Vol. 7, No. 5 Printed in U.S.A.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1975, p. 629-635 Copyright O 1975 American Society for Microbiology
Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa: Binding of Penicillin to Pseudomonas aeruginosa KM 338 HIDEKAZU SUGINAKA,* AKIRA ICHIKAWA,
AND
SHOZO KOTANI
Department of Microbiology, Osaka University Dental School, Joan-cho, Kita-ku, Osaka 530, Japan Received for publication 30 December 1974
A comparison of the binding of radioactive penicillin G to whole cells and the membrane fraction derived from Pseudomonas aeruginosa KM 338 was made. This organism has intrinsic resistance to penicillin. The binding to the membrane fraction which catalyzed peptidoglycan synthesis followed saturation type kinetics and saturation was achieved at approximately 2 nmol of penicillin G per ml, whereas binding to the whole cells was entirely of the nonsaturation type. The binding of carbenicillin to the membrane fraction was determined by competition between radioactive penicillin G and unlabeled carbenicillin for the binding sites. It was bound at the same sites in almost the same manner. When whole cells were pretreated with high concentration of unlabeled penicillin G or carbenicillin, the subsequent binding of radioactive penicillin G to the membrane fraction from carbenicillin-treated cells was entirely nonspecific, but with penicillin G-pretreated cells it was still specific. There was apparently specific binding of radioactive penicillin G to ethylenediaminetetraacetate-treated cells. P. aeruginosa KM 338 had an extremely low activity of f3-lactamase compared with other enzyme-producing organisms. This enzyme from P. aeruginosa KM 338 was of the cephalosporinase type. These data indicate that penicillin resistance of P. aeruginosa KM 338 may be a consequence of the development of a permeability barrier which prevents the antibiotic from reaching its sites of action in the cytoplasmic membrane.
In general, most strains of Pseudomonas aeruginosa are resistant to penicillins. The intrinsic mechanisms of resistance to penicillin in this organism have been investigated (27). The peptidoglycan isolated from cell walls of this organism (12, 18) is similar to that of penicillin-susceptible organisms (10, 23, 25). In a previous paper (27) it was reported that the membrane fraction derived from P. aeruginosa KM 338 catalyzes peptidoglycan synthesis, including the cross-linking reaction. This transpeptidase is inhibited by a low concentration of penicillin G or carbenicillin in a similar manner (27), whereas the intact cells are unsusceptible to penicillin G and are relatively susceptible to carbenicillin (22). It has been suggested that penicillin is irreversibly bound to transpeptidase in the cytoplasmic membrane of penicillin-susceptible organisms (16, 17, 25, 26). On the other hand, it has also been thought that penicillinase is an important factor of penicillin-resistance mechanisms in P. aeruginosa, similar to penicillinase-producing organisms, such as Staphylococcus aureus and Escherichia coli (3).
In the present experiments, the comparative binding abilities of penicillin G and carbenicillin were studied with whole cells and the membrane fraction from a strain of P. aeruginosa. Also, the j-lactamase activities produced by this strain was compared with those from the enzyme-producing organisms. (A summary of this work was presented at the 46th Annual Meeting of the Japanese Society for Microbiology, Tokyo, 8 April 1973.) MATERIALS AND METHODS Organisms. The organism used was Pseudomonas aeruginosa KM 338, derived from ATCC 17653. This strain has been used in the previous study (27) of peptidoglycan synthesis in our laboratory. The organisms used as reference for ,B-lactamase activities were Staphylococcus aureus 235, Escherichia coli 386 and 3032, and Citrobacter 817. All these organisms were furnished by Research Laboratories, Fujisawa Pharaceutical Co., Ltd. (Osaka, Japan) and had been isolated from clinical materials. Minimal inhibitory concentrations (MIC) of penicillin G and carbenicillin
for these strains are shown in Table 1. Antibiotics. Potassium 6-phenyl( acet- 1[14C])aminopenicillanate, abbreviated [14C]penicillin 629
630
SUGINAKA, ICHIKAWA, AND KOTANI
TABLE 1. Minimal inhibitory concentrations of penicillin G and carbenicillin Antibiotic
P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817
MIC (Ag/ml) Penicillin G
Carbenicillin
30,000 2,000 16,000 16,000 8,000
125 25 25 800 800
G, was obtained from Amersham Searle (Buckinghamshire, England). It had a specific activity of 45 M4Ci/Mgmol. [14C]benzylpenicilloic acid was prepared from [14C]penicillin G by treatment with commercial penicillinase. The reaction mixture contained, in a total volume of 0.4 ml, 40,000 U of penicillinase (Tokyo Kasei Co., Ltd., Tokyo, Japan), 20 Mmol of tris(hydroxymethyl)aminomethane-hydrochloride (Tris) buffer (pH 7.5), and 10 smol of [14C]penicillin G. After incubation at 37 C for 4 h, the reaction mixture was boiled for 10 min for inactivation of the enzyme activity. The resulting compound was not contaminated by penicillin G when examined by thin-layer chromatography on Silica Gel G in nbutanol-acetic acid-water (200:5:200, vol/vol, upper phase). Unlabeled penicillin G and benzylpenicilloic acid were supplied by Meiji Seika Co., Ltd. (Osaka, Japan). Carbenicillin and cephalosporin C were furnished from Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan), and ampicillin was obtained from Takeda Chemical Industries Co., Ltd. (Osaka, Japan). Assays for the binding of radioactive penicillin G to whole cells and the membrane fraction of P. aeruginosa KM 338. The methods used were almost the same as described in the previous study (26). The organism was routinely grown at 37 C to mid- or late-log phase in Trypticase soy broth (BBL) as described previously (27). The membrane fraction (particulate fraction) was prepared by differential centrifugation from disrupted cells after alumina grinding (27). The incubation mixture, containing 25 ,1 of the membrane preparation which catalyzed peptidoglycan synthesis (approximately 20 mg of protein per ml), 10 il of 1 M Tris-hydrochloride buffer (pH 7.5), 2 gl of 1 M MgCl2, 3 Ml of water, and 10 ,ul of ['4C]penicillin G solution varying in concentration from 0.5 to 50 nmol/ml, was incubated for 30 min at 30 C. After incubation, the reaction was terminated by the addition of 10 ul of 0.6 M unlabeled penicillin G solution, and then either of two methods were used for the assay of bound penicillin. In one case, the incubation mixture was spotted on Toyo filter paper no. 51 (Toyo Roshi Kaisha Co., Ltd., Osaka, Japan) and then developed in n-butanol-acetic acid-water (200:5:200, vol/vol, upper phase; ascending) for 4 h, and then the process was repeated with the same solvent. The unbound penicillin G and benzylpenicilloic acid moved near the solvent front, and penicillin G bound to the membrane protein remained at the origin of the chromatogram. The origin was cut out and counted in scintillation fluid containing 15.5 g of
ANTIMICROB. AGENTS CHEMOTHER. 2,5-diphenyloxazole and 114 g of 1,4-bis[2-(4-methyl5-phenyloxazole) ]benzene in 3.8 liters of toluene. In the second assay, the membrane fraction-bound [14Cjpenicillin G was pelleted by centrifugation at 45,000 x g for 20 min, and the pellet was then washed four times by centrifugation with 8 ml of 0.05 M Tris-hydrochloride (pH 7.5) containing 0.001 M MgCl2. Finally, the pellet was dissolved in 0.4 ml of 4% sodium dodecyl sulfate. To the sample in a glass scintillation vial, 1 ml of Biosolv (formula BBS-3, Beckman Instruments Inc., Fullerton, Calif.) was added, followed by 9 ml of the above scintillation fluid. Both the chromatographic and washing methods were employed for most of the experiments described in this paper. The former procedure yielded somewhat lower values for radioactive penicillin binding, but both of the above methods were found to be reproducible and reliable. In the assay for binding to whole cells, the cells of a logarithmic phase growth were harvested by centrifugation, washed with 0.05 M Tris-hydrochloride buffer (pH 7.5) or phosphate buffer (pH 7.2) containing 0.001 M MgCl2, and finally resuspended in the above buffer in 1/20 of the volume of the growth medium. Five hundred microliters of the cell suspension (about 2 x 109 cells/ml) was incubated with 200 Ml of 1 M Tris buffer (pH 7.5) or phosphate buffer (pH 7.2), 40 ul of 1 M MgCl2, 60 Ml of water, and 200 gl of [14C]penicillin G solution, varying in concentration from 2.5 to 3,750 nmol per ml, for 30 min at 30 C. After incubation, the cells were recovered by centrifugation at 10,000 x g for 10 min and washed with adequate amounts of the buffer of the above composition. Finally, they were suspended in 0.4 ml of 4% sodium dodecyl sulfate and counted as described above. Counting was carried out in a Packard liquid scintillation spectrometer. Assay for 3-lactamase activities. All the organisms tested were grown in Trypticase soy broth (BBL) at 37 C for 18 h. These broth cultures were disrupted with an ultrasonic disintegrator (60 W, 20 kcycle/s. Ohtake Seisakusho Co., Ltd., Tokyo, Japan) for 10 min, and the resulting suspensions of each disrupted culture were used as the enzyme source. ,B-Lactamase activities were estimated by a modification of the bioassay method of Humphrey et al. (13), using Bacillus subtilis ATCC 6633 (obtained from the Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. [Osaka, Japan]) as test organism. All assays for ,B-lactamase were carried out with a substrate concentration of 50 Ag/ml. The diluted sonic extracts were incubated with test antibiotics in 0.067 M phosphate buffer (pH 7.0) at 37 C for 1 h. After incubation, the reaction was terminated by boiling for 3 min, and the enzyme activity was measured by cup-plate bioassay of the residual antibiotic.
RESULTS Binding of radioactive penicillin G to the membrane fraction from P. aeruginosa KM 338. When a suspension of the membrane fraction from P. aeruginosa KM 338 was incubated at 30 C with varying concentrations of [I4C]penicillin G for 30 min, the ['4C]penicillin
VOL. 7, 1975
BINDING OF PENICILLIN TO P. AERUGINOSA
G was irreversibly bound to the membrane fraction which catalyzes peptidoglycan synthesis as previously described (27) (Fig. 1). The binding of ['4C]penicillin G followed saturation type kinetics as had been shown earlier in the binding of penicillin G to whole cells (4, 5, 8, 15, 20, 24, 26) and membrane fractions (16, 26) from various penicillin-susceptible organisms. The binding was biphasic, showing a relatively high initial binding at low penicillin concentrations (to 2 nmol/ml), followed by an apparent secondary binding which was linearly related to penicillin concentration. At the point of maximum specific binding, about 3.2 nmol of [I4C ]penicillin G were bound per g of membrane protein, and the concentration required for half-maximum binding under the condition of the experiment was approximately 0.5 nmol of penicillin G per ml, with saturation of the penicillin-specific receptor sites achieved at approximately 2 nmol/ml. The binding value given was obtained by extrapolating the linear secondary binding curve to zero concentration. The secondary phase was also seen with boiled membrane fractions with inactivated transpep.C
6
0 S.-
0) 0
2 CZ o0
631
tidase activity, and is presumably due to some nonspecific binding or trapping of the radioactive compound. Benzylpenicilloic acid (open ,B-lactam ring) was also bound with a linear dependence on its concentration, as in the case with penicillin G binding to the boiled membrane fraction above. As previously observed with other organisms (16), the bound penicillin was not washed out of the membrane fraction from P. aeruginosa KM 338 with buffer or high concentrations of unlabeled penicillin and was not removed on treatment with penicillinase. This binding seems to be through a relatively tight covalent linkage (irreversible binding). Treatment with 1 M neutral hydroxylamine, however, resulted in virtual reversal of the binding of [4C ]penicillin G to the membrane fraction from P. aeruginosa KM 338, the same as that from B. subtilis (15) (data not shown). Treatment with trypsin or Pronase destroyed the binding components of the membrane fraction, suggesting that it is protein in nature (Table 2). Binding of radioactive penicillin G to whole cells of P. aeruginosa KM 338. When suspensions of whole cells of P. aeruginosa KM 338 were incubated with varying concentrations of [4C ]penicillin G, the antibiotic was also irreversibly bound to the cells, as with the membrane fraction (Fig. 2). However, the binding was entirely nonspecific. The amount bound to whole cells was directly proportional to the concentration up to a level of at least 750 nmol/ml, although binding to the membrane fraction from this organism and to the whole cells of penicillin-susceptible organisms was specific as described above and previously (4, 5, 8, 15, 20, 24, 26). The binding of [14C]benzylpenicilloic acid to the cells was also nonspecific and of lower affinity compared with penicillin G. Inhibition binding of radioactive penicillin TABLE 2. Effects of trypsin and Pronase on binding of G to the membrane fraction from P. aeruginosa KM 338a
[14C]penicillin
10 q O ~~~~5 ~ 14C-PC-G or PA Conc. (nmoles/ml) FIG. 1. Binding of [l4C]penicillin G or [14C]benzylpenicilloic acid to the membrane fraction from P. aeruginosa KM 338. Assays were carried out with the washing method as described. Data are expressed as moles of bound radioactivity per gram of membrane protein. Where boiled membrane fraction was employed, this fraction in buffer was placed in a boiling water bath for 5 min prior to addition of [14C]penicillin G solution (x). Symbols: 0, penicillin G; 0, benzylpenicilloic acid.
Treatment None Trypsin (1 mg/ml) Pronase (1 mg/ml)
[P4C]penicillin G bound Counts/min
%
3,582 886 640
100 24.7 17.9
a The membrane fraction (20 mg of protein/ml} was treated with trypsin or Pronase at pH 7.5 for 30 min at 37 C before [14C]penicillin G was added. Assays were carried out with the paper chromatographic method as described.
632
SUGINAKA, ICHIKAWA, AND KOTANI 1 .5
100
ANTIMICROB. AGENTS CHEMOTHER.
The resulting membrane fraction was incubated with varying concentrations of [14C]penicillin G, and control cells were handled in a similar 751.0 I~ manner except that the initial incubation contained only cells and buffer. The binding of S_~~~~~~~~~~~ [14C ]penicillin G to the membrane fraction from the penicillin G-treated cells was still of the biphasic saturation type, whereas the binding to that from the carbenicillin-treated cells was entirely of the nonsaturation type (Fig. 4). 14C-PCG or 14C-PA Conc. (nmoles/ml) Effect of ethylenediaminetetraacetate FIG. 2. Binding of ['4C]penicillin G or ['4C]benzyl- treatment on binding of radioactive penicillin penicilloic acid to the whole cells of P. aeruginosa KM G to the whole cells of P. aeruginosa KM 338. 338. Assays were carried out as described with the A logarithmic phase culture of P. aeruginosa addition of ["4C]penicillin G solutions varying in KM 338 was centrifuged, and the cells were concentrations from 0.5 to 10 (left) to 750 (right) washed and resuspended at ¼io of the original nmol/ml. Data are expressed as moles of bound concentration in 0.05 M phosphate buffer (pH radioactivity per gram (dry weight) of whole cells. 7.2) containing 0.5 M sucrose. This preparation Symbols: 0, penicillin G; 0, benzylpenicilloic acid. was divided into two portions, to one of which G to the membrane fraction from P. aerugin- was added ethylenediaminetetraacetate osa KM 338 by unlabeled penicillin G, ben- (EDTA) to a final concentration of 10 mM. The zylpenicilloic acid, and carbenicillin. The second acted as a control. These preparations between competition G and un[o4C4penicillin 0 labeled penicillins for specific binding sites was determined by incubating the membrane fraction from P. aeruginosa KM 338 with 2.5 nmol pe Gand various concenof [m4Cpe ]penicillin trations of the test antibiotic (8) (Fig. 3). The unlabeled antibiotics tested, if irreversibly _25 bound to the same binding sites as radioactive penicillin G, would prevent binding of the 0 labeled penicillin G. The binding of .-o [14C]penicillin G was inhibited by unlabeled penicillin G or carbenicillin in the almost same .c50 manner and extent, but benzylpenicilloic acid did not prevent the binding of [14C]penicillin G. 4-The inhibition of [14C]penicillin G by car- 0 benicillin suggests that the two substances are I)75 bound at the same sites. Binding of radioactive penicillin G to the membrane fraction from penicillin G or carbenicillin-treated cells from P. aeruginosa KM 338. Penetration of penicillin into its target 100 site was indirectly measured by determining the 10 20 0 reduction in ('4C]penicillin G-binding capacity of the membrane fraction prepared from P. Antibiotics Conc. (nmoles/ml) aeruginosa cells previously incubated with unFIG. 3. Competitive inhibition of binding of labeled penicillin G or carbenicillin. The cells were incubated with a high concentration (500 [I4C]penicillin G to the membrane fraction from P. KM 338 by unlabeled penicillin G, carnmol/ml) of unlabeled penicillin G or car- aeruginosa benicillin, and benzylpenicilloic acid. The membrane benicillin and then washed with adequate fraction was incubated with varying concentrations of amounts of the buffer by centrifugation. The test antibiotics and 2.5 nmol of G per washed cells were disrupted with sonic treat- ml. Assays were carried out with [14C]penicillin the washing method ment (Super sonic vibrator, UR-150, Tominaga as described. Data are expressed as percent inhibition Works, Ltd., Tokyo, Japan), and the broken cell of bound radioactivity by test antibiotics. Symbols: preparation was fractionated by differential 0, penicillin G; 0, carbenicillin; A, benzylpenicilloic centrifugations as described previously (27). acid.
BINDING OF PENICILLIN TO P. AERUGINOSA
VOL. 7, 1975
633
aeruginosa KM 338, whereas from penicillinaseproducing Staphylococcus aureus 235 and Escherichia coli 3032 it was hydrolyzed at a high rate. The MIC for penicillin G for the former was 30 mg/ml, and those for the latter strains were 0.3 and 16 mg/ml, respectively (Table 1). P. aeruginosa also had little activity against cephalosporin C compared with cephalosporinase-producing Citrobacter 817 and the other organisms tested.
0D
-o n C)
s
6
cL 0~
6
co3 C-)
CD C-
0
,E Q. CD _
0
1
3
5
1 4C-PC-G Conc. (nmol es/ml FIG. 4. Binding of [14C]penicillin G to the membrane fraction from penicillin G- or carbenicillintreated cells of P. aeruginosa KM 338. Washed cells, harvested at logarithmic phase, were treated with unlabeled penicillin G (0) or carbenicillin (A) (500 nmol/ml) or without antibiotic (A) at 30 C for 30 min as described. The treated cells were disrupted by sonic treatment. The broken cell suspension was separated by differential centrifugation; the resulting membrane fraction was incubated with varying concentrations of [14Cjpenicillin G; and assays were carried out with the washing method as described. Data are expressed as moles of bound radioactivity per gram of membrane protein.
incubated at 37 C for 15 min, and the cells then washed in the same buffer by centrifugation (10,000 x g for 10 min). The resulting cells were incubated with varying concentrations of ['4C]penicillin G in 0.05 M phosphate buffer (pH 7.2) without MgCl2 and in the presence of 0.5 M sucrose. Results of the binding ability are shown in Fig. 5. It can be seen that there was apparently specific binding of [4C ]penicillin G to the EDTA-treated cells. Activities of $-lactamases from P. aeruginosa KM 338. Relative activities and substrate profiles of #-lactamases in sonically treated cultures from P. aeruginosa KM 338 and from several clinically isolated organisms were examined. A comparison of the activity for a range of substrates (Table 3) shows that ampicillin was little hydrolyzed by the enzyme from P. were were
_
0
co CD
10-
C-)
0
1
14C-PC-G
2
3
4
5
Conc. (nmoles/ml)
FIG. 5. Effect of EDTA treatment on binding of [14C]penicillin G to the whole cells of P. aeruginosa KM 338. Washed cells, harvested at logarithmic phase, were treated with (0) or without (0) 10 mM EDTA in the presence of 0.5 M sucrose. After washing, the treated cells were incubated with varying concentrations of [14Cjpenicillin G as described. Data are expressed as counts per minute of bound radioactivity per tube sample. TABLE 3. Activities of ,3-lactamases against ampicillin and cephalosporin C Relative activity
Substrate
Ampicillin
Ampicllin P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817
0.3 300 0.8 850 22
Cephalosporin C
1.0 11 39 24 740
634
SUGINAKA, ICHIKAWA, AND KOTANI
TABLE 4. Substrate profiles of j-lactamases Relative activity
Substrate
P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817 a
Car- Cephcilnbn-IalosAmpi- Peni-
cillin
Gili
10 100 19 100 3
85 15 110
48 8 52
-
-
ceii
porin
-
100 5 100 5 100
-a
-, Activity too low to be determined.
The substrate profiles of the enzyme from these organisms were determined against ampicillin, penicillin G, carbenicillin, and cephalosporin C. The rate of hydrolysis of these substrates was calculated with an arbitrary value of 100 for the rate of ampicillin or cephalosporin C hydrolysis (Table 4). The profile of the enzyme from P. aeruginosa KM 338 was clearly different from those from S. aureus 235 and E. coli 3032; it is characterized by its relatively high rate of hydrolysis of cephalosporin C being the same as those from cephalosporinase-producing Citrobacter 817 and penicillinase-negative E. coli 386. Ampicillin was 10-fold less rapidly hydrolyzed than cephalosporin C, and both penicillin G and carbenicillin were very poorly or not hydrolyzed by P. aeruginosa KM 338.
DISCUSSION As previously reported (27), the membrane fraction derived from P. aeruginosa KM 338 catalyzes peptidoglycan synthesis, including the cross-linking reaction. This transpeptidase is inhibited by low concentration of penicillins, similar to E. coli (14), whereas growth of this organism is extremely unsusceptible to penicillin G. It has been suggested that penicillin is irreversibly bound to the transpeptidase in the cytoplasmic membrane of penicillin-susceptible organisms (16, 25, 26). Penicillin G and carbenicillin were also irreversibly bound to the membrane fraction derived from P. aeruginosa KM 338, which catalyzed peptidoglycan synthesis (27). The binding followed saturation type kinetics (specific binding), and its saturation was achieved at low concentration (2 nmol/ mil) of the antibiotic, as has been shown (16, 26) for penicillin-susceptible organisms. However, the binding to whole cells of this organism was entirely of the nonsaturation type (nonspecific), although binding to penicillin-susceptible organisms was specific as previously desciKbed (4, 5,
ANTIMICROB. AGENTS CHEMOTHER.
8, 15, 20, 24, 26), and saturation was achieved at low concentration of penicillin G, as with the membrane fractions (16, 17). About 3 nmol of penicillin G was specifically bound per g of the membrane protein at saturation. The amount of binding of penicillin G to the membrane fraction from P. aeruginosa KM 338 was lower than that of gram-positive organisms (26, 27) (S.
aureus and B. subtilis, which bind 28 and 36
nmol/g of protein, respectively). After treatment of the whole cells of P. aeruginosa KM 338 with a high concentration of unlabeled penicillin, the specific binding of radioactive penicillin G to the resulting membrane fraction from the treated cells still remained with penicillin G pretreatment, but was eliminated by a carbenicillin pretreatment. This result suggests that penicillin G cannot penetrate into the target enzyme (transpeptidase), whereas carbenicillin can penetrate into its sites. It has been reported that a permeability barrier can be damaged by EDTA without affecting the viability of the bacteria (11). Eagon (6) showed that the cell walls of P. aeruginosa contain comparatively large amounts of calcium and magnesium. This action of EDTA is attributed to the chelation of divalent metals which are required for the structural integrity of the cell envelope (1, 7). Rogers et al. (21) demonstrated that a proteinlipopolysaccharide complex is released from the cell envelope by EDTA. Repaske (19) found that EDTA allows lysozyme to attack cell walls of certain bacteria. By EDTA treatment of P. aeruginosa KM 338, penicillin G was specifically bound to the whole cells. This phenomenon is also seen after polymyxin treatment, as will be described in a subsequent paper. It had been reported that EDTA reverses the resistance of P. aeruginosa to several antibiotics in vitro (2, 29). This may depend on the increase in bacterial permeability to penicillins with EDTA, enabling penicillin to reach its specific binding sites. On the other hand, ,B-lactamase produced from P. aeruginosa might also be considered as a resistance activity for penicillins. P. aeruginosa KM 338 (MIC of penicillin G, 30 mg/ml), however, has an extremely low level of penicillinase compared with penicillinase-producing S. aureus and E. coli (MIC of penicillin G, 2 and 16 mg/ml, respectively). ,B-Lactamase does not appear to be the basis of intrinsic resistance to penicillins of this organism, although in some cases (9, 28) the nature of resistance can be attributed to ,B-lactamase. These results suggest that intrinsic resistance to
BINDING OF PENICILLIN TO P. AERUGINOSA
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635
nine carboxypeptidase: penicillin-sensitive enzymatic penicillin in P. aeruginosa may be attributed to reaction in strains of Escherichia coli. J. Biol. Chem. surface (outer of the cell barrier a permeability 243:3180-3192. layer) which prevents the antibiotics from 15. Lawrence, P. J., M. Rogolsky, and V. T. Hanh. 1971. Binding of radioactive benzylpenicillin to sporulating reaching their sites of action in the cytoplasmic Bacillus cultures: chemistry and fluctuations in spemembrane. cific binding capacity. J. Bacteriol. 108:662-667.
ACKNOWLEDGMENTS We thank M. Nishida and T. Murakawa (Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) for the assay of ,B-lactamase activities. LITERATURE CITED 1. Asbell, M. A., and R. G. Eagon. 1966. Role of multivalent cations in the organization, structure, and assembly of the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 92:380-387. 2. Barrett, E., and A. W. Asscher. 1972. Action of
3.
4. 5.
6. 7.
8.
9.
10.
11. 12.
13. 14.
ethylenediaminetetra-acetic acid (EDTA) on carbenicillin-resistant strains of Pseudomonas aeruginosa. J. Med. Microbiol. 5:355-359. Citri, N., and M. P. Pollock. 1966. The biochemistry and function of ,-lactamase (penicillinase) p. 237-323. In F. F. Nord (ed.), Advances in enzymology, vol. 28. John Wiley & Sons, New York. Cooper, P. D. 1956. Site of action of radiopenicillin. Bacteriol. Rev. 20:28-48. Duerksen, J. D. 1964. Localization of the site of fixation of the inducer, penicillin, in Bacillus cereus. Biochim. Biophys. Acta 87:123-140. Eagon, R. G. 1969. Cell wall-associated inorganic substances from Pseudomonas aeruginosa. Can. J. Microbiol. 15:235-236. Eagon, R. G., and K. J. Carson. 1965. Lysis of cell walls and intact cells of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid and lysozyme. Can. J. Microbiol. 11:193-201. Edwards, J. R., and J. T. Park. 1969. Correlation between growth inhibition and the binding of various penicillins and cephalosporins to Staphylococcus aureus. J. Bacteriol. 99:459-462. Fullbrook, P. D., S. W. Elson, and B. Slocombe. 1970. R-factor mediated ,B-lactamase in Pseudomonas aeruginosa. Nature (London) 226:1054-1056. Ghuysen, J. M., J. L. Strominger, and D. J. Tipper. 1968. Bacterial cell walls, p. 53-104. In M. Florkin and E. H. Stotz (ed.), Comprehensive biochemistry, vol. 26A. American Elsevier Publishing Co., New York. Hamilton-Miller, J. M. T. 1965. Effect of EDTA upon bacterial permeability to benzylpenicillin. Biochem. Biophys. Res. Commun. 20:688-691. Heilmann, H. D. 1972. On the peptidoglycan of the cell walls of Pseudomonas aeruginosa. Eur. J. Biochem. 31:456-463. Humphrey, J. H., and J. W. Lightbown. 1952. A general theory for plate assay of antibiotics with some practical applications. J. Gen. Microbiol. 7:129-143. Izaki, K., M. Matsuhashi, and J. L. Strominger. 1968. Biosynthesis of the peptidoglycan of bacterial cell walls. XIII. Peptidoglycan transpeptidase and D-ala-
16. Lawrence, P. J., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XV. The binding of radioactive penicillin to the particulate enzyme preparation of Bacillus subtilis and its reversal with hydroxylamine or thiols. J. Biol. Chem. 245:3653-3659. 17. Lawrence, P. J., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XVI. The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis. J. Biol. Chem. 245:3660-3666. 18. Martin, H. H., H. D. Heilmann, and H. J. Preusser. 1972. State of the rigid-layer in cell walls of some gram-negative bacteria. Arch. Mikrobiol. 83:332-346. 19. Repaske, R. 1956. Lysis of gram-negative bacteria by lysozyme. Biochim. Biophys. Acta 22:189-197. 20. Rogers, H. J. 1967. The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of the antibiotic by staphylococci. Biochem. J. 103:90-102. 21. Rogers, S. W., H. E. Gilleland, Jr., and R. G. Eagon. 1969. Characterization of a protein-lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid. Can. J. Microbiol. 15:743-748. 22. Rolinson, G. N., and R. Sutherland. 1968. Carbenicillin, a new semisynthetic penicillin active against Pseudomonas aeruginosa, p. 609-613. Antimicrob. Agents Chemother. 1967. 23. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477. 24. Schmid, R., and R. Plapp. 1972. Binding of "4C-penicillin G to Proteus mirabilis. Arch. Mikrobiol. 83:246-260. 25. Strominger, J. L., P. M. Blumberg, H. Suginaka, J. Umbreit, and G. G. Wickus. 1971. How penicillin kills bacteria: progress and problems. Proc. R. Soc. London Ser. B 179:369-383. 26. Suginaka, H., P. M. Blumberg, and J. L. Strominger. 1972. Multiple penicillin-binding components in Bacillus subtilis. Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J. Biol. Chem. 247:5279-5288. 27. Suginaka, H., A. Ichikawa, and S. Kotani. 1974. Penicillin-resistant mechanisms in Pseudomonas aeruginosa: effects of penicillin G and carbenicillin on transpeptidase and D-alanine carboxypeptidase activities. Antimicrob. Agents Chemother., 6:672-675. 28. Sykes, R. B., and M. H. Richmond. 1971. R-factor, beta-lactamase, and carbenicillin-resistant Pseudomonas aeruginosa. Lancet 2:342-344. 29. Weiser, R., A. W. Asscher, and J. Wimpenny. 1968. In vitro reversal of antibiotic resistance by ethylenediaminetetraacetic acid. Nature (London) 219:1365-1366.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1975, p. 629-635 Copyright O 1975 American Society for Microbiology
Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa: Binding of Penicillin to Pseudomonas aeruginosa KM 338 HIDEKAZU SUGINAKA,* AKIRA ICHIKAWA,
AND
SHOZO KOTANI
Department of Microbiology, Osaka University Dental School, Joan-cho, Kita-ku, Osaka 530, Japan Received for publication 30 December 1974
A comparison of the binding of radioactive penicillin G to whole cells and the membrane fraction derived from Pseudomonas aeruginosa KM 338 was made. This organism has intrinsic resistance to penicillin. The binding to the membrane fraction which catalyzed peptidoglycan synthesis followed saturation type kinetics and saturation was achieved at approximately 2 nmol of penicillin G per ml, whereas binding to the whole cells was entirely of the nonsaturation type. The binding of carbenicillin to the membrane fraction was determined by competition between radioactive penicillin G and unlabeled carbenicillin for the binding sites. It was bound at the same sites in almost the same manner. When whole cells were pretreated with high concentration of unlabeled penicillin G or carbenicillin, the subsequent binding of radioactive penicillin G to the membrane fraction from carbenicillin-treated cells was entirely nonspecific, but with penicillin G-pretreated cells it was still specific. There was apparently specific binding of radioactive penicillin G to ethylenediaminetetraacetate-treated cells. P. aeruginosa KM 338 had an extremely low activity of f3-lactamase compared with other enzyme-producing organisms. This enzyme from P. aeruginosa KM 338 was of the cephalosporinase type. These data indicate that penicillin resistance of P. aeruginosa KM 338 may be a consequence of the development of a permeability barrier which prevents the antibiotic from reaching its sites of action in the cytoplasmic membrane.
In general, most strains of Pseudomonas aeruginosa are resistant to penicillins. The intrinsic mechanisms of resistance to penicillin in this organism have been investigated (27). The peptidoglycan isolated from cell walls of this organism (12, 18) is similar to that of penicillin-susceptible organisms (10, 23, 25). In a previous paper (27) it was reported that the membrane fraction derived from P. aeruginosa KM 338 catalyzes peptidoglycan synthesis, including the cross-linking reaction. This transpeptidase is inhibited by a low concentration of penicillin G or carbenicillin in a similar manner (27), whereas the intact cells are unsusceptible to penicillin G and are relatively susceptible to carbenicillin (22). It has been suggested that penicillin is irreversibly bound to transpeptidase in the cytoplasmic membrane of penicillin-susceptible organisms (16, 17, 25, 26). On the other hand, it has also been thought that penicillinase is an important factor of penicillin-resistance mechanisms in P. aeruginosa, similar to penicillinase-producing organisms, such as Staphylococcus aureus and Escherichia coli (3).
In the present experiments, the comparative binding abilities of penicillin G and carbenicillin were studied with whole cells and the membrane fraction from a strain of P. aeruginosa. Also, the j-lactamase activities produced by this strain was compared with those from the enzyme-producing organisms. (A summary of this work was presented at the 46th Annual Meeting of the Japanese Society for Microbiology, Tokyo, 8 April 1973.) MATERIALS AND METHODS Organisms. The organism used was Pseudomonas aeruginosa KM 338, derived from ATCC 17653. This strain has been used in the previous study (27) of peptidoglycan synthesis in our laboratory. The organisms used as reference for ,B-lactamase activities were Staphylococcus aureus 235, Escherichia coli 386 and 3032, and Citrobacter 817. All these organisms were furnished by Research Laboratories, Fujisawa Pharaceutical Co., Ltd. (Osaka, Japan) and had been isolated from clinical materials. Minimal inhibitory concentrations (MIC) of penicillin G and carbenicillin
for these strains are shown in Table 1. Antibiotics. Potassium 6-phenyl( acet- 1[14C])aminopenicillanate, abbreviated [14C]penicillin 629
630
SUGINAKA, ICHIKAWA, AND KOTANI
TABLE 1. Minimal inhibitory concentrations of penicillin G and carbenicillin Antibiotic
P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817
MIC (Ag/ml) Penicillin G
Carbenicillin
30,000 2,000 16,000 16,000 8,000
125 25 25 800 800
G, was obtained from Amersham Searle (Buckinghamshire, England). It had a specific activity of 45 M4Ci/Mgmol. [14C]benzylpenicilloic acid was prepared from [14C]penicillin G by treatment with commercial penicillinase. The reaction mixture contained, in a total volume of 0.4 ml, 40,000 U of penicillinase (Tokyo Kasei Co., Ltd., Tokyo, Japan), 20 Mmol of tris(hydroxymethyl)aminomethane-hydrochloride (Tris) buffer (pH 7.5), and 10 smol of [14C]penicillin G. After incubation at 37 C for 4 h, the reaction mixture was boiled for 10 min for inactivation of the enzyme activity. The resulting compound was not contaminated by penicillin G when examined by thin-layer chromatography on Silica Gel G in nbutanol-acetic acid-water (200:5:200, vol/vol, upper phase). Unlabeled penicillin G and benzylpenicilloic acid were supplied by Meiji Seika Co., Ltd. (Osaka, Japan). Carbenicillin and cephalosporin C were furnished from Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan), and ampicillin was obtained from Takeda Chemical Industries Co., Ltd. (Osaka, Japan). Assays for the binding of radioactive penicillin G to whole cells and the membrane fraction of P. aeruginosa KM 338. The methods used were almost the same as described in the previous study (26). The organism was routinely grown at 37 C to mid- or late-log phase in Trypticase soy broth (BBL) as described previously (27). The membrane fraction (particulate fraction) was prepared by differential centrifugation from disrupted cells after alumina grinding (27). The incubation mixture, containing 25 ,1 of the membrane preparation which catalyzed peptidoglycan synthesis (approximately 20 mg of protein per ml), 10 il of 1 M Tris-hydrochloride buffer (pH 7.5), 2 gl of 1 M MgCl2, 3 Ml of water, and 10 ,ul of ['4C]penicillin G solution varying in concentration from 0.5 to 50 nmol/ml, was incubated for 30 min at 30 C. After incubation, the reaction was terminated by the addition of 10 ul of 0.6 M unlabeled penicillin G solution, and then either of two methods were used for the assay of bound penicillin. In one case, the incubation mixture was spotted on Toyo filter paper no. 51 (Toyo Roshi Kaisha Co., Ltd., Osaka, Japan) and then developed in n-butanol-acetic acid-water (200:5:200, vol/vol, upper phase; ascending) for 4 h, and then the process was repeated with the same solvent. The unbound penicillin G and benzylpenicilloic acid moved near the solvent front, and penicillin G bound to the membrane protein remained at the origin of the chromatogram. The origin was cut out and counted in scintillation fluid containing 15.5 g of
ANTIMICROB. AGENTS CHEMOTHER. 2,5-diphenyloxazole and 114 g of 1,4-bis[2-(4-methyl5-phenyloxazole) ]benzene in 3.8 liters of toluene. In the second assay, the membrane fraction-bound [14Cjpenicillin G was pelleted by centrifugation at 45,000 x g for 20 min, and the pellet was then washed four times by centrifugation with 8 ml of 0.05 M Tris-hydrochloride (pH 7.5) containing 0.001 M MgCl2. Finally, the pellet was dissolved in 0.4 ml of 4% sodium dodecyl sulfate. To the sample in a glass scintillation vial, 1 ml of Biosolv (formula BBS-3, Beckman Instruments Inc., Fullerton, Calif.) was added, followed by 9 ml of the above scintillation fluid. Both the chromatographic and washing methods were employed for most of the experiments described in this paper. The former procedure yielded somewhat lower values for radioactive penicillin binding, but both of the above methods were found to be reproducible and reliable. In the assay for binding to whole cells, the cells of a logarithmic phase growth were harvested by centrifugation, washed with 0.05 M Tris-hydrochloride buffer (pH 7.5) or phosphate buffer (pH 7.2) containing 0.001 M MgCl2, and finally resuspended in the above buffer in 1/20 of the volume of the growth medium. Five hundred microliters of the cell suspension (about 2 x 109 cells/ml) was incubated with 200 Ml of 1 M Tris buffer (pH 7.5) or phosphate buffer (pH 7.2), 40 ul of 1 M MgCl2, 60 Ml of water, and 200 gl of [14C]penicillin G solution, varying in concentration from 2.5 to 3,750 nmol per ml, for 30 min at 30 C. After incubation, the cells were recovered by centrifugation at 10,000 x g for 10 min and washed with adequate amounts of the buffer of the above composition. Finally, they were suspended in 0.4 ml of 4% sodium dodecyl sulfate and counted as described above. Counting was carried out in a Packard liquid scintillation spectrometer. Assay for 3-lactamase activities. All the organisms tested were grown in Trypticase soy broth (BBL) at 37 C for 18 h. These broth cultures were disrupted with an ultrasonic disintegrator (60 W, 20 kcycle/s. Ohtake Seisakusho Co., Ltd., Tokyo, Japan) for 10 min, and the resulting suspensions of each disrupted culture were used as the enzyme source. ,B-Lactamase activities were estimated by a modification of the bioassay method of Humphrey et al. (13), using Bacillus subtilis ATCC 6633 (obtained from the Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. [Osaka, Japan]) as test organism. All assays for ,B-lactamase were carried out with a substrate concentration of 50 Ag/ml. The diluted sonic extracts were incubated with test antibiotics in 0.067 M phosphate buffer (pH 7.0) at 37 C for 1 h. After incubation, the reaction was terminated by boiling for 3 min, and the enzyme activity was measured by cup-plate bioassay of the residual antibiotic.
RESULTS Binding of radioactive penicillin G to the membrane fraction from P. aeruginosa KM 338. When a suspension of the membrane fraction from P. aeruginosa KM 338 was incubated at 30 C with varying concentrations of [I4C]penicillin G for 30 min, the ['4C]penicillin
VOL. 7, 1975
BINDING OF PENICILLIN TO P. AERUGINOSA
G was irreversibly bound to the membrane fraction which catalyzes peptidoglycan synthesis as previously described (27) (Fig. 1). The binding of ['4C]penicillin G followed saturation type kinetics as had been shown earlier in the binding of penicillin G to whole cells (4, 5, 8, 15, 20, 24, 26) and membrane fractions (16, 26) from various penicillin-susceptible organisms. The binding was biphasic, showing a relatively high initial binding at low penicillin concentrations (to 2 nmol/ml), followed by an apparent secondary binding which was linearly related to penicillin concentration. At the point of maximum specific binding, about 3.2 nmol of [I4C ]penicillin G were bound per g of membrane protein, and the concentration required for half-maximum binding under the condition of the experiment was approximately 0.5 nmol of penicillin G per ml, with saturation of the penicillin-specific receptor sites achieved at approximately 2 nmol/ml. The binding value given was obtained by extrapolating the linear secondary binding curve to zero concentration. The secondary phase was also seen with boiled membrane fractions with inactivated transpep.C
6
0 S.-
0) 0
2 CZ o0
631
tidase activity, and is presumably due to some nonspecific binding or trapping of the radioactive compound. Benzylpenicilloic acid (open ,B-lactam ring) was also bound with a linear dependence on its concentration, as in the case with penicillin G binding to the boiled membrane fraction above. As previously observed with other organisms (16), the bound penicillin was not washed out of the membrane fraction from P. aeruginosa KM 338 with buffer or high concentrations of unlabeled penicillin and was not removed on treatment with penicillinase. This binding seems to be through a relatively tight covalent linkage (irreversible binding). Treatment with 1 M neutral hydroxylamine, however, resulted in virtual reversal of the binding of [4C ]penicillin G to the membrane fraction from P. aeruginosa KM 338, the same as that from B. subtilis (15) (data not shown). Treatment with trypsin or Pronase destroyed the binding components of the membrane fraction, suggesting that it is protein in nature (Table 2). Binding of radioactive penicillin G to whole cells of P. aeruginosa KM 338. When suspensions of whole cells of P. aeruginosa KM 338 were incubated with varying concentrations of [4C ]penicillin G, the antibiotic was also irreversibly bound to the cells, as with the membrane fraction (Fig. 2). However, the binding was entirely nonspecific. The amount bound to whole cells was directly proportional to the concentration up to a level of at least 750 nmol/ml, although binding to the membrane fraction from this organism and to the whole cells of penicillin-susceptible organisms was specific as described above and previously (4, 5, 8, 15, 20, 24, 26). The binding of [14C]benzylpenicilloic acid to the cells was also nonspecific and of lower affinity compared with penicillin G. Inhibition binding of radioactive penicillin TABLE 2. Effects of trypsin and Pronase on binding of G to the membrane fraction from P. aeruginosa KM 338a
[14C]penicillin
10 q O ~~~~5 ~ 14C-PC-G or PA Conc. (nmoles/ml) FIG. 1. Binding of [l4C]penicillin G or [14C]benzylpenicilloic acid to the membrane fraction from P. aeruginosa KM 338. Assays were carried out with the washing method as described. Data are expressed as moles of bound radioactivity per gram of membrane protein. Where boiled membrane fraction was employed, this fraction in buffer was placed in a boiling water bath for 5 min prior to addition of [14C]penicillin G solution (x). Symbols: 0, penicillin G; 0, benzylpenicilloic acid.
Treatment None Trypsin (1 mg/ml) Pronase (1 mg/ml)
[P4C]penicillin G bound Counts/min
%
3,582 886 640
100 24.7 17.9
a The membrane fraction (20 mg of protein/ml} was treated with trypsin or Pronase at pH 7.5 for 30 min at 37 C before [14C]penicillin G was added. Assays were carried out with the paper chromatographic method as described.
632
SUGINAKA, ICHIKAWA, AND KOTANI 1 .5
100
ANTIMICROB. AGENTS CHEMOTHER.
The resulting membrane fraction was incubated with varying concentrations of [14C]penicillin G, and control cells were handled in a similar 751.0 I~ manner except that the initial incubation contained only cells and buffer. The binding of S_~~~~~~~~~~~ [14C ]penicillin G to the membrane fraction from the penicillin G-treated cells was still of the biphasic saturation type, whereas the binding to that from the carbenicillin-treated cells was entirely of the nonsaturation type (Fig. 4). 14C-PCG or 14C-PA Conc. (nmoles/ml) Effect of ethylenediaminetetraacetate FIG. 2. Binding of ['4C]penicillin G or ['4C]benzyl- treatment on binding of radioactive penicillin penicilloic acid to the whole cells of P. aeruginosa KM G to the whole cells of P. aeruginosa KM 338. 338. Assays were carried out as described with the A logarithmic phase culture of P. aeruginosa addition of ["4C]penicillin G solutions varying in KM 338 was centrifuged, and the cells were concentrations from 0.5 to 10 (left) to 750 (right) washed and resuspended at ¼io of the original nmol/ml. Data are expressed as moles of bound concentration in 0.05 M phosphate buffer (pH radioactivity per gram (dry weight) of whole cells. 7.2) containing 0.5 M sucrose. This preparation Symbols: 0, penicillin G; 0, benzylpenicilloic acid. was divided into two portions, to one of which G to the membrane fraction from P. aerugin- was added ethylenediaminetetraacetate osa KM 338 by unlabeled penicillin G, ben- (EDTA) to a final concentration of 10 mM. The zylpenicilloic acid, and carbenicillin. The second acted as a control. These preparations between competition G and un[o4C4penicillin 0 labeled penicillins for specific binding sites was determined by incubating the membrane fraction from P. aeruginosa KM 338 with 2.5 nmol pe Gand various concenof [m4Cpe ]penicillin trations of the test antibiotic (8) (Fig. 3). The unlabeled antibiotics tested, if irreversibly _25 bound to the same binding sites as radioactive penicillin G, would prevent binding of the 0 labeled penicillin G. The binding of .-o [14C]penicillin G was inhibited by unlabeled penicillin G or carbenicillin in the almost same .c50 manner and extent, but benzylpenicilloic acid did not prevent the binding of [14C]penicillin G. 4-The inhibition of [14C]penicillin G by car- 0 benicillin suggests that the two substances are I)75 bound at the same sites. Binding of radioactive penicillin G to the membrane fraction from penicillin G or carbenicillin-treated cells from P. aeruginosa KM 338. Penetration of penicillin into its target 100 site was indirectly measured by determining the 10 20 0 reduction in ('4C]penicillin G-binding capacity of the membrane fraction prepared from P. Antibiotics Conc. (nmoles/ml) aeruginosa cells previously incubated with unFIG. 3. Competitive inhibition of binding of labeled penicillin G or carbenicillin. The cells were incubated with a high concentration (500 [I4C]penicillin G to the membrane fraction from P. KM 338 by unlabeled penicillin G, carnmol/ml) of unlabeled penicillin G or car- aeruginosa benicillin, and benzylpenicilloic acid. The membrane benicillin and then washed with adequate fraction was incubated with varying concentrations of amounts of the buffer by centrifugation. The test antibiotics and 2.5 nmol of G per washed cells were disrupted with sonic treat- ml. Assays were carried out with [14C]penicillin the washing method ment (Super sonic vibrator, UR-150, Tominaga as described. Data are expressed as percent inhibition Works, Ltd., Tokyo, Japan), and the broken cell of bound radioactivity by test antibiotics. Symbols: preparation was fractionated by differential 0, penicillin G; 0, carbenicillin; A, benzylpenicilloic centrifugations as described previously (27). acid.
BINDING OF PENICILLIN TO P. AERUGINOSA
VOL. 7, 1975
633
aeruginosa KM 338, whereas from penicillinaseproducing Staphylococcus aureus 235 and Escherichia coli 3032 it was hydrolyzed at a high rate. The MIC for penicillin G for the former was 30 mg/ml, and those for the latter strains were 0.3 and 16 mg/ml, respectively (Table 1). P. aeruginosa also had little activity against cephalosporin C compared with cephalosporinase-producing Citrobacter 817 and the other organisms tested.
0D
-o n C)
s
6
cL 0~
6
co3 C-)
CD C-
0
,E Q. CD _
0
1
3
5
1 4C-PC-G Conc. (nmol es/ml FIG. 4. Binding of [14C]penicillin G to the membrane fraction from penicillin G- or carbenicillintreated cells of P. aeruginosa KM 338. Washed cells, harvested at logarithmic phase, were treated with unlabeled penicillin G (0) or carbenicillin (A) (500 nmol/ml) or without antibiotic (A) at 30 C for 30 min as described. The treated cells were disrupted by sonic treatment. The broken cell suspension was separated by differential centrifugation; the resulting membrane fraction was incubated with varying concentrations of [14Cjpenicillin G; and assays were carried out with the washing method as described. Data are expressed as moles of bound radioactivity per gram of membrane protein.
incubated at 37 C for 15 min, and the cells then washed in the same buffer by centrifugation (10,000 x g for 10 min). The resulting cells were incubated with varying concentrations of ['4C]penicillin G in 0.05 M phosphate buffer (pH 7.2) without MgCl2 and in the presence of 0.5 M sucrose. Results of the binding ability are shown in Fig. 5. It can be seen that there was apparently specific binding of [4C ]penicillin G to the EDTA-treated cells. Activities of $-lactamases from P. aeruginosa KM 338. Relative activities and substrate profiles of #-lactamases in sonically treated cultures from P. aeruginosa KM 338 and from several clinically isolated organisms were examined. A comparison of the activity for a range of substrates (Table 3) shows that ampicillin was little hydrolyzed by the enzyme from P. were were
_
0
co CD
10-
C-)
0
1
14C-PC-G
2
3
4
5
Conc. (nmoles/ml)
FIG. 5. Effect of EDTA treatment on binding of [14C]penicillin G to the whole cells of P. aeruginosa KM 338. Washed cells, harvested at logarithmic phase, were treated with (0) or without (0) 10 mM EDTA in the presence of 0.5 M sucrose. After washing, the treated cells were incubated with varying concentrations of [14Cjpenicillin G as described. Data are expressed as counts per minute of bound radioactivity per tube sample. TABLE 3. Activities of ,3-lactamases against ampicillin and cephalosporin C Relative activity
Substrate
Ampicillin
Ampicllin P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817
0.3 300 0.8 850 22
Cephalosporin C
1.0 11 39 24 740
634
SUGINAKA, ICHIKAWA, AND KOTANI
TABLE 4. Substrate profiles of j-lactamases Relative activity
Substrate
P. aeruginosa KM 338 S. aureus 235 E. coli 386 E. coli 3032 Citrobacter 817 a
Car- Cephcilnbn-IalosAmpi- Peni-
cillin
Gili
10 100 19 100 3
85 15 110
48 8 52
-
-
ceii
porin
-
100 5 100 5 100
-a
-, Activity too low to be determined.
The substrate profiles of the enzyme from these organisms were determined against ampicillin, penicillin G, carbenicillin, and cephalosporin C. The rate of hydrolysis of these substrates was calculated with an arbitrary value of 100 for the rate of ampicillin or cephalosporin C hydrolysis (Table 4). The profile of the enzyme from P. aeruginosa KM 338 was clearly different from those from S. aureus 235 and E. coli 3032; it is characterized by its relatively high rate of hydrolysis of cephalosporin C being the same as those from cephalosporinase-producing Citrobacter 817 and penicillinase-negative E. coli 386. Ampicillin was 10-fold less rapidly hydrolyzed than cephalosporin C, and both penicillin G and carbenicillin were very poorly or not hydrolyzed by P. aeruginosa KM 338.
DISCUSSION As previously reported (27), the membrane fraction derived from P. aeruginosa KM 338 catalyzes peptidoglycan synthesis, including the cross-linking reaction. This transpeptidase is inhibited by low concentration of penicillins, similar to E. coli (14), whereas growth of this organism is extremely unsusceptible to penicillin G. It has been suggested that penicillin is irreversibly bound to the transpeptidase in the cytoplasmic membrane of penicillin-susceptible organisms (16, 25, 26). Penicillin G and carbenicillin were also irreversibly bound to the membrane fraction derived from P. aeruginosa KM 338, which catalyzed peptidoglycan synthesis (27). The binding followed saturation type kinetics (specific binding), and its saturation was achieved at low concentration (2 nmol/ mil) of the antibiotic, as has been shown (16, 26) for penicillin-susceptible organisms. However, the binding to whole cells of this organism was entirely of the nonsaturation type (nonspecific), although binding to penicillin-susceptible organisms was specific as previously desciKbed (4, 5,
ANTIMICROB. AGENTS CHEMOTHER.
8, 15, 20, 24, 26), and saturation was achieved at low concentration of penicillin G, as with the membrane fractions (16, 17). About 3 nmol of penicillin G was specifically bound per g of the membrane protein at saturation. The amount of binding of penicillin G to the membrane fraction from P. aeruginosa KM 338 was lower than that of gram-positive organisms (26, 27) (S.
aureus and B. subtilis, which bind 28 and 36
nmol/g of protein, respectively). After treatment of the whole cells of P. aeruginosa KM 338 with a high concentration of unlabeled penicillin, the specific binding of radioactive penicillin G to the resulting membrane fraction from the treated cells still remained with penicillin G pretreatment, but was eliminated by a carbenicillin pretreatment. This result suggests that penicillin G cannot penetrate into the target enzyme (transpeptidase), whereas carbenicillin can penetrate into its sites. It has been reported that a permeability barrier can be damaged by EDTA without affecting the viability of the bacteria (11). Eagon (6) showed that the cell walls of P. aeruginosa contain comparatively large amounts of calcium and magnesium. This action of EDTA is attributed to the chelation of divalent metals which are required for the structural integrity of the cell envelope (1, 7). Rogers et al. (21) demonstrated that a proteinlipopolysaccharide complex is released from the cell envelope by EDTA. Repaske (19) found that EDTA allows lysozyme to attack cell walls of certain bacteria. By EDTA treatment of P. aeruginosa KM 338, penicillin G was specifically bound to the whole cells. This phenomenon is also seen after polymyxin treatment, as will be described in a subsequent paper. It had been reported that EDTA reverses the resistance of P. aeruginosa to several antibiotics in vitro (2, 29). This may depend on the increase in bacterial permeability to penicillins with EDTA, enabling penicillin to reach its specific binding sites. On the other hand, ,B-lactamase produced from P. aeruginosa might also be considered as a resistance activity for penicillins. P. aeruginosa KM 338 (MIC of penicillin G, 30 mg/ml), however, has an extremely low level of penicillinase compared with penicillinase-producing S. aureus and E. coli (MIC of penicillin G, 2 and 16 mg/ml, respectively). ,B-Lactamase does not appear to be the basis of intrinsic resistance to penicillins of this organism, although in some cases (9, 28) the nature of resistance can be attributed to ,B-lactamase. These results suggest that intrinsic resistance to
BINDING OF PENICILLIN TO P. AERUGINOSA
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635
nine carboxypeptidase: penicillin-sensitive enzymatic penicillin in P. aeruginosa may be attributed to reaction in strains of Escherichia coli. J. Biol. Chem. surface (outer of the cell barrier a permeability 243:3180-3192. layer) which prevents the antibiotics from 15. Lawrence, P. J., M. Rogolsky, and V. T. Hanh. 1971. Binding of radioactive benzylpenicillin to sporulating reaching their sites of action in the cytoplasmic Bacillus cultures: chemistry and fluctuations in spemembrane. cific binding capacity. J. Bacteriol. 108:662-667.
ACKNOWLEDGMENTS We thank M. Nishida and T. Murakawa (Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) for the assay of ,B-lactamase activities. LITERATURE CITED 1. Asbell, M. A., and R. G. Eagon. 1966. Role of multivalent cations in the organization, structure, and assembly of the cell wall of Pseudomonas aeruginosa. J. Bacteriol. 92:380-387. 2. Barrett, E., and A. W. Asscher. 1972. Action of
3.
4. 5.
6. 7.
8.
9.
10.
11. 12.
13. 14.
ethylenediaminetetra-acetic acid (EDTA) on carbenicillin-resistant strains of Pseudomonas aeruginosa. J. Med. Microbiol. 5:355-359. Citri, N., and M. P. Pollock. 1966. The biochemistry and function of ,-lactamase (penicillinase) p. 237-323. In F. F. Nord (ed.), Advances in enzymology, vol. 28. John Wiley & Sons, New York. Cooper, P. D. 1956. Site of action of radiopenicillin. Bacteriol. Rev. 20:28-48. Duerksen, J. D. 1964. Localization of the site of fixation of the inducer, penicillin, in Bacillus cereus. Biochim. Biophys. Acta 87:123-140. Eagon, R. G. 1969. Cell wall-associated inorganic substances from Pseudomonas aeruginosa. Can. J. Microbiol. 15:235-236. Eagon, R. G., and K. J. Carson. 1965. Lysis of cell walls and intact cells of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid and lysozyme. Can. J. Microbiol. 11:193-201. Edwards, J. R., and J. T. Park. 1969. Correlation between growth inhibition and the binding of various penicillins and cephalosporins to Staphylococcus aureus. J. Bacteriol. 99:459-462. Fullbrook, P. D., S. W. Elson, and B. Slocombe. 1970. R-factor mediated ,B-lactamase in Pseudomonas aeruginosa. Nature (London) 226:1054-1056. Ghuysen, J. M., J. L. Strominger, and D. J. Tipper. 1968. Bacterial cell walls, p. 53-104. In M. Florkin and E. H. Stotz (ed.), Comprehensive biochemistry, vol. 26A. American Elsevier Publishing Co., New York. Hamilton-Miller, J. M. T. 1965. Effect of EDTA upon bacterial permeability to benzylpenicillin. Biochem. Biophys. Res. Commun. 20:688-691. Heilmann, H. D. 1972. On the peptidoglycan of the cell walls of Pseudomonas aeruginosa. Eur. J. Biochem. 31:456-463. Humphrey, J. H., and J. W. Lightbown. 1952. A general theory for plate assay of antibiotics with some practical applications. J. Gen. Microbiol. 7:129-143. Izaki, K., M. Matsuhashi, and J. L. Strominger. 1968. Biosynthesis of the peptidoglycan of bacterial cell walls. XIII. Peptidoglycan transpeptidase and D-ala-
16. Lawrence, P. J., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XV. The binding of radioactive penicillin to the particulate enzyme preparation of Bacillus subtilis and its reversal with hydroxylamine or thiols. J. Biol. Chem. 245:3653-3659. 17. Lawrence, P. J., and J. L. Strominger. 1970. Biosynthesis of the peptidoglycan of bacterial cell walls. XVI. The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis. J. Biol. Chem. 245:3660-3666. 18. Martin, H. H., H. D. Heilmann, and H. J. Preusser. 1972. State of the rigid-layer in cell walls of some gram-negative bacteria. Arch. Mikrobiol. 83:332-346. 19. Repaske, R. 1956. Lysis of gram-negative bacteria by lysozyme. Biochim. Biophys. Acta 22:189-197. 20. Rogers, H. J. 1967. The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of the antibiotic by staphylococci. Biochem. J. 103:90-102. 21. Rogers, S. W., H. E. Gilleland, Jr., and R. G. Eagon. 1969. Characterization of a protein-lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylenediaminetetraacetic acid. Can. J. Microbiol. 15:743-748. 22. Rolinson, G. N., and R. Sutherland. 1968. Carbenicillin, a new semisynthetic penicillin active against Pseudomonas aeruginosa, p. 609-613. Antimicrob. Agents Chemother. 1967. 23. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol. Rev. 36:407-477. 24. Schmid, R., and R. Plapp. 1972. Binding of "4C-penicillin G to Proteus mirabilis. Arch. Mikrobiol. 83:246-260. 25. Strominger, J. L., P. M. Blumberg, H. Suginaka, J. Umbreit, and G. G. Wickus. 1971. How penicillin kills bacteria: progress and problems. Proc. R. Soc. London Ser. B 179:369-383. 26. Suginaka, H., P. M. Blumberg, and J. L. Strominger. 1972. Multiple penicillin-binding components in Bacillus subtilis. Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J. Biol. Chem. 247:5279-5288. 27. Suginaka, H., A. Ichikawa, and S. Kotani. 1974. Penicillin-resistant mechanisms in Pseudomonas aeruginosa: effects of penicillin G and carbenicillin on transpeptidase and D-alanine carboxypeptidase activities. Antimicrob. Agents Chemother., 6:672-675. 28. Sykes, R. B., and M. H. Richmond. 1971. R-factor, beta-lactamase, and carbenicillin-resistant Pseudomonas aeruginosa. Lancet 2:342-344. 29. Weiser, R., A. W. Asscher, and J. Wimpenny. 1968. In vitro reversal of antibiotic resistance by ethylenediaminetetraacetic acid. Nature (London) 219:1365-1366.
