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Antibiotic uptake by bacteria as measured by partition in polymer aqueous phase systems
ANALYTICAL
BIOCHEMISTRY
Antibiotic
NICOLE CNRS-CERCOA
141, 94-1~
(1984)
Uptake by Bacteria as Measured by Partition in Polymer Aqueous Phase Systems MOREAU,PATRICIA
LACROIX,ANDLAURENTFOURNEL
(laboratoire associh ri I’ENSCP) 2 ci 8, rue Henri Dunant 94320 Thiais, France Received January 13, 1984
Polyetbyleneglycol/dextran and polyethyleneglycol/salt two-phase systemsare used to measure the entry of antibiotics into Escherichia coli and Staphylococcus aureus cells. Aminoglycosides, macrolides, a quinolone, and a cycline were assayed. The method is simple and rapid and eliminates the problems encountered with filters, especially nonspecific binding; it allows rapid uptake kinetics to be measured.
The ability of antibiotics to enter bacterial cells is an important factor in the antimicrobial activity of drugs. The amount of drugs taken up by bacteria has generally been evaluated by filtration on glass fiber, cellulose nitrate, polycarbonate filters, etc., depending on the kind of antibiotic assayed (1,2); alternatively, cells may be centrifuged. Although widely used, these methods present some disadvantages. It is difficult to make more than one or two measurements per minute: with the centrifugation technique, 1 min is the minimum time required to obtain a cell pellet; with the filtration technique, one needs to take into account the time of filtration of the sample and of 2 or 3 washings. For a slow uptake kinetics, such as that of aminoglycosides, this is not a drawback, but for rapid uptake kinetics (pristinamycins, quinolones) it is a limitation. Furthermore, in the case of the filtration method, the preparation and washing of the filter are critical to avoid variable binding of antibiotic to filter, variable amounts of trapped medium, and variable times of filtration. Phase partition is a method used for purification and analysis of proteins, cells, cell organelles, and membrane vesicles (for reviews, see (3-5)). It has also been used as a tool to measure interactions between biomolecules (6,7) or for quantitation of bacterial cells (8). The purpose of this paper is to show CW3-2697184 $3.00 Copyright 8 1984 by Academic FVes, Inc. All rights of repmduction in any form reserved.
how phase partition can be used as a very convenient method to measure the uptake of antibiotics by bacterial cells. MATERIALS
AND METHODS
Chemicals
Dextran T500 was purchased from Pharmacia; polyethylene glycol (PEG)’ 6000 was purchased from Touzart et Matignon. Dihydrostreptomycin and pristinamycins were gifts from Rhhne-Poulenc; rosaramicin was a gift from Unilabo; pefloxacin was a gift from Roger Bellon; fortimicin A was a gift from Abbott; tetracycline was a gift from Pfizer. [3H]Dihydrostreptomycin and [3H]acetylcoenzyme A were purchased from Amersham; dihydrorosaramicin and [3H]dihydrorosaramicin (9), [3H]pefloxacin (unpublished results) and [3H]pristinamycins (10) were synthesized in our laboratory. All other chemicals were of analytical grade. Bacterial Strains, Media, and Phase Systems Escherichia coli K 12 and Staphylococcus aureus 209P were from our laboratory collection. Escherichia coli R135 (11) synthesizes ’ Abbreviation used: PEG, polyethylene glycol. 94
ANTIBIOTIC
UPTAKE
MEASURED
the enzyme AAC3(1) which is capable of acetylating gentamicin and fortimicins. Medium A was Davis and Mingioli minimum medium; it contained, per liter, 7 g of K2HP04, 3 g of KH2P04, 0.5 g of sodium citrate * 3 H20, 0.1 g of MgSO4 7Hz0, 0.1 g of ammonium sulfate, 2 g of glucose (the latter being autoclaved separately), pH 7. Medium B contained per liter, 0.7 g of ammonium sulfate, 1 g of KH2PO4, 6 g of Na2HP04. 12 HzO, 0.2 g of magnesium sulfate. 7H20, pH 7.2. Medium C contained per liter, 0.25 g of magnesium sulfate. 7H20, 7.1 g of KI-I2P@, 2 g of glucose; the pH was adjusted to 6 with KOH. l
Uptake Assays E. coli. Stock solutions of 20% (w/w) dextran and 40% (w/w) PEG were prepared in water. The phase system was prepared by mixing 25 g of 20% dextran, 10 g of 40% PEG, 50 g of 2X concentrated medium A, 15 g of water. The mixture was shaken and the phases were allowed to separate either through overnight settling at 4°C or by centrifugation at 1OOOgfor 5 min at 4°C. Cells were grown at 37°C in medium A up to middle to late log phase (turbidity of 100, measured with an Hach turbidimeter Model 2100A). They were centrifuged, washed with dextran phase, and resuspended in the same phase, at a three-times higher concentration. After 10 to 15 min of preincubation at 37°C with shaking, the antibiotic was added as specified and shaking was continued. At different times after antibiotic addition, l-ml samples were removed and added to 1 ml of PEG phase kept in small tubes at 0°C. The mixtures were stirred with a vortex-type mixer and left for 1 to 2 h at 0°C. An aliquot from one or both phases was sampled for antibiotic analysis. For radioactive antibiotics, 200 ~1 of each phase was mixed with 200 ~1 of water and 3 ml of Beckman HPb scintillation fluid. In the case of fortimicin A, a radioenzymatic assay was carried out (12). A 25-~1 volume of a purified extract of E. coli R 135 ( 13) and 10
BY
95
PARTITION
~1 of an aqueous solution of AcCoA (0.2 pm/ ml, 54 Ci/mol) were added to 50 ~1 of the phase to be tested (the PEG phase was boiled a few minutes before assay to release the fortimicin from the bacteria). The mixtures were incubated for 45 min at 37’C, 50 ~1 were placed on Whatman P8 1 paper and processed as previously described ( 12). A standard curve was obtained with phases containing known concentrations of fortimicin. S. aureus. The uptake of antibiotics was assessedas for E. cob, with the following modifications: cells were grown in tryptic soy broth to 100 Klett units (Klett Summerson, blue filter, 400-420 nm). After centrifugation at 3000g for 10 min, they were washed with medium B and resuspended in the same medium at a concentration corresponding to 300 Klett units (7 X lo8 bacteria per ml). The stock solutions were 40% PEG (w/w) in 20 mM Na2HP04 ; and 40% MgS04 - 7H20 (w/w) in 20 IIIM Na2HP04. Tubes containing 375 ~1 of PEG stock solution and 1125 ~1 of MgS04 stock solution were kept at 0°C. To determine the uptake, bacteria plus antibiotic were shaked at the desired temperature. At different times, 500~~1 samples were removed and added to the cold phases. After stirring, the mixture was left for 20 to 30 min at 0°C. The resulting volumes were 0.5 and 1.5 ml for the upper PEG-rich phase and the lower salt-rich phase, respectively. Medium C was used in the case of tetracycline uptake. All the volumes were doubled to allow aliquots of 500 ~1 to be sampled. The fluorescence was measured by excitation at 365 nm and emission at 510 nm. For both microorganisms, the partition of antibiotic and bacteria alone were determined under the same conditions. The zero-time value was measured at 0°C. RESULTS
AND
DISCUSSION
The principle of the method is schematized in Fig. 1. The following operations are carried out to assay the uptake of an antibiotic by bacteria:
MOREAU,
LACROIX,
AND FOURNEL
bacteria
plus antibiotic
\
time 1 d the uptake kit-&a m&ii
FIG. 1. Principle of the method. Bacteria and antibiotic are partitioned separately or together in the two phase system. If there is no interaction between them, they partition independently of each other. As soon as there is fixation or entry of the antibiotic into the bacteria, the partition will be perturbed. As the phase system is chosen to partition bacteria into one phase, the variation of the apparent partition coefficient of antibiotic or the increase of the amount of antibiotic into the bacteriacontaining phase allows to determine the quantity of antibiotic taken up by the celis.
( 1) The choosing of a suitable phase system. (2) The preparation of tubes containing the phase system and kept at 0°C. (3) The determination of the partition of antibiotic. (4) The determination of the partition of bacteria. (5) The mixing at 0°C of the phase system, the bacteria and the antibiotic. Allowing it to settle followed by the measurement of the quantity of antibiotic in one or both phases (zero time). (6) The performing of the uptake assay at the desired temperature. (7) The addition at various time intervals of cells plus antibiotic aliquots to tubes at 0°C followed by mixing, permitting to settle, and then analysis. Partition
dextran system, partition coefficients are roughly of 1 for the four drugs assayed. In the PEG/MgS04 system, large variations are observed; examples of values are as follows: 25.5 for HzPIA pristinamycin, 23.2 for H2PII,+ pristinamycin, 0.35 for pefloxacin, 4 for rosaramicin, 0.35 for streptomycin, and 1.2 for tetracycline. Hydrophobic drugs, pristinamycins and rosaramicin preferentially move to the upper hydrophobic phase. Partition
of Bacteria
In the PEG/dextran system, using either rich medium (tryptic soy broth), minimum medium A or medium C, E. coli cells gather in the upper PEG phase. In the PEG/salt system using,medium B, S. aweus cells are found in the lower salt phase.
of Antibiotics
Uptake Experiments As partition can vary with the concentrations of drug and phases, it is necessary to measure it for each series of assay. In the PEG/
The uptakes of dihydrostreptomycin, dihydrorosaramicin, pefloxacin, and fortimicin
ANTIBIOTIC antlbdc uptake
UPTAKE
MEASURED ROSARAMICIN
97
BY PARTITION
a
37-C
(cpm I
i
I+
5
0
10
15
20
f~ 3H
DHRM
0 ‘t’
DHRM +RM
25
alone
30 Tm-w (mm)
b
0 0
5
10
15
20
TllW(lMl
25
FIG. 2. Uptake of antibiotics by E. co/i. Bacteria and antibiotics were incubated at different temperatures. At various times, aliquots of the mixture were withdrawn and partitioned as described under Material and Methods. The concentration of antibiotics in the top and bottom phases was then determined, the uptake was calculated and plotted as a function of time. External concentrations of antibiotics were: (2a) dihydrorosaramicin (DHRM) alone 2 pg/mI, 3.4 pM (20 Ci/mol). Dihydrorosaramicin in competition with rosaramicin (RM): DHRM 1.7 j.tM (40 Ci/mol), RM 1.7 pM. (2b) Fortimicin A 10 &ml, 25 pM (2~) Dihydrostreptomycin 5 &ml, 8.5 j&M (25 Ci/mol), streptomycin as an inductor, 8.5 @M or dinitrophenol as an inhibitor, 1 mM were added 15 min before dihydrostreptomycin.
were measured with E. coli. The uptakes of pefloxacin, pristinamycin IA, pristinamycin IIA, and tetracycline were evaluated with S. aureus. The kinetics of uptake are shown on Figs. 2 and 3. Whenever the uptake was also measured by the filtration method (dihydro-
streptomycin, dihydrorosaramicin, pristinamycin, pefloxacin) the same results were essentially found with either technique. If the following symbols are used: E;A, K = partition coefficients for antibiotic alone and in the presence of bacteria at time
98
MOREAU, A uptake
(cpm
LACROIX,
1
DIHYDROSTREPTOMYCIN 0
rtductlon
0
no induction
A Inhibition
5
AND FOURNEL
with
streptomycin
with
DNP
10
37°C
0
15
20
25
30 Time (min)
FIG. 2-Continued.
t, respectively. (K is the ratio of concentration
in the upper phase to concentration in the lower phase). BA, TA = total quantity of antibiotic alone in lower and upper phase, respectively. B, T = total quantity of antibiotic in the ,presence of bacteria at time t, in lower and upper phase, respectively. i = total quantity of antibiotic inside the bacteria. We can write the following: --In the case of E. culi (or bacteria in the upper phase): i = (K - K,)B or i = T - TA, depending on whether both or only upper phase are analyzed. -In the case of 5’. aureu.s (or bacteria in the lower phase): i = (l/K - l/K,)H or i = B - BA. One can express i as the concentration of antibiotic within the cells assuming an internal volume of 1.14 X lo-” ml per bacterium for S. aureu~ (14) and of 2.3 X lo-I2 ml for E. coli (2). Certain comments can be made concerning the choice of phase system. If, in theory, the technique applies even when bacterial partition is in both phases, it is therefore of great importance to gather the bacteria into only one phase thus enhancing accuracy. This pre-
sents generally no problem since the partition of cells usually takes place between one of the phases and the interface. The smaller the concentration of bacteria at the interface, the greater is the accuracy of the measurement. Another important point is the partition of the antibiotic: if the difference in partition behavior between bacteria and antibiotic is great, for instance if the cells are in the bottom phase and most of the antibiotic in the top phase, the variation of the quantity of antibiotic in the bottom phase will be easy to detect, and analysis of the bottom phase could be sufficient to estimate the penetration. Whenever it is possible, it is of interest to be in a such situation. It is the case for hydrophobic drugs such as pristinamycins ( 15), chloramphenicol, and in some degree rosaramicin, in PEG/salt system. The PEG/dextran system does not offer this advantage, and we recommend use of the former. If the partition coefficient K of the antibiotic is near to 1, it is necessary to analyse both phases to estimate the penetration with enough precision. The main drawback of the phase partition technique is that it requires some exploratory work to find a suitable phase system for the separation of the bacteria in one of the two phases. But there are numerous advantages to
Concntntlon
PRISTINAMYCINS
wthln
5
+’Concentration cdl
within
p, 0°C
0
o’H
HPIA
(nor)
o’H
HP&
(00
10
(pg /ml 1
a
25°C
0
15
Ttmc (mm)
b
PEFLOXACIN
0 1l.Y 0 20-v 0 37oc
0
0
O 0
8
0 0
0 0
0
n 0
0
t
Concentntmn (pg/ml)
withm
cell
TETRACYCLINE
37OC
C
FIG. 3. Uptake of antibiotics by S. aweus. The assay was carried out as described in Fig. 2 and under Materials and Methods. The concentration of antibiotics was determined either in bottom or both phases for pristinamycins, in both phases for pefloxacin and tetracyclin. External concentrations of drugs were 1.8 pM (650 Ci/mol) for pristinamycin PIA (3a); I .S PM (360 Ci/mol) for pristinamycin PIIA (3a): 0.1 JL~J ml, 0.5 j.tM (100 Ci/mol), for pefloxacin (3b); and 10 &ml, 22.5 pM, for tetracycline (3~). 99
100
MOREAU,
LACROIX,
AND FOURNEL
the technique. The partition technique can The method also allows competition exfacilitate uptake assays when rapid kinetics periments to be carried out (some such exare involved. The only operation to carry out periments have successfully been performed is to take samples of bacteria and antibiotic between rosaramicin and dihydrorosaramicin suspension, add them to already prepared (Fig. 2a), and between pristinamycins IA and tubes, and mix. It can easily be done every IIA (15). Furthermore, the method is not re10 to 15 s. All the other manipulations (phase stricted to the two bacterial species used here, separation, sampling of each phase, analysis) providing suitable phase system can be found can be done afier the end of the uptake process. for each particular microorganism. Successful Furthermore, it is not absolutely necessary to experiments have been carried out for chloruse radioactive drugs for analysis since liquid amphenicol uptake by Klebsiella, Serratia, aliquots are available. Two examples are given and Enterobacter species using a PEG/salt to demonstrate this possibility: the penetration system (to be published elsewhere). of fortimicin into E. co/i measured by a radioenzymatic assay, and the penetration of ACKNOWLEDGMENTS tetracycline into S. aureus, measured by fluWe are grateful to Dr. M. L. Capmau for the synthesis orescence. In the latter case, however, there of labeled pristinamycins. appear some difficulties because, as previously observed (16), fluorescence depends on a REFERENCES number of factors and only approximate valHoeller, A. H., Spector, R., and Aalyson, M. (1980) ues can be obtained. With our technique, it 3. Antibiot. 33, 604-613. is possible to measure with great accuracy the McMuny, L., and Levy, S. B. (1978) Antimicrob. Agents Chemother. 14, 20 I-209. binding of drugs to bacteria before the actual Fisher, D. (1981) Biochem. J. 196, l-10. penetration takes place. The assay is simply Albertsson, P. A., Andersson, B., Larsson, C., and performed at 0°C where no penetration of Akerlund, H. E. (1981) Methods Biochem. Anal. this drug can take place, at least in the present 28, 115-150. cases(results shown for pefloxacin, Fig. 3b, 5. Albertsson, P. A. ( 1982) Methods Biochem. Anal. 29, 1-24. not shown for other drugs, but checked up to 6. Walter, H. ( 1978) Trends B&hem. Sci. 3, 97- 100. time of 30 min). Furthermore, there is no need I. Le Goffic, F., Momau, N., Langrene, S., and Pasquier, to wash the bacteria and hence no risk to lose A. (1980) Anal. B&hem. 107,417-423. any of the drug accumulated, since the mea8. Mattiasson, B., Ling, T. G. I., and Ramstorp, M. surement can be done in the presence of sur(1981) .I. Immunol. Methods 41, 105-I 14. 9. Siegrist, S., Lagouardat, J., Moreau, N., and Le Goffic, rounding antibiotic. Finally, this technique F. (1981) Eur. J. B&hem. 115, 323-327, completely eliminates the problems encoun10. Abbe, J., Vindimian, E., Capmau, M. L., and Lx tered with filters: nonspecific binding of drugs, Goffic, F. (1981) Eur. J. Med. Chem. 16,69-70. slowness of fikatiOn, obstruction of filters at 11. Umezawa, EL,Yagkawa,M., Matsuhashi, Y., Nahigh antibiotic inputs when bacteria form agganawa, H., Yamamoto, H., Kondo, S., Takeuchi, T., and Chabbert, Y. A. (1973) J. Antibiot 26, gregates or assume elongated forms. 612-614. Some cares must be taken. During mixing 12. Haas, M. J., and Dowding, J. E. (1975) in Methods and settling of the two-phase system, the temin Enzymology (Hash, J. H., ed.), Vol. 43, pp. perature is critical and must be kept low to 612-615, Academic Press, New York. avoid loss or penetration of the drug and all 13. Le Goffic, F., Moreau, N., and Chevereau, M. (1973) Biochimie 55, 1183-I 186. operations after sampling must be carried out in ice. The sampling must be as accurate as 14. Collins, S. H., and Allan Hamilton, N. (1976) J. Bacteriol. 126, 1224-1231. possible; when experiments have to be re- 15. Lacroix, P., Capmau, M. L., and Ix Gothc, F., subpeated, it is important to use exactly the same mitted for publication. proportions of each phase and to take several 16. Smith, M. C. M., and Chapra, I. (1983) Antimrcrob. Agents Chemother. 23, 175-l 78. “zero-time” samples.
BIOCHEMISTRY
Antibiotic
NICOLE CNRS-CERCOA
141, 94-1~
(1984)
Uptake by Bacteria as Measured by Partition in Polymer Aqueous Phase Systems MOREAU,PATRICIA
LACROIX,ANDLAURENTFOURNEL
(laboratoire associh ri I’ENSCP) 2 ci 8, rue Henri Dunant 94320 Thiais, France Received January 13, 1984
Polyetbyleneglycol/dextran and polyethyleneglycol/salt two-phase systemsare used to measure the entry of antibiotics into Escherichia coli and Staphylococcus aureus cells. Aminoglycosides, macrolides, a quinolone, and a cycline were assayed. The method is simple and rapid and eliminates the problems encountered with filters, especially nonspecific binding; it allows rapid uptake kinetics to be measured.
The ability of antibiotics to enter bacterial cells is an important factor in the antimicrobial activity of drugs. The amount of drugs taken up by bacteria has generally been evaluated by filtration on glass fiber, cellulose nitrate, polycarbonate filters, etc., depending on the kind of antibiotic assayed (1,2); alternatively, cells may be centrifuged. Although widely used, these methods present some disadvantages. It is difficult to make more than one or two measurements per minute: with the centrifugation technique, 1 min is the minimum time required to obtain a cell pellet; with the filtration technique, one needs to take into account the time of filtration of the sample and of 2 or 3 washings. For a slow uptake kinetics, such as that of aminoglycosides, this is not a drawback, but for rapid uptake kinetics (pristinamycins, quinolones) it is a limitation. Furthermore, in the case of the filtration method, the preparation and washing of the filter are critical to avoid variable binding of antibiotic to filter, variable amounts of trapped medium, and variable times of filtration. Phase partition is a method used for purification and analysis of proteins, cells, cell organelles, and membrane vesicles (for reviews, see (3-5)). It has also been used as a tool to measure interactions between biomolecules (6,7) or for quantitation of bacterial cells (8). The purpose of this paper is to show CW3-2697184 $3.00 Copyright 8 1984 by Academic FVes, Inc. All rights of repmduction in any form reserved.
how phase partition can be used as a very convenient method to measure the uptake of antibiotics by bacterial cells. MATERIALS
AND METHODS
Chemicals
Dextran T500 was purchased from Pharmacia; polyethylene glycol (PEG)’ 6000 was purchased from Touzart et Matignon. Dihydrostreptomycin and pristinamycins were gifts from Rhhne-Poulenc; rosaramicin was a gift from Unilabo; pefloxacin was a gift from Roger Bellon; fortimicin A was a gift from Abbott; tetracycline was a gift from Pfizer. [3H]Dihydrostreptomycin and [3H]acetylcoenzyme A were purchased from Amersham; dihydrorosaramicin and [3H]dihydrorosaramicin (9), [3H]pefloxacin (unpublished results) and [3H]pristinamycins (10) were synthesized in our laboratory. All other chemicals were of analytical grade. Bacterial Strains, Media, and Phase Systems Escherichia coli K 12 and Staphylococcus aureus 209P were from our laboratory collection. Escherichia coli R135 (11) synthesizes ’ Abbreviation used: PEG, polyethylene glycol. 94
ANTIBIOTIC
UPTAKE
MEASURED
the enzyme AAC3(1) which is capable of acetylating gentamicin and fortimicins. Medium A was Davis and Mingioli minimum medium; it contained, per liter, 7 g of K2HP04, 3 g of KH2P04, 0.5 g of sodium citrate * 3 H20, 0.1 g of MgSO4 7Hz0, 0.1 g of ammonium sulfate, 2 g of glucose (the latter being autoclaved separately), pH 7. Medium B contained per liter, 0.7 g of ammonium sulfate, 1 g of KH2PO4, 6 g of Na2HP04. 12 HzO, 0.2 g of magnesium sulfate. 7H20, pH 7.2. Medium C contained per liter, 0.25 g of magnesium sulfate. 7H20, 7.1 g of KI-I2P@, 2 g of glucose; the pH was adjusted to 6 with KOH. l
Uptake Assays E. coli. Stock solutions of 20% (w/w) dextran and 40% (w/w) PEG were prepared in water. The phase system was prepared by mixing 25 g of 20% dextran, 10 g of 40% PEG, 50 g of 2X concentrated medium A, 15 g of water. The mixture was shaken and the phases were allowed to separate either through overnight settling at 4°C or by centrifugation at 1OOOgfor 5 min at 4°C. Cells were grown at 37°C in medium A up to middle to late log phase (turbidity of 100, measured with an Hach turbidimeter Model 2100A). They were centrifuged, washed with dextran phase, and resuspended in the same phase, at a three-times higher concentration. After 10 to 15 min of preincubation at 37°C with shaking, the antibiotic was added as specified and shaking was continued. At different times after antibiotic addition, l-ml samples were removed and added to 1 ml of PEG phase kept in small tubes at 0°C. The mixtures were stirred with a vortex-type mixer and left for 1 to 2 h at 0°C. An aliquot from one or both phases was sampled for antibiotic analysis. For radioactive antibiotics, 200 ~1 of each phase was mixed with 200 ~1 of water and 3 ml of Beckman HPb scintillation fluid. In the case of fortimicin A, a radioenzymatic assay was carried out (12). A 25-~1 volume of a purified extract of E. coli R 135 ( 13) and 10
BY
95
PARTITION
~1 of an aqueous solution of AcCoA (0.2 pm/ ml, 54 Ci/mol) were added to 50 ~1 of the phase to be tested (the PEG phase was boiled a few minutes before assay to release the fortimicin from the bacteria). The mixtures were incubated for 45 min at 37’C, 50 ~1 were placed on Whatman P8 1 paper and processed as previously described ( 12). A standard curve was obtained with phases containing known concentrations of fortimicin. S. aureus. The uptake of antibiotics was assessedas for E. cob, with the following modifications: cells were grown in tryptic soy broth to 100 Klett units (Klett Summerson, blue filter, 400-420 nm). After centrifugation at 3000g for 10 min, they were washed with medium B and resuspended in the same medium at a concentration corresponding to 300 Klett units (7 X lo8 bacteria per ml). The stock solutions were 40% PEG (w/w) in 20 mM Na2HP04 ; and 40% MgS04 - 7H20 (w/w) in 20 IIIM Na2HP04. Tubes containing 375 ~1 of PEG stock solution and 1125 ~1 of MgS04 stock solution were kept at 0°C. To determine the uptake, bacteria plus antibiotic were shaked at the desired temperature. At different times, 500~~1 samples were removed and added to the cold phases. After stirring, the mixture was left for 20 to 30 min at 0°C. The resulting volumes were 0.5 and 1.5 ml for the upper PEG-rich phase and the lower salt-rich phase, respectively. Medium C was used in the case of tetracycline uptake. All the volumes were doubled to allow aliquots of 500 ~1 to be sampled. The fluorescence was measured by excitation at 365 nm and emission at 510 nm. For both microorganisms, the partition of antibiotic and bacteria alone were determined under the same conditions. The zero-time value was measured at 0°C. RESULTS
AND
DISCUSSION
The principle of the method is schematized in Fig. 1. The following operations are carried out to assay the uptake of an antibiotic by bacteria:
MOREAU,
LACROIX,
AND FOURNEL
bacteria
plus antibiotic
\
time 1 d the uptake kit-&a m&ii
FIG. 1. Principle of the method. Bacteria and antibiotic are partitioned separately or together in the two phase system. If there is no interaction between them, they partition independently of each other. As soon as there is fixation or entry of the antibiotic into the bacteria, the partition will be perturbed. As the phase system is chosen to partition bacteria into one phase, the variation of the apparent partition coefficient of antibiotic or the increase of the amount of antibiotic into the bacteriacontaining phase allows to determine the quantity of antibiotic taken up by the celis.
( 1) The choosing of a suitable phase system. (2) The preparation of tubes containing the phase system and kept at 0°C. (3) The determination of the partition of antibiotic. (4) The determination of the partition of bacteria. (5) The mixing at 0°C of the phase system, the bacteria and the antibiotic. Allowing it to settle followed by the measurement of the quantity of antibiotic in one or both phases (zero time). (6) The performing of the uptake assay at the desired temperature. (7) The addition at various time intervals of cells plus antibiotic aliquots to tubes at 0°C followed by mixing, permitting to settle, and then analysis. Partition
dextran system, partition coefficients are roughly of 1 for the four drugs assayed. In the PEG/MgS04 system, large variations are observed; examples of values are as follows: 25.5 for HzPIA pristinamycin, 23.2 for H2PII,+ pristinamycin, 0.35 for pefloxacin, 4 for rosaramicin, 0.35 for streptomycin, and 1.2 for tetracycline. Hydrophobic drugs, pristinamycins and rosaramicin preferentially move to the upper hydrophobic phase. Partition
of Bacteria
In the PEG/dextran system, using either rich medium (tryptic soy broth), minimum medium A or medium C, E. coli cells gather in the upper PEG phase. In the PEG/salt system using,medium B, S. aweus cells are found in the lower salt phase.
of Antibiotics
Uptake Experiments As partition can vary with the concentrations of drug and phases, it is necessary to measure it for each series of assay. In the PEG/
The uptakes of dihydrostreptomycin, dihydrorosaramicin, pefloxacin, and fortimicin
ANTIBIOTIC antlbdc uptake
UPTAKE
MEASURED ROSARAMICIN
97
BY PARTITION
a
37-C
(cpm I
i
I+
5
0
10
15
20
f~ 3H
DHRM
0 ‘t’
DHRM +RM
25
alone
30 Tm-w (mm)
b
0 0
5
10
15
20
TllW(lMl
25
FIG. 2. Uptake of antibiotics by E. co/i. Bacteria and antibiotics were incubated at different temperatures. At various times, aliquots of the mixture were withdrawn and partitioned as described under Material and Methods. The concentration of antibiotics in the top and bottom phases was then determined, the uptake was calculated and plotted as a function of time. External concentrations of antibiotics were: (2a) dihydrorosaramicin (DHRM) alone 2 pg/mI, 3.4 pM (20 Ci/mol). Dihydrorosaramicin in competition with rosaramicin (RM): DHRM 1.7 j.tM (40 Ci/mol), RM 1.7 pM. (2b) Fortimicin A 10 &ml, 25 pM (2~) Dihydrostreptomycin 5 &ml, 8.5 j&M (25 Ci/mol), streptomycin as an inductor, 8.5 @M or dinitrophenol as an inhibitor, 1 mM were added 15 min before dihydrostreptomycin.
were measured with E. coli. The uptakes of pefloxacin, pristinamycin IA, pristinamycin IIA, and tetracycline were evaluated with S. aureus. The kinetics of uptake are shown on Figs. 2 and 3. Whenever the uptake was also measured by the filtration method (dihydro-
streptomycin, dihydrorosaramicin, pristinamycin, pefloxacin) the same results were essentially found with either technique. If the following symbols are used: E;A, K = partition coefficients for antibiotic alone and in the presence of bacteria at time
98
MOREAU, A uptake
(cpm
LACROIX,
1
DIHYDROSTREPTOMYCIN 0
rtductlon
0
no induction
A Inhibition
5
AND FOURNEL
with
streptomycin
with
DNP
10
37°C
0
15
20
25
30 Time (min)
FIG. 2-Continued.
t, respectively. (K is the ratio of concentration
in the upper phase to concentration in the lower phase). BA, TA = total quantity of antibiotic alone in lower and upper phase, respectively. B, T = total quantity of antibiotic in the ,presence of bacteria at time t, in lower and upper phase, respectively. i = total quantity of antibiotic inside the bacteria. We can write the following: --In the case of E. culi (or bacteria in the upper phase): i = (K - K,)B or i = T - TA, depending on whether both or only upper phase are analyzed. -In the case of 5’. aureu.s (or bacteria in the lower phase): i = (l/K - l/K,)H or i = B - BA. One can express i as the concentration of antibiotic within the cells assuming an internal volume of 1.14 X lo-” ml per bacterium for S. aureu~ (14) and of 2.3 X lo-I2 ml for E. coli (2). Certain comments can be made concerning the choice of phase system. If, in theory, the technique applies even when bacterial partition is in both phases, it is therefore of great importance to gather the bacteria into only one phase thus enhancing accuracy. This pre-
sents generally no problem since the partition of cells usually takes place between one of the phases and the interface. The smaller the concentration of bacteria at the interface, the greater is the accuracy of the measurement. Another important point is the partition of the antibiotic: if the difference in partition behavior between bacteria and antibiotic is great, for instance if the cells are in the bottom phase and most of the antibiotic in the top phase, the variation of the quantity of antibiotic in the bottom phase will be easy to detect, and analysis of the bottom phase could be sufficient to estimate the penetration. Whenever it is possible, it is of interest to be in a such situation. It is the case for hydrophobic drugs such as pristinamycins ( 15), chloramphenicol, and in some degree rosaramicin, in PEG/salt system. The PEG/dextran system does not offer this advantage, and we recommend use of the former. If the partition coefficient K of the antibiotic is near to 1, it is necessary to analyse both phases to estimate the penetration with enough precision. The main drawback of the phase partition technique is that it requires some exploratory work to find a suitable phase system for the separation of the bacteria in one of the two phases. But there are numerous advantages to
Concntntlon
PRISTINAMYCINS
wthln
5
+’Concentration cdl
within
p, 0°C
0
o’H
HPIA
(nor)
o’H
HP&
(00
10
(pg /ml 1
a
25°C
0
15
Ttmc (mm)
b
PEFLOXACIN
0 1l.Y 0 20-v 0 37oc
0
0
O 0
8
0 0
0 0
0
n 0
0
t
Concentntmn (pg/ml)
withm
cell
TETRACYCLINE
37OC
C
FIG. 3. Uptake of antibiotics by S. aweus. The assay was carried out as described in Fig. 2 and under Materials and Methods. The concentration of antibiotics was determined either in bottom or both phases for pristinamycins, in both phases for pefloxacin and tetracyclin. External concentrations of drugs were 1.8 pM (650 Ci/mol) for pristinamycin PIA (3a); I .S PM (360 Ci/mol) for pristinamycin PIIA (3a): 0.1 JL~J ml, 0.5 j.tM (100 Ci/mol), for pefloxacin (3b); and 10 &ml, 22.5 pM, for tetracycline (3~). 99
100
MOREAU,
LACROIX,
AND FOURNEL
the technique. The partition technique can The method also allows competition exfacilitate uptake assays when rapid kinetics periments to be carried out (some such exare involved. The only operation to carry out periments have successfully been performed is to take samples of bacteria and antibiotic between rosaramicin and dihydrorosaramicin suspension, add them to already prepared (Fig. 2a), and between pristinamycins IA and tubes, and mix. It can easily be done every IIA (15). Furthermore, the method is not re10 to 15 s. All the other manipulations (phase stricted to the two bacterial species used here, separation, sampling of each phase, analysis) providing suitable phase system can be found can be done afier the end of the uptake process. for each particular microorganism. Successful Furthermore, it is not absolutely necessary to experiments have been carried out for chloruse radioactive drugs for analysis since liquid amphenicol uptake by Klebsiella, Serratia, aliquots are available. Two examples are given and Enterobacter species using a PEG/salt to demonstrate this possibility: the penetration system (to be published elsewhere). of fortimicin into E. co/i measured by a radioenzymatic assay, and the penetration of ACKNOWLEDGMENTS tetracycline into S. aureus, measured by fluWe are grateful to Dr. M. L. Capmau for the synthesis orescence. In the latter case, however, there of labeled pristinamycins. appear some difficulties because, as previously observed (16), fluorescence depends on a REFERENCES number of factors and only approximate valHoeller, A. H., Spector, R., and Aalyson, M. (1980) ues can be obtained. With our technique, it 3. Antibiot. 33, 604-613. is possible to measure with great accuracy the McMuny, L., and Levy, S. B. (1978) Antimicrob. Agents Chemother. 14, 20 I-209. binding of drugs to bacteria before the actual Fisher, D. (1981) Biochem. J. 196, l-10. penetration takes place. The assay is simply Albertsson, P. A., Andersson, B., Larsson, C., and performed at 0°C where no penetration of Akerlund, H. E. (1981) Methods Biochem. Anal. this drug can take place, at least in the present 28, 115-150. cases(results shown for pefloxacin, Fig. 3b, 5. Albertsson, P. A. ( 1982) Methods Biochem. Anal. 29, 1-24. not shown for other drugs, but checked up to 6. Walter, H. ( 1978) Trends B&hem. Sci. 3, 97- 100. time of 30 min). Furthermore, there is no need I. Le Goffic, F., Momau, N., Langrene, S., and Pasquier, to wash the bacteria and hence no risk to lose A. (1980) Anal. B&hem. 107,417-423. any of the drug accumulated, since the mea8. Mattiasson, B., Ling, T. G. I., and Ramstorp, M. surement can be done in the presence of sur(1981) .I. Immunol. Methods 41, 105-I 14. 9. Siegrist, S., Lagouardat, J., Moreau, N., and Le Goffic, rounding antibiotic. Finally, this technique F. (1981) Eur. J. B&hem. 115, 323-327, completely eliminates the problems encoun10. Abbe, J., Vindimian, E., Capmau, M. L., and Lx tered with filters: nonspecific binding of drugs, Goffic, F. (1981) Eur. J. Med. Chem. 16,69-70. slowness of fikatiOn, obstruction of filters at 11. Umezawa, EL,Yagkawa,M., Matsuhashi, Y., Nahigh antibiotic inputs when bacteria form agganawa, H., Yamamoto, H., Kondo, S., Takeuchi, T., and Chabbert, Y. A. (1973) J. Antibiot 26, gregates or assume elongated forms. 612-614. Some cares must be taken. During mixing 12. Haas, M. J., and Dowding, J. E. (1975) in Methods and settling of the two-phase system, the temin Enzymology (Hash, J. H., ed.), Vol. 43, pp. perature is critical and must be kept low to 612-615, Academic Press, New York. avoid loss or penetration of the drug and all 13. Le Goffic, F., Moreau, N., and Chevereau, M. (1973) Biochimie 55, 1183-I 186. operations after sampling must be carried out in ice. The sampling must be as accurate as 14. Collins, S. H., and Allan Hamilton, N. (1976) J. Bacteriol. 126, 1224-1231. possible; when experiments have to be re- 15. Lacroix, P., Capmau, M. L., and Ix Gothc, F., subpeated, it is important to use exactly the same mitted for publication. proportions of each phase and to take several 16. Smith, M. C. M., and Chapra, I. (1983) Antimrcrob. Agents Chemother. 23, 175-l 78. “zero-time” samples.