Proc. Natl. Acad. Sci. USA

Vol. 73, No. 9, pp. 3293-3297, September 1976 Microbiology

Suppression of lytic effect of beta lactams on Escherichia coli and other bacteria (penicillin/pH dependence of lysis/murein hydrolases/E. coli autolysins)

E. W. GOODELL*, RUBIN LOPEZt, AND ALEXANDER TOMASZ* * The Rockefeller University, New York, N.Y. 10021; and t Instituto de Immunologia y Biologia Microbiana, Madrid, Spain Communicated by William Trager, June 17,1976 Growth of E coli 't pH 5 protected the bacteria ABSIRACT against the lytic effect of beta lactam antibiotics typically observed when the cells are grown at pH 7 or 7.5, i.e., the H values routinely used in laboratory experiments. In contrast, the typical effects of beta lactam antibiotics on cellular shape and elongation and cell division appeared to be similar in cultures grown under neutral and acid pH conditions. The pH-dependent antibiotic tolerance can also be demonstrated with pneumococci, staphylococci, streptococci, and Bacillus subtilis. We suggest that the mechanism of the pH-dependent antibiotic tolerance may involve either the production of a more stable plasma membrane or the suppression of the activity of a murein hydrolase(s) that catalyzes the antibiotic-induced lysis; at least a fraction of these enzyme molecules may be localized at the cell surface and be accessible to experimental manipulation.

Penicillins and cephalosporins are beta lactam antibiotics; at sufficiently high concentration these antibiotics are bactericidal and they cause the physical disintegration (lysis) of the cells. At lower concentration individual beta lactams have a variety of biological effects, which may include inhibition of cell division and elongation, and alteration of cell shape. The specific effect of any given antibiotic on the cell appears to be related to its capacity to bind to and inhibit one or more of three proteins present in Escherichia coli. These three proteins exhibit a high degree of specificity in binding to the various beta lactam antibiotics and seem to play distinct roles in the regulation of cell shape, elongation, and division (1). As to the bactericidal and lytic effects of beta lactams, it has been suggested repeatedly in the literature (2) that these pharmacologically most important effects of the penicillins are due to their inhibition of several enzymes involved in the murein metabolism of bacteria (3). One of these enzymes, a transpeptidase, catalyzes the crosslinking of peptide side chains of neighboring glycan units in the cell wall. (4). It is, however, not obvious why inhibition of any of these enzymes alone should cause cellular lysis, since cell lysis presumably requires breaking of chemical bonds in the completed portion of the cell wall through the activity of hydrolytic enzymes (5-7). In addition, structural damage to the plasma membrane must also accompany the penicillin-induced lysis (8, 9). The importance of cell wall hydrolyzing enzymes (autolysins) in the lytic process has been demonstrated in several species of Gram-positive bacteria (10-12); in cells with suppressed autolytic activities penicillin caused inhibition of growth without lysis ("penicillin tolerance") (13). In this communication we describe a growth condition (low pH) at which cultures of the Gram-negative bacterium E. coli exhibit antibiotic tolerance. We have also found pH values at which several other bacterial species show tolerance to beta lactam antibiotics. The mechanism of this phenomenon is not known yet. One of two mechanisms seems most plausible: (a) the suppression 3293

of a cell-wall-degrading enzyme (autolysin); or (b) production of a more stable plasma membrane at the protective pH.

MATERIALS AND METHODS E. coli W7 (Dap-, Lys-) requiring both 2,6-diaminopimelic acid and lysine for growth (14) was cultured with aeration in complete medium (Antibiotic Medium no. 3; Difco) buffered with 0.1 M tris(hydroxymethyl)aminomethane (Tris) and 0.01 M citric acid and containing 40,ug/ml of diaminopimelic acid. The culture medium was used at two pH values: pH 7.5 ("permissive" for lysis) and pH 5.0 ("nonpermissive" for lysis). Anaerobic cultures of pneumococci (Diploous pneumWniae, strain R36A) and aerobic cultures of Bacillus subtilis 168 (obtained from Dr. L. Mindich of the Public Health Research Institute, New York) were both grown in a casein hydrolysate medium ("C-medium") (15) at 370. The pH of the media was set with 0.1 M buffers at the following values: 5.5 (Tris-maleate), 7.0 (potassium phosphate) and 8.0 (Tris or potassium phosphate) (16). Staphylo cs aureus (strain 209P, obtained from Dr. Leon Sabath of the Department of Medicine, University of Minnesota School of Medicine, Minneapolis, Minn.) and Streptococcus faeczum durans (ATCC 9790, from the erican Type Culture Collection) were cultured aerobically in Antibiotic Medium no. 3 (Difco). The media were buffered with 0.1 M Tris containing 0.01 M citric acid and 0.05 M Na2HPO4. All cells were grown at 370.

Growth of the cultures was monitored by measurin'g the light scattering of the cell suspensions (Coleman nephelometer) (15). Viable titers of the cultures were assayed by the routine procedures. Benzylpenicillin (Squibb), mecillinam (Leo Pharmaceutical Products, Ballerup, Denmark) and cloxacillin (Wyeth Laboratories, Philadelphia) were commercial products. Cephalexin and cephaloridine were obtained through the courtesy of Dr. F. Price of Bristol Laboratories, Syracuse, N.Y. RESULTS Fig. 1 shows the effect of four beta lactam antibiotics (benzylpenicillin, mecillinam, cephalexin, and cephaloridine) on E. coli cultures grown at pH 5 and 7.5. The bacteria grew at normal rates at both pH values and exhibited identical dose responses. Yet, with each of the four antibiotics, the characteristic lytic effect at pH 7.5 was replaced by a simple growth inhibition in the cultures at pH 5.0. Table 1 shows that the morphological effects characteristic of the various beta lactams (1) remained essentially the same at the two pH values. Bacteria grown in cephalexin (10-200 ,ug/ml) and in benzylpenicillin (10-100 Ag/ml) became long filaments. In addition, penicillin-induced filaments at pH 5

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N

1

0

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2

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FIG. 1. Effect of pH on the response of E. coli W7 to increasing concentrations of beta lactam antibiotics. Bacteria were inoculated into complete medium buffered at eitherpH 5.0 or pH 7.5; growth was monitored by nephelometry (Coleman nephelometer). Benzylpenicillin (A), cephaloridine (B), mecillinam (C), or cephalexin (D) was added to the culture (O hr) when the cells were growing exponentially. The final concentrations of the antibiotics were 1 ,ug/ml (X), 10 ug/ml (@), 25 Mg/ml (0), 50 Mg/ml (U), 100 ,g/ml (A), and 1000 Mzg/ml (A). Open circles (0) represent growth in the absence of antibiotics. The lines in the figures serve only to simplify interpretation of the data. Upper panels: cultures at pH 7.5; lower panels: cultures at pH 5.0. A

B

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C

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FIG. 2. Bactericidal action of beta lactam antibiotics at pH 5.0 and pH 7.5. E. coli cells growing in complete medium buffered at pH 5.0 (0) or pH 7.5 (0) were treated (at 0 hr) with 25 Mug/ml of benzylpenicillin (A), 25 Mug/ml of cephaloridine (B), 1000 ,ug/ml mecillinam (C), or 100 Mg/ml of cephalexin (D). Samples were periodically removed, diluted, and plated out to determine the number of viable cells. The medium used to dilute the cells and the agar medium onto which they were plated were both complete media of the same pH in which the cells had grown.

Proc. Natl. Acad. Sci. USA 73 (1976)

microbiology: Goodell et al.

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Table 1. The effect of beta lactam antibiotics on cell shape at permissive and nonpermissive conditions

I

BENZYLPENICILLIN

I

MECILLINAM

CEPHALEXIN

CEPHALORIDINE

p 9/m I

pH 5.0

1 pH 5.0

pH 7.0

pH 7.5

pH 5.0

pH 7.5

pH 7.5

pH 5.0

LYSI S

1 0

25

N. D.

LYSIS

1 0 0 I

'

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0

~~~lYSIS

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1 0 00 I..

I

I

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-

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:Ak

c e

.

I

I

E. coli was grown at pH 5.0 and at pH 7.5 in the presence of four beta lactams for 3 hr. Phase contrast micrographs were then made of the cells. The drawings depict typical bacteria at each growth condition. N.D., not determined.

showed "bulges" (see photographs). Higher concentrations of these two beta lactams inhibited cell elongation. Cephaloridine inhibited cell growth at all concentrations; mecillinam caused the cells to become spherical. The rapid loss of viability caused by mecillinam and cephalexin in cultures grown at pH 7.5 was dramatically inhibited at pH 5.0. In contrast, viability loss caused by benzylpenicillin and cephaloridine only decreased somewhat at the lower pH (Fig. 2). The unusual response to penicillin at low pH was not caused by the degradation or metabolism of beta lactams to less effective antibiotic derivatives, since the tolerant response was independent of penicillin concentration (at least up to 200 ttg/ml of benzylpenicillin and 1 mg/ml of mecillinam). In addition, the specific morphological effects of the drugs did appear even at the low pH. An experiment specifically designed to detect a possible inactivation of penicillin gave negative results. In this experiment cultures of E. colh were incubated with benzylpenicillin at both pH values and with antibiotic concentrations ranging from 10 to 200 ag/ml. After incubation at 370 for 3 hr, the bacteria were removed by filtration and the penicillin concentration in the sterile filtrates was determined using pneumococcal cultures as a very sensitive bioassay. The

concentrations of penicillin that had "survived" the pre-incubation with E. coli at the two different pH values were found to be virtually identical. Furthermore, the benzylpenicillin in the sterile filtrates caused the typical lytic effect on the pneumococcal cultures. In an attempt to find an in vitro correlate to the pH dependence of pencillin lysis, we determined the rates of "lysis" of E. coli suspensions damaged by repeated freezing and thawing. A culture (25 ml) of bacteria was grown at pH 7.5. At a cell titer of ca. 109 cells per ml, the culture was centrifuged and the cells were resuspended in 1 ml of distilled water. After two cycles of freezing (dry ice-acetone bath) and thawing (370), 0.1 ml volumes of the cell suspension were diluted 10-fold into a series of buffers (0.1 M Tris-maleate) ranging in pH from 5 to 8. The rate of decline in the turbidity (OD 660, Zeiss spectrophotometer) was monitored during incubation at 37°. It can be seen that optimal lysis occurred at pH values higher than 7.0 (Fig. 3). The pH-dependent penicillin tolerance is not restricted to E. coli, but is also exhibited by such diverse species as Diplococcus pneumoniae, Streptococcus faecium, Staphylococcus aureus, and Bacillus subtilis (Fig. 4). The antibiotic tolerance of pneumococci and Bacillus subtilis will be described in more

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Proc. Natl. Acad. Sci. USA 73 (1976)

the specific morphological effects (1) and the dose responses are similar at the lysis-permissive and lysis-nonpermissive pH values; and (iv) loss of viability is also greatly reduced in the case of some antibiotics. The mechanism of penicillin-induced lysis of bacteria is most likely to involve at least two distinct stages corresponding to damage to the two structural components of the bacterial cell: (I) the cell wall and (It) the plasma membrane. While the penicillin tolerance in pneumococci was clearly correlated with reduced autolysin activity (10, 13), the mechanism of pHdependent lysis resistance in E. colh is not yet known. At this time we are considering two alternative mechanisms: (a) the low pH may suppress the activity of a cell-wall-hydrolyzing enzyme(s); (b) the stability of the plasma membrane may change at the nonpermissive condition. Op den Kamp and his colleagues have shown (17) that protoplasts formed from B. subtilis grown at low pH are less sensitive to lysis under hypotonic conditions and are often rod-shaped rather than spherical. This bacterium, as well as others, synthesizes new types of phospholipids (18) when the pH of the culture is lowered. On the other hand, two observations suggest that the suppression of murein hydrolase activity may be important in the pH-dependent tolerance: (i) lysis of frozen-thawed cell suspensions shows a pH dependence similar to that of penicillininduced lysis of live bacteria; (II) pH-dependent penicillin tolerance has also been observed in Staph. aureus, pneumococci, Strep. faecium, and B. subtills. In each one of these species the pH values permitting lysis were found to be near the in vitro pH optima of the corresponding major autolytic enzymes [pH 8.5 in B. subtilis (19), 6.9 in pneumococcus (20), 7.0 in staphylococcus (21), and 6.5 in Streptococcus faecium (22)]. The bacteria showed penicillin tolerance in media with pH values at which the activities of the corresponding autolysins are suppressed (pH 6.6 in B. subtilis, pH 6.0 in staphylococcus, pH 5.5 and 8.0 in pneumococcus, and pH 5.0 and 8.0 in streptococcus). E. coil extracts contain several murein hydrolase activities: an endopeptidase (23), an amidase (24), a transglycosylase (25), and, possibly, a muramidase (26). However, at present it is not clear which if any of these enzymes are responsible for penicillin-induced lysis in E. coli. The low pH optimum (pH 4.5) of transglycosylase (25) makes its participation in penicillin lysis unlikely. Endopeptidase has a pH optimum near 7.5; however,

control

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1 2 HOURS FIG. 3. The pH dependence of lysis of E. coli suspensions damaged by freezing and thawing. Bacteria were grown in complete medium at pH 7.5 to the middle of exponential phase. After centrifugation, the cells were resuspended in buffer solutions (0.1 M Trismaleate) with pH values ranging from 5 to 8. A portion of the bacterial culture (8 + p) was treated with benzylpenicillin (1000 units/ml) for 10 min prior to centrifuigation and these cells were resuspended in pH 8 buffer containing benzylpenicillin (1000 units/ml). The bacterial suspensions in buffers were nex* exposed to two cycles of freezing (-40°) and thawing (+370) and the lysis (decline in the turbidity, measured as OD 660) at 370 was followed using a Zeiss spectrophotometer. The numbers next to the curves represent the pH of the' suspensions. A control suspension received formaldehyde (0.2% final concentration) before the freezing and thawing.

detail in a forthcoming publication (Lopez et al., manuscript in preparation).

DISCUSSION The observations described indicate that, for E. colh, growth at pH 5 constitutes a "nonpermissive" condition for the lytic and, in the case of mecillinam and cephalexin, for the bactericidal effects of beta lactam antibiotics. The phenomenon described is reminiscent of the antibiotic "tolerance" of autolysin-defective pneumococci (13) in several respects: (I) the lytic response of E. coli to the antibiotics is changed to simple growth inhibition; (il) the effect is not drug'specific and is observed over wide dose ranges; (ill) the beta lactam antibiotics seem to reach (and inhibit) their normal metabolic targets at pH 5, since both Staph. aureus

Strep. faecium

B. subtilis

D. pneumoniae

,6.0 i 8.0

8.0 50

7.0

6.5

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FIG. 4. The pH-dependent penicillin tolerance of four Gram-positive bacterial species. Bacteria were grown in the appropriate culture media (see Materials and Methods) at the pH values indicated. Antibiotic was added at 0 min of incubation. Benzylpenicillin was used for pneumococci (0.1 ;g/ml) and streptococci (10 Mg/ml); staphylococci and B. subtilis received cloxacillin at 1 Ag and 0.2 jg/ml, respectively. Growth and lysis were monitored by nephelometry (Coleman nephelometer).

Microbiology: Goodell et al. this enzyme should be inhibited by the beta lactams at the concentration used in our experiments (23). Fig. 3 shows that the lysis of frozen-thawed E. colh suspensions was not inhibited by 1000 units/ml of benzylpenicillin. The E. colh amidase appears to be active only on low-molecular-weight fragments of murein and essentially inactive on intact cell walls (24). While bacteria are known to regulate their intracellular pH (27), the activity of enzymes that function outside the main permeability barrier of the cells, such as autolysins, may be a more direct function of the pH of the extracellular medium. The preliminary studies described in this paper demonstrate that the pH dependence of penicillin sensitivity may be a general phenomenon applicable to many species of microorganisns. It should provide a useful experimental tool for studies on the mechanisms of antibiotic-induced lysis and killing of bacteria. The observations described indicate that, as in pneumococci, the primary effect of beta lactams in E. colh is the inhibition of growth and that this primary effect is not necessarily followed by the pharmacologically important lytic and cytocidal effects of these drugs. Similarly, the beta lactam effects on cell shape, elongation, and division are clearly separable from the lytic effect and, in part, from the cytocidal effect. The findings suggest that in E. colh antibiotic-induced lysis and killing occur via a distinct pathway that may involve the activity of an autolytic enzyme(s) and-possibly-a triggering mechanism similar to the one described recently in pneumococci (28). This investigation has been supported by a grant from the National Institutes of Health (Biomedical Support Grant to the Rockefeller University). E.W.G. is the recipient of a National Research Service Award. 1. Spratt, B. G. (1975) Proc. Nati. Acad. Sd. USA 72,% 29993003. . 2. Blumberg, P. M. & Strominger, J. L. (1974) Bacteriol. Rev. 38, 291-35. 3. Tamura, T., Imae, Y. & Strominger, J. L. (1976) J. Biol. Chem.

251,414-423.

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4. Park, J. T. & Strominger, J. L. (1957) Science 125,99-101. 5. Schwarz, U. & Weidel, W. (1965) Z. Naturforsch. 20b, 147153. 6. Weidel, W. & Pelzer, H. (1964) Ado. Enzymol. 2, 193-232. 7. Rogers, H. J. (1967) Nature 213,31-3. 8. Lederberg, J. & St. Clair, J. (1958) . Bacteriol. 75, 143-160. 9. Prestidge, L. & Pardee, A. (1957) J. Bacterlol. 74,48-59. 10. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature 227,138140. 11. Rogers, H. J. & Forsberg, C. W. (1971) J. Bacteriol. 108, 1235-1243. 12. Ayusawa, D., Yoneda, Y., Yamane, K. & Maruo, B. (1975) J. Bacteriol. 124, 459-465. 13. Tomasz, A. (1974) Ann. N.Y. Acad. Sci. 235,439-447. 14. Schwarz, U., Asmus, A. & Frank, H. (1969) J. Mol. Blol. 41, 419-429. 15. Tomasz, A. (1970) J. Bacteriol. 101, 860-871. 16. Gomori, G. (1955) in Methods in Enzymology, eds., Colowick, S. P. & Kaplan, N. 0. (Academic Press, New York), Vol. 1, pp. 138-146. 17. Op den Kamp, J. A. F., van Iterson, W. & van Deenen, L. L. M. (1967) Biochlm. Blophys. Acta 135,862-884. 18. Houtsmuller, U. & van Deenen, L (1965) Blochim. Biophys. Acta 106,564-576. 19. Herbold, D. R. & Glaser, L. (1975) J. Biol. Chem. 250,16761682. 20. H6ltje, J. V. & Tomasz, A. (1976) J. Biol. Chem. 251,41994207. 21. Takebe, I., Singer, H. J., Wise, E. M. & Park, J. T. (1970) J. Bacterfol. 102, 14-19. 22. Shockman, G. D., Thompson, J. S. & Conover, M. J. (1967) Biochemistry 6,1054-1065. 23. Hartmann, R., H6Itje, J. V. & Schwarz, U. (1972) Nature 235, 426-429. 24. van Heijenoort, J., Parquet, C., Flouret, B. & van Heijenoort, Y. (1975) Eur. J. Biochem. 58,611-619. 25. H6ltje, J. V., Mirelman, D., Sharon, N. & Schwarz, U. (1975) J. Bacteriol. 124, 1067-1076. 26. Pelzer, H. (1963) Z. Naturforsch. 18b, 950-956. 27. Kadish, L. J. & Pardee, A. (1963) Biochim. Biophys. Acta 78, 764-766. 28. Tomasz, A. & Waks, S. (1975) Proc. NatI. Acad. Sci. USA 72, 4162-4166.

Suppression of lytic effect of beta lactams on Escherichia coli and other bacteria.

Proc. Natl. Acad. Sci. USA Vol. 73, No. 9, pp. 3293-3297, September 1976 Microbiology Suppression of lytic effect of beta lactams on Escherichia col...
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