ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1979, p.513-517 0066-4804/79/04-0513/05$02.00/0

Vol. 15, No. 4

Peptidoglycan Transpeptidase Inhibition in Pseudomonas aeruginosa and Escherichia coli by Penicillins and Cephalosporins BRIAN A. MOORE,* SIDNEY JEVONS, AND KEITH W. BRAMMER Pfizer Central Research, Sandwich, Kent, England

Received for publication 4 January 1979

Peptidoglycan transpeptidase activity has been studied in cells of Escherichia coli 146 and Pseudomonas aeruginosa 56 made permeable to exogenous, nucleotide-sugar peptidoglycan precursors by ether treatment. Transpeptidase activity was inhibited, in both organisms, by a range of penicillins and cephalosporins, the Pseudomonas enzyme being more sensitive to inhibition in each case. Conversely, growth of E. coli 146 was more susceptible to these antibiotics than growth of P. aeruginosa 56. Furthermore, similar transpeptidase inhibition values were obobtained for the four penicillins examined against the Pseudomonas enzyme, although only two of these (carbenicillin and pirbenicillin) inhibited the growth of this organism. We therefore conclude that the high resistance of P. aeruginosa 56 to growth inhibition by most fl-lactam antibiotics cannot be due to an insensitive peptidoglycan transpeptidase.

The ,B-lactam antibiotics, which inhibit the growth of many gram-positive and gram-negative bacteria, exert their lethal action by interfering with bacterial cell wall biosynthesis (1, 4). The target site of penicillins is thought to be the enzyme peptidoglycan transpeptidase (18), which catalyzes the final reaction in bacterial cell wall biosynthesis, i.e., the formation of an interpeptide linkage between the amino acid side chains of the peptidoglycan polymer (1). This enzymatic reaction has been shown to occur in both gram-positive and gram-negative bacteria (2, 5, 9, 10, 21), and is thought to be common to most, if not all, bacteria. Pseudomonas aeruginosa however, is characteristically resistant to the majority of /-lactam antibiotics (15). To investigate the role of peptidoglycan transpeptidase in this resistance, we have examined the effect of various penicillins and cephalosporins on this enzymatic reaction in a strain of P. aeruginosa which shows typical resistance to these antibiotics. The effects have been compared with those produced by the same antibiotics on the transpeptidase activity of a susceptible strain of Escherichia coli.

5'-diphospho-N-acetylmuramyl-L-alanyl-Dglutamyl-meso-diaminopimelyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide) was prepared by a Uridine

method based on that of Lugtenberg et al. (8). A 9liter amount of CGPY broth (7) was seeded with an overnight culture of B. cereus ATCC 11778 and grown, with aeration, at 37°C until the culture reached an optical density at 540 nm (ODrAo) of approximately 0.4. Chloramphenicol (50 ,ug/ml) was added to the broth culture, followed after 15 min by vancomycin (12.5 ,ug/ml) and incubation was continued for a further 40 to 60 min. Cells were then harvested in a continuous action rotor (MSE Scientific Instruments, Sussex, England) at 18,000 rpm (21,000 x g), using a throughput flow rate of 200 to 400 ml/min. The cells were finally suspended in 40 ml of ice-cold distilled water. Trichloroacetic acid was added to give a final concentration of 5% (wt/vol) and, after 30 min at 00C, the precipitate was removed by centrifugation. The resulting pellet was extracted twice with ice-cold 5% trichloroacetic acid by resuspension and centrifugation. The supernatants were pooled and extracted three times with'an equal volume of diethyl ether. The aqueous phase was retained and neutralized with 1 N NaOH. Residual ether was removed from the aqueous phase under a stream of air, and the volume of the extract was reduced to approximately 5 ml by evaporation under vacuum. The sample was then desalted by elution from a MATERIALS AND METHODS Sephadex G10 column (400 by 30 mm) with distilled Bacterial strains. P. aeruginosa 56 and E. coli water. The ultraviolet (UV)-absorbing (260 nm) frac146 were both f8-lactamase-producing clinical isolates. tions were retained, pooled, and applied to an AG1Both exhibited smooth-colony morphology on cystine- X4, 200- to 400-mesh (Bio-Rad Laboratories) anion lactose electrolyte-deficient agar (Oxoid Ltd.). Bacil- exchange column (100 by 10 mm) and eluted with a lus cereus ATCC 11778 (NCIB 9231) was obtained 200-ml linear gradient of 0 to 0.3 M NaCl in 0.01 M from the National Collection of Industrial Bacteria. HCI. The fractions from the major ultraviolet-absorbPreparation of UDP-MurNAc-pentapeptide. ing peak were retained, pooled, evaporated under vac513

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MOORE, JEVONS, AND BRAMMER

uum, and desalted as before. The UV-absorbing fractions from the desalting column were pooled, evaporated to dryness under vacuum and suspended in a small volume of 0.05 M sodium phosphate buffer (pH 7.0). UDP-MurNAc-pentapeptide concentration was determined by N-acetylamino-sugar estimation (17) and ultraviolet absorption at 262 nm relative to a uridine diphosphate (UDP) standard. The two methods gave comparable results. The UDP-MurNAc-pentapeptide solution was adjusted to approximately 5 mM in 0.05 M sodium phosphate buffer (pH 7.0) and stored in 100-pl portions at -200C. Characterization of the UDP-MurNAc-pentapeptide. A smaller-scale preparation, using identical methods, was further purified by descending paper chromatography. The material was applied as a thin band to a sheet of Whatman 3MM paper, together with marker spots at both ends of the band. The chromatogram was developed in isobutyric acid-I N ammonia (5:3) and the marker spots were identified as single ninhydrin-positive species on the acetonewashed chromatogram. The corresponding band was cut from the paper and eluted by the method of Eshdat and Mirelman (3). Amino acid analysis of the freezedried eluate showed that alanine, glutamic acid, and meso-diaminopimelic acid, respectively, were present in the molar ratio 3:1:1. This material was used as a reference marker to check the identity of subsequent preparations by cellulose thin-layer chromatography. Two solvent systems were used; isobutyric acid/l N ammonia (5:3) and ethanol/i M ammonium acetate at pH 7.2 (5:2), giving Rf values for the UDP-MurNAcpentapeptide of approximately 0.2 and 0.03, respec-

ANTIMICROB. AGENTS CHEMOTHER. vials containing Instagel scintillation fluid (Packard) and counted in a Nuclear Chicago liquid scintillation counter. Determination of MICs of antibiotics. Minimal inhibitory concentrations (MICs) were determined by usinf a standard agar plate technique. Organisms were grown in Iso-sensitest broth (Oxoid Ltd.) at 37°C for 18 h. Cultures were diluted 100-fold in Iso-sensitest broth and inoculated onto Iso-sensitest agar containing serial doubling dilutions of antibiotic, using a multipoint inoculator (Denley Instruments), and gave an inoculum of approximately 104 colony-forming units. After incubation at 37°C for 18 h, the MIC was recorded as the lowest concentration of antibiotic which completely inhibited visible growth of the organism. Chemicals and antibiotics. Adenosine triphosphate, UDP, and vancomycin were obtained from Sigma Chemical Co. Chloramphenicol (Grade B) was obtained from Calbiochem. UDP-['4C]GlnNAc (300 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Ampicillin, carbenicillin, pirbenicillin (Pfizer), cephalexin (Glaxo Research Ltd.), and cephalothin (Eli Lilly) were laboratory reference samples. Benzylpenicillin (Glaxo), cephaloridine (Eli Lilly), and cefazolin (Fujisawa) were obtained commercially.

RESULTS Incorporation of N-acetyl-['4C]glucosamine by ETB into either SDS-insoluble or trichloroacetic acid-insoluble material was dependent upon the presence of UDP-MurNAc-pentapeptide. The requirement for UDP-MurNAc-pentapeptide suggests that [14C]GlnNAc is incorporated only into peptidoglycan in the manner described by Mirelman et al. (11). Transpeptidase activity in both E. coli 146 and P. aeruginosa 56 ETB was measured by the incorporation of radiolabel into SDS-insoluble peptidoglycan. This incorporation was linear for at least 90 min, being about 450 to 550 pmol of [I4C]GlnNAc per mg of ETB protein per h for E. coli 146 and 400 to 1,000 pmol for P. aeruginosa 56. Transpeptidase activity in ETB of both P. aeruginosa 56 and E. coli 146 was subject to inhibition by a number of 8-lactam antibiotics (Fig. 1-4). In any single transpeptidase inhibition experiment, the uninhibited (control) level of ['4C]GlnNAc incorporation was estimated at least in duplicate, and replicate values were normally within 5% of the mean value. The level of ['4C]-GlnNAc incorporation at each inhibitor concentration was again derived from at least two determinations. In each case, the inhibited reactions have been expressed in terms of percentage of the control reaction. In this way, the data from many such experiments (encompassing the range of control incorporation values

tively. Preparation of ETB. P. aeruginosa 56 and E. coli 146 were grown in 1 liter of either brain heart infusion broth (Difco Laboratories) or CGPY broth at 37°C until the ODso was approximately 0.4. The cells were harvested by centrifugation and rendered permeable by ether treatment as described by Mirelman et al. (11). The protein content of ether-treated bacteria (ETB) was determined by the method of Lowry et al. (6). ETB were stored in 100-pl portions at -20°C. Estimation of transpeptidase activity. Reaction mixtures routinely contained 10 p1 of 1 M tris(hydroxymet,byl)methylamine (pH 8.3) 10 i1 of 1 M NH4Cl, 5 p1 of 1 M MgCl2, 5 Al of 20 mM 2mercaptoethanol, 20 pl of 0.1 M disodium adenosine triphosphate, 10 pl of uridine-5'-diphospho-N-acetylD-[U-_4C]glucosamine (UDP-['4C]GInNAc; 15.8 mCi/ mmol, 12.5 ,uCi/ml), 10 id of UDP-MurNAc-pentapeptide (1.0 or 5.0 mM), 60 i1 of distilled water, 20 p1 of antibiotic (or distilled water), and 50 ud of ETB (2 to 10 mg/ml of protein). Reactions were initiated by the addition of ETB and incubated at 370C for 1 h. The reactions were terminated by the addition of 1 ml of 4% sodium dodecyl sulfate (SDS), and the samples were placed in a boiling water bath for 30 min. SDSinsoluble material was collected on 0.45-pum membrane filters (Milhipore Corp.) and washed with two volumes of 2% SDS followed by five volumes of distilled water. The filters were allowed to dry, placed in scintillation above) have been normalized and compared,

TRANSPEPTIDASE INHIBITION BY /?-LACTAMS

VOL. 15, 1979

515

70

60-

c

50-

*400

30-

x

20-

w

10-

05 01

10 100 Concentration of}?- lactom (pg/mi)

1000

FIG. 1. Inhibition of transpeptidase activity by ampicillin and benzylpenicillin in ETB of P. aeruginosa 56 and E. coli 146. The percent incorporation of N-acetyl-["C]glucosamine at a range of antibiotic concentrations, relative to the control reaction (as described in the text) is shown. Symbols: 0, ampicillin and, A, benzylpenicillin against P. aeruginosa 56; 0, ampicillin and A, benzylpenicillin against E. coli 146.

relative to the uninhibited reactions expressed as 100%. The graphs show the average, normalized percent values from several separate experiments. Each point on a graph is the average of between 2 and 18 separate estimations. The range about these average percent values varied, but was typically of the order of ±5% at high levels of inhibition and around ±10% at lower levels of inhibition. The 50% inhibitory concentrations (IC5os) derived from these graphs are shown in Table 1 together with the MICs of the antibiotics against both organisms. The four penicillins examined inhibited the E. coli 146 transpeptidase reaction with 05s08 ranging from 2.0 to 5.2 jig/ml (Table 1). The MICs, however, did not parallel these activities. The IC50 and MIC of benzylpenicillin, for example, were 2.1 and 25 jig/ml, respectively, compared with those of pirbenicillin (5.2 and 1.6 jg/ml, respectively). Ampicillin and benzylpenicillin were both antibacterially inactive against P. aeruginosa 56 (MICs greater than 100 jig/ml) despite considerably lower transpeptidase IC5os (0.10 and 0.17 ,ug/ml, respectively) than were seen with E. coli 146. Carbenicillin and pirbenicillin, however, were antibacterially active against P. aeruginosa 56, with ICwos very similar to those of ampicillin and benzylpenicillin. All four penicillins were considerably more effective (approximately 10- to 30-fold) in inhibiting the

O1

10

100

Concentration of,3-toctom (jig/mi)

FIG. 2. Inhibition of transpeptidase activity by carbenicillin and pirbenicillin in ETB of P. aeruginosa 56 and E. coli 146. The percent incorporation of N-acetyl-/rCJglucosamine at a range of antibiotic concentrations, relative to the control reaction (as described in the text) i8 shown. Symbols: 0, carbenicillin and A, pirbenicillin against P. aeruginosa 56; 0, carbenicillin and A, pirbenicillin against E. coli 146. 110-

100 90 800

70

E

60-

50 0

20-

101 0

01

10

100

100 0 200 0

Concentration ot R-Ioctom (jug/ml)

FIG. 3. Inhibition of transpeptidase activity by cephalexin and cephalothin in ETB of P. aeruginosa 56 and E. coli 146. The percent incorporation of N. acetyl-"4CJglucosamine at a range of antibiotic concentrations, relative to the control reaction (as described in the text) is shown. Symbols: 0, cephalexin and A, cephalothin against P. aeruginosa 56; *, cephalexin and A, cephalothin against E. coli 146.

transpeptidase activity of P. aeruginosa 56 than that of E. coli 146. E. coli 146 was susceptible to the four cephalosporins examined, with MICs similar to those

516

MOORE, JEVONS, AND BRAMMER 105100-

90-

o 800

70-

0

FE

60-

Z 50cr - 40-

20 10., wr

0

16o

01 10 Concentration of B-lactom (pg/ml)

FIG. 4. Inhibition of transpeptidase activity by cefazolin and cephaloridine in ETB of P. aeruginosa 56 and E. coli 146. The percent incorporation of Nacetyl- L4C]glucosamine at a range of antibiotic concentrations, relative to the control reaction (as described in the text) is shown. Symbols: 0, cefazolin and 4, cephaloridine against P. aeruginosa 56; *, cefazolin and A, cephaloridine against E. coli 146.

TABLE 1. Comparison of MICs of bacterial growth with transpeptidase IC5os for several ,-lactams against E. coli 146 and P. aeruginosa 56 E. coli 146 P. aeruginosa 56 Antibiotic

Ampicillin Benzylpenicillin Carbenicillin Pirbenicillin Cephalexin Cephalothin Cefazolin

Cephaloridine

IC50a

MICb

ICsoa

MICb

(Ag/

(4g/

(Ag/

(pg/

2.1 3.0 5.2 >200 32.0 22.0 4.6

3.1 25 12.5 1.6 6.25 6.25 1.6 3.1

ml) 2.0

nil)

ml) 0.10 0.17 0.2 0.17 48.0 13.0 6.2 4.1

ml) >100 >100 50 6.25 >100 >100 >100 >100

a IC50s were obtained from Fig. 1-4. h MICs were derived from at least two estimations.

of ampicillin and pirbenicillin. P. aeruginosa 56, however, was resistant to all four cephalosporins (MICs greater than 100 ,Ag/ml) although their transpeptidase ICsos for this organism were lower than the corresponding IC5os for the susceptible E. coli 146.

DISCUSSION Mirelman et al. (11) have demonstrated that ether-treated cells of E. coli PAT 84 utilize exogenous UDP-GlnNAc and UDP-MurNAcpentapeptide in peptidoglycan biosynthesis. This synthesis proceeds via macromolecular

ANTIMICROB. AGENTS CHEMOTHER.

peptidoglycan precursors which are cross-linked to preexisting peptidoglycan almost exclusively by penicillin-sensitive transpeptidation. Our results show that similar reactions occur in E. coli 146 and in P. aeruginosa 56 and that the incorporation of precursors is inhibited, in both organisms, by,8-lactam antibiotics. The observed transpeptidase inhibition in ETB may possibly be affected by factors other than the intrinsic sensitivity of this enzyme. For example, the 8)-lactams tested could bind to Dalanine carboxypeptidase present in the ETB (1, 11, 12), leaving less antibiotic available for transpeptidase inhibition, and thus falsely elevating the transpeptidase ICQos. Alternatively, falsely high IC5>s may be the consequence of the ,-lactamase content of ETB. We have prepared crude extracts of P. aeruginosa 56 and E. coli 146 cells and have assayed these for f,-lactamase activity by using the microiodometric assay (20). Preliminary results indicate that both organisms possess a cephalosporinase-like enzyme activity, (B. A. Moore, unpublished data). Also using the chromogenic cephalosporin substrate, 87/312 (13), (Glaxo), we have detected /?-lactamase activity in ETB of P. aeruginosa 56 and E. coli 146 and have shown that both types of ETB contain similar amounts of enzyme, (B. A. Moore, unpublished data). Certainly, the ICros of cephalosporin are higher than those of the penicillins, possibly due to higher cephalosporinase than penicillinase activity in ETB. Nevertheless, the comparison of P. aeruginosa 56 and E. coli 146 transpeptidase IC..ws remains valid, since the type and content of ETB fl-lactamase are similar in both organisms. An alternative explanation for the higher cephalosporin IC.0s could be simply that cephalosporins are intrinsically weaker transpeptidase inhibitors than penicillins, and that other, more sensitive enzymes may be their site of lethal action in the whole cell (1, 18). One further possibility, albeit unlikely, is that the P. aeruginosa 56 transpeptidase is modified in some way after ether treatment and is thus rendered more sensitive to ,B-lactams than the E. coli 146 enzyme. Despite these considerations, which we have yet to fully investigate, the E. coli 146 transpeptidase is clearly sensitive to inhibition by /B-lactam antibiotics, and the P. aeruginosa 56 enzyme is apparently even more sensitive. In contrast, cells of E. coli are more susceptible to growth inhibition by these antibiotics than are cells of P. aeruginosa. Furthermore, the two /3lactams active against P. aeruginosa, carbenicillin and pirbenicillin, do not exhibit greater transpeptidase inhibition than ampicillin and

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benzylpenicillin against P. aeruginosa 56 ETB. Thus the degree of sensitivity of the P. aeruginosa 56 transpeptidase enzyme is clearly not a major determinant of the susceptibility of the intact organism to f8-lactam antibiotics. This conclusion differs from that reached by other workers (14) who used a different strain of P. aeruginosa. Since the transpeptidase activity of P. aeruginosa 56 is sensitive to inhibition by /-lactams, the reason for the resistance of this organism to the majority of this class of antibiotics must reside elsewhere. Although alternative killing sites cannot be excluded (1, 18), the most likely mechanisms are high intrinsic resistance resulting from poor penetration of the antibiotic to the site of action (15), or the presence of 8lactamases capable of hydrolyzing the antibiotic before it can inhibit the transpeptidase enzyme (15, 19). It has been suggested elsewhere (16) that resistance may be due to a combination of B-lactamases and intrinsic resistance. Our direct demonstration of the sensitivity of P. aeruginosa 56 transpeptidase to ,B-lactam antibiotics emphasizes the major role played by other factors in the antibacterial susceptibility of this bacterial strain. As P. aeruginosa 56 appears typical of many strains of this species, it is probable that this conclusion is true for P. aeruginosa strains in general. ACKNOWLEDGMENTS We thank K. A. Holmes and J. A. Pegg for their excellent technical assistance. The peptide analysis of UDP-MurNAc-pentapeptide was performed by M. Szelke, Hammersmith Hospital Medical School, London.

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