directly compared the ability of the

susceptible and resistant cultures to take up95 2.31 oxytetracycline. The susceptible strain sorbed8.3 1 .25 36% of the total antibiotic available, whereas

the resistant strain accumulated only 1 %. The5.7 1 .10 chemical profile of the oxytetracycline sorbed

by the resistant strain was studied chromato-ions as de- graphically. Only one significant fluorophor was

found, the oxytetracycline. No evidence of cellulartransformation of tetracyclines by resistant or

of filtration, susceptible strains was ever detected. Transforma-cold special tions to nonfluorescent moieties were ruled outxtractedwith since recoveries measured by fluorescence were

Iethen spotted essentially complete.Istems which In similar studies in which the initial concen-itemperature tration of oxytetracycline was 20 m, 18.7 ith water, de- 1.6 mAmoles (94%) of each ml was accountedethane, pyri- for in the cell extract plus the filtrate.by filtration The totality of the extraction of the filter-ie solvent of impinged cells by the barbital-calcium reagent0.5 N ammo- was further investigated by use of tritium-,stem no. 33, labeled tetracycline and the resistant cultureifer (pH 3.5) (Table 4). The amounts of radioactivity in cellas used wet. extracts and cell digests were comparable, ver-

per by their ifying that the reagent effectively extracts lowlevels of cell-associated tetracyclines.

line-7-3H hy- Representative uptake determination. The rawuclear Corp. fluorescence data from a typical 2-hr uptake

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TABLE 3. Recovery of oxytetracycline after uptake experiments

Oxytetracycline per ml of 2-hr incubation mixture'

Escherichia coli strain and conditionMembrane filterb Filtrate (m,umoles) Totalc (m,umoles) Recoveryc

Susceptible, resting. 71.5 128.8 200.3 i 5.15d 100.2Resistant, resting................ 2.02 193.1 195.1 :1: 4.71 97.6Susceptible, nonviablee .......... 2.09 185.9 188.0 O±i4.60 94.0Resistant, nonviable.1.95 192.0 194.0 i 2.89 97.0

a Protocol as described in Materials and Methods with 200 jAM oxytetracycline in make-up. Eachvalue is the average of five determinations.

b Antibiotic found after 1 ml of mixture was impinged.c The average total was 195.0 + 6.31 m,umoles; the average recovery was 97.5%zj.d Standard deviation.e Resting-cell population heated in a boiling-water bath for 15 min.

TABLE 4. Efficacy of barbital-calcium reagent as ani ext ractant

Uptake6by DP-\I/mg of cells ~~DPM ratiosSpecific activity of

fluorescenceDPmg of cells

Expt tetracycline solution fursec(Dp\jam,AOle (MAMcelS)M Of

Expected by I Found in Found in Extract Extractfluorescence extract cell digest Digest Expected

1 3.46 X 104 2.56 8.86 X 104 7.63 X 104 8.35 X 104 0.914 0.8612 4.09 X 104 3.10 12.68 X 104 10.91 X 104 11.39 X 104 0.958 0.860

Avg 2.83 - 0.936 0.861

a DPM = disintegrations per minute.b Protocol as described in Methods and Materials with 50 AM tetracycline in the make-up and with

use of the resistant culture.

determination experiment are shown in Table 5.In the absence of cells, a small but readily meas-urable quantity of oxytetracycline becameinsoluble in the incubation mixture during theexperimental time course. This material, im-pinged on the membrane filter, could not beremoved by washing, but it was extracted byreagent. This was true of all tetracyclines studied.No other procedural problems were encountered.In agreement with Franklin and Godfrey (5),we found little loss of cell-associated tetracyctinesduring the washings. Therefore, tetracyclineuptake was defined as the quantity of antibioticretained by the washed cells and corrected for (i)the zero-time fluorescence of the complete in-cubation mix, and (ii) the quantity of nonsorbedantibiotic which became filter impingeable duringthe time course of uptake.

Mathematical relationship of uptake as a func-tion of concentration. To partition the multipleeffects of antibiotic concentration on net uptake,use was made of Arima and Izaki's (1) observa-tion that 10-2 M sodium azide significantly hin-dered, but did not abolish, cellular accumulation

of the tetracyclines. Over the entire concentra-tion range evaluated, the portion of uptake notsensitive to azide inhibition could be expressedbest by a Freundlich equation for an adsorptionisotherm. While the Freundlich equation is anempirically derived relationship, it is excellentfor use in compiling information and interpola-tion. In its logarithmic form it is written: loguptake = log a + f log concentration, whereuptake has the units of millimicromoles permilligram of cells, and the concentration is thenonsorbed antibiotic in millimicromoles permilliliter. With this relationship established, thecellular accumulation of tetracyclines understandard conditions was recognized to be bi-phasic. From low to high antibiotic concentra-tions, the initial mode is indistinguishable fromthe uptake observed in the presence of azide,and it is expressed by the same isotherm equa-tion. But, abruptly at a concentration charac-teristic for each tetracycline and each culture,there is the onset of a second mode of uptakewhich is azide-sensitive. This second phase isspecified also by a Freundlich equation but byone with a much steeper slope (Fig. 2, 3).

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Specification ofcellular accumulation ofdifferenttetracyclines. The level of uptake and the par-ticular concentration at which the azide-sensitivecomponent of the process is initiated was es-tablished for a series of tetracyclines by use ofboth the susceptible and resistant strains. Theindividual isotherm equations were determinedby ascertaining the uptakes in the presence andabsence of 10-2 M azide over the one-log con-centration range just after the respective minimalinhibitory concentrations. The individual inter-cepts were then calculated (Table 6, Fig. 4, 5).

Tetracycline adsorption by nonviable cells.Early studies suggested that the portion of tetra-cycline uptake which is not abolished by azidewas synonymous with the uptake characteristic

TABLE 5. Raw fluorescence data from a 50 ,.umoxytetracycline study with susceptible

Escherichia coli

Fluorescence units perml of extract"

Antibiotic used No cells SusceptibleE. coli

0 time 2 hr 0 time 2 hr

None............... 23 24 40 3750SM oxytetracycline.... 53 152 95 9,742

Corrected only for the background fluores-cence of the barbital-calcium reagent (25 fluores-cence units).

2.4-

0

E 1.21FIG. 2. aZIDE

E

0.6-

0

0.

0.0----

1.2 1.8 2.4 3.0 3.6

LOG1O CONCENTRATION (pM)

FIG. 2. Tetracycline uptake by the resistant Escher-

ichia coli as a function of concentration. Each point isthe average of three determinations. Symbols: 0 =

resultsfrom incubations supplemented with 1" M azide;E = results from standard conditions.

S

Ie.

EE 1.2

1.2 1.8 2.4

LOGIO CONCENTRATION (puM)FIG. 3. Oxytetracycline uptake by the sensitive

Escherichia coli as a function of concentration. Eachpoint is the average of two determinations. Symbols:o = results from incubations supplemented with 1O2M azide; i = results from standard conditions.

of heat-killed cells. To test this hypothesis, experi-ments were conducted with both the resistantand susceptible strains; oxytetracycline levelswere examined at pre- and postintercept concen-trations (Table 7). Clearly, the cellular uptake ofoxytetracycline in the presence of azide is equiva-lent to the quantity of antibiotic adsorbed bynonviable cells.

DIscUSSIONProgress toward understanding the physiolog-

ical basis for the development of bacterial re-sistance to the tetracyclines has been limited by(i) the lack of a simple assay procedure which isaccurate and precise at the minimal bacteriostaticlevels of these antibiotics, and (ii) by the absenceof a specific biological model which may besubjected to experiment. This work was addressedto both of these problems.The adaptation of Kohn's fluorometric pro-

cedure (9) proved a solution to the first problem.The method was convenient and applicable toall tetracycines studied. The measurementswere sufficiently sensitive to allow determinationof cellular uptakes when the initial antibioticconcentrations were less than the minimal in-hibitory levels. The recovery experiments and theisotope study of sampling effectiveness attestto the accuracy of the method.The recovery studies and the chromatograms

of the cell extracts showed that no significantquantity of tetracycline was degraded or trans-

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TABLE 6. Compilation of sorption data for various tetracyclines

Intercept of isotherms Total uptake AzideuipnsenitiveEscherichia coli Tetracycline Logiostrain

uptake Logio Isotherm ra Isotherm a(mnAmoles/ concn (jIm) slope slopemg)

Resistant 5-Oxytetracycline 0.93 2.93 2.68 1.00 0.435 0.9596-Deoxy-5-oxytetracycline 0.84 2.11 2.52 0.990 1.27 0.982Tetracycline 0.90 2.67 1.58 0.986 0.558 0.9936-Demethyltetracycline 1.56 2.38 2.23 0.991 0.524 0.9816-Deoxytetracycline 0.68 1.41 1.35 0.990 0.439 0.9846-Demethyl-6-deoxytetracycline 0.22 0.63 1.43 0.983 1.08 0.997

Susceptible 5-Oxytetracycline 0.33 1.05 3.30 0.998 0.389 0.9856-Deoxy-5-oxytetracycline 0.72 1.54 3.48 0.996 0.418 0.971Tetracycline 0.25 1.00 0.867 0.997 0.212 0.9536-Demethyltetracycline 0.38 0.87 0.827 0.987 0.127 0.9856-Deoxytetracycline 0.82 0.99 2.53 0.975 0.345 0.9786-Demethyl-6-deoxytetracycline 0.84 0.98 1.76 0.995 0.179 0.979

a r is the correlation coefficient for the "best" line calculated by the minimizing of the uptake com-ponents of deviation.

SENSITIVE E. COLI

0.5 I.Q 1.5 2.0 2.5

LOGIO CONCENTRATION (jUM)

FIG. 4. Sorption isotherms of the sensitive Escher-ichia coli for the different tetracyclines (T, tetracycline;OT, 5-oxytetracycline; DMT, 6-demethyltetracycline;DOT, 6-deoxytetracycline; DOOT, 6-deoxy-5-oxy-tetracycline; DMDOT, 6-demethyl-6-deoxytetracy-cline). The lines with the steeper slope are plots ofthetotal uptake. The azide-insensitive portions of the up-take are shown by the lines with the shallower slopes.

formed during the course of the experimentswith either culture. These data are evidencethat the resistant strain does not achieve itsparticular physiological state through metabolicmodification of the tetracycline during its trans-port and accumulation processes. This knowledge,plus the crypticity phenomenon cited earlier,

and Franklin and Godfrey's (5) observationthat resistance is not attributable to super excre-tion of the antibiotic by resistant strains, com-pletes a body of indirect evidence that tetracyclineresistance results from decreased penetration ofthe drug to its inhibition sites.The comparative data presented document, in

detail, the several general reports which showedthat, at concentrations which allow E. colistrains to be classified as resistant or susceptibleto a particular tetracycline, the resistant cultureaccumulates much less of the antibiotic than doesthe susceptible one. However, the accumulationof a tetracycline as a function of concentrationwas found to follow an identical pattern whetherthe culture was resistant or susceptible. Only theplacement of the pattern in two-dimensionalspace was highly specific for each culture andeach compound. The discovery that the uptakedata for a given tetracycline and culture could beexpressed by two distinctive Freundlich equa-tions allowed ready compilation and interpre-tation. The dependence of uptake on concentra-tion is biphasic, and it changes from the firstmode to the second very abruptly. The firstphase is not dependent on functional cell ma-chinery but the second is. The second mode isthe principal mechanism of uptake at relativelyhigh antibiotic concentrations and is undoubtedlythe energy-dependent process described by Arimaand Izaki (1).The data which compare the two cultures have

led to the conclusion that tetracycline-resistancedevelopment actually consists of the shift of the

1.75

1.50-

0= 1.25-

EE

0.75-w

I0-

200. 0.25-

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bimodal uptake pattern into a region of higherconcentration. Conceptually, a strain that be-comes resistant simply requires higher externalconcentrations of tetracycline before the secondmode of uptake is triggered to become functional.Although resistance to the tetracycline is clearlya transport anomaly, the genome-mediated

I.75

1.50

7o 1.25

IV 1.00

E

,, 0.75

D 0.500

0-i

0.25

RESISTANT E. COLI

DO'

DMDOT

DMT

/r DOOT

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5LOGIO CONCENTRATION (YM)

FIG. 5. Sorption isotherms of the resistant Escher-ichia coli for the different tetracyclines (see legend toFig. 4). The lines with the steeper slope are plots of thetotal uptake. The azide-insensitive portions of the up-take are shown by the lines with the shallower slopes.

TABLE 7. Influence of cell conditiont on the azide-insensitive oxytetracycline (OT) uptake

OT uptakea. b(mpmoles per mg of cells)

LoginCulture OT ial Resting cells Nonviable cellsc

No azide 10-2 M No 10-2 Mazide azide azide

Susceptible 0.60 1.29 1.28 1.34 1.271.40 10.3 4.01 4.58 4.11

Resistant 2.20 5.35 5.15 4.85 4.753.40 875 35.4 35.8 35.7

a The geometric means for the four conditions were15.8 (resting, no azide), 5.53 (resting, 10-2 M azide),5.73 (nonviable, no azide), and 5.46 (nonviable,10-2 M azide).bStandard procedure, single flask per determi-

nation. The calculated log,o intercept concentra-tion for the isotherms for the susceptible culturewas 1.05; for the resistant culture it was 2.93.

c Resting-cell population heated 15 min in aboiling-water bath.

modification of the cell is not the simple loss orreduction of the active transport mechanism.Another and more obvious conclusion stems

from the relationship observed between the con-centration at which the uptake mode changes andthe organism's susceptibility to the antibiotic.There is essentially a one-for-one correlationbetween the minimal inhibitory concentrationand the concentration at which energy-dependenttransport becomes functional. From a samplesize of 12 (2 cultures x 6 tetracyclines), thecorrelation coefficient was 0.934 between thelogarithms of these two concentrations. Thecorrelation is remarkably good when one con-siders the dissimilarities of the two physicalenvironments used for the respective determina-tions.Based on the foregoing, and on the assump-

tion that the double phasing of uptake is a mani-festation of cell compartmentation, a model isadvanced which suggests the sequence of eventswhich occur in transport of tetracyclines and theconsequences of genetic change toward resistance.The first interaction between a tetracycline and

E. coli is proposed to be a sorption process byactive centers located in cell wall material andto be based on physical forces only. For a givenstrain and compound, the number and affinityof the active centers specifies the concentrationeffect. A particular level of tetracycline satura-tion or penetration of the cell by this first uptakemode must be achieved before molecules arespatially available for participation in the secondmode. This second process, when it occurs, bringsantibiotic molecules inside the permeabilitymembrane by an energy-dependent push or pulltransport mechanism and makes them availablefor binding at the sites inhibiting protein bio-synthesis. These latter are the bacteriostaticallyeffective tetracycline molecules. Those boundto cell wall centers are essentially irrelevant.The model, as far as it has gone, is consistent

with the facts. Available data support the con-cept that the first process is simply physicaladsorption by a bulk constituent of the cell,specifically the cell wall. Franklin and Godfrey(5) showed that, at minimal bacteriostatic levels,the majority of the sorbed chlortetracycine wasin the fraction consisting mainly of cell wall ma-terial. In my work, the process was shown to beazide-insensitive. It was also observed that up-take by this mode was identical whether the cellswere viable or heat-killed. All the evidence sup-ports the contention that no metabolic machineryis required for this first sorption process to occur,and that the adsorbent is exterior to the cyto-plasmic membrane.The proposition that not all tetracycline uptake

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TETRACYCLINE ACCUMULATION BY E. COLI

is relevant to bacteriostatic action is supportedby studies which investigate the mechanism ofaction of these antibiotics. The tetracyclines mostprobably interfere with the transfer of amino acidsfrom the aminoacyl-transfer ribonucleic acids topolypeptides on the ribosomes (4). L. E. Day(unpublished data) has shown that 25 ,ug per mg ofribosomal protein of the different tetracyclinesused in these experiments was roughly equivalentin effectiveness. All inhibited 40 to 70% of poly-uridylic acid-directed polyphenylalanine forma-tion in a cell-free E. coli system. While the quanti-tative meaning of this effect is difficult to extrap-olate to the intact cell, it is reasonable to assumethat, within the cell, all of the tetracycines underexamination do not differ by more than twofoldin their efficacy as inhibitors of protein biosyn-thesis. Yet, the uptake levels for the differenttetracyclines at the MIC values vary sevenfold,which indicates that not all sorbed antibiotic isfunctional in the inhibitory process.

Since the tetracyclines exert their effect at theribosomal level of cell organization, a secondprocess must become operative to enable theseantibiotics to penetrate beyond the cell wall. Theobserved second mode of uptake is energy-de-pendent. This satisfies at least one of the require-ments for an active transport of tetracyclinesacross the cytoplasmic membrane.The suggestion that the second mode represents

the accumulation of bacteriostatically effectivemolecules is compatible with the observed effectof the tetracyclines on bacterial growth. The con-centration correlation has already been men-tioned. The onset of that portion of antibioticaccumulation which is azide-sensitive is veryabrupt. This particular accumulation also risesextremely rapidly with small increases in externalconcentration (Fig. 6). The abrupt and rapid in-crease of this class of molecules as a function ofconcentration has a direct counterpart in thesharp and sudden diminution in growth responsewhich was observed during the titration of mini-mal inhibitory concentrations.The model also permits a mechanistic interpre-

tation of the multistep, cumulative resistancepattern found for tetracyclines (see 14). Resist-ance development is viewed as a series of changesin cell wall chemistry which increase the quantityor alter the affinity, or both, of those sites whichadsorb the tetracycline. These modifications ex-terior to the cytoplasmic membrane lead to re-quirements for higher extracellular antibiotic con-centrations in order to initiate the second uptakeprocess.

Additional genetic studies coupled with uptakedeterminations are warranted. In our uptake ex-periments with resting cells, resistant cells

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OT CONCENTRATION (mj, moles/ml)FIG. 6. Azide-sensitive portion of oxytetracycline

uptake by the susceptible Escherichia coli. The curvewas determined from the difference between the iso-therms for the total and the azide-insensitive uptakesdetailed in Table 6.

accumulated less antibiotic than susceptible oneseven though they were grown in a medium withno tetracycline supplementation. This contrastswith the report of Izaki, Kiuchi, and Arima (8),who showed that, for a strain whose resistancefactor was demonstrably extrachromosomal, thedecreased uptake of tetracycline is dependent onthe growth of the organism in the presence of thedrug. The genetic factor in the resistant isolateused in my work could not be transferred to E.coli K-12 by cell-to-cell contact (A. R. English,unpublished data).Obvious kinetic and cell fractionation data are

necessary to prove and modify the model.

ACKNOWLEDGMENTI thank Irene Stasko for her skilled technical assist-

ance.

LITERATURE CITED

1. ARIMA, K., AND K. IZAKI. 1963. Accumulation ofoxytetracycline relevant to its bactericidal ac-tion in the cells of Escherichia coli. Nature 200:192-193.

2. DAVIDSON, J. D., AND P. FEIGELSON. 1957. Prac-tical aspects of internal-sample liquid-scintilla-tion counting. Intern. J. Appl. Radiation Iso-topes 2:1-18.

3. DAvIs, B. D., AND W. K. MAS. 1952. Analysis

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of the biochemical mechanism of drug resist-ance in certain bacterial mutants. Proc. Natl.Acad. Sci. U.S. 38:775-785.

4. FRANKLIN, T. J. 1966. Mode of action of thetetracycines. Symp. Soc. Gen. Microbiol. 16:192-212.

5. FRANKLIN, T. J., AND A. GODFREY. 1965. Re-sistance of Escherichia coli to tetracyclines.Biochem. J. 94:54-60.

6. IzAuK, K., AND K. ARIMA. 1963. Disappearanceof oxytetracycline accumulation in the cellsof multiple drug-resistant Escherichia coli.Nature 200:384-385.

7. IZAKI, K., AND K. ARIMA. 1965. Effect of variousconditions on accumulation of oxytetracyclinein Escherichia coli. J. Bacteriol. 89:1335-1339.

8. IZAKI, K., K. KIUCHI, AND K. ARIMA. 1966.Specificity and mechanism of tetracyclineresistance in a multiple drug resistant strainof Escherichia coli. J. Bacteriol. 91:628-633.

9. KOHN, K. W. 1961. Determination of tetracy-clines by extraction of fluorescent complexes.

J. BACTERIOL.

Application to biological materials. Anal.Chem. 33:862-866.

10. LASKIN, A. I., AND W. M. CHAN. 1964. Inhibitionby tetracyclines of polyuridylic acid directedphenylalanine incorporation in Escherichia colicell-free systems. Biochem. Biophys. Res.Commun. 14:137-142.

11. LAST, J. A., AND J. F. SNELL. 1966. Microbiolog-ical transformation of oxytetracycline. Nature211:1002-1003.

12. NEUJAHR, H. Y., AND B. EWALDSSON. 1964.Counting of weak ,B-emitters in bacterial cellsby means of the liquid scintillation method.Anal. Biochem. 8:487-494.

13. OKAMOTO, S., AND D. MIZUNO. 1964. Mechanismof chloramphenicol and tetracycline resistancein Escherichia coli. J. Gen. Microbiol. 35:125-133.

14. POLSINELLI, M. 1964. Genetic analysis of oxy-tetracycline resistance in Bacillus subtilisby means of transformation. J. Gen. Microbiol.36:423-428.

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