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Understanding penicillin-binding proteins
ANNLDO 3(7)47-54, 1986
ISSN 0738-1751
VOLUME 3, NUMBER 7, JULY 1986
EDITORIAL BOARD Editor
Associate Editors
DANIEL AMSTERDAM, PhD,
RONALD N. JONES, MD,
State University of New York
at Buffalo and Erie County Laboratory
CLYDE THORNSBERRY,PhD,
Kaiser-Permanente Medical Care Program
Center for Infectious Diseases, Centers for Disease Control
HAROLD C. NEU, MD,
LOWELL S. YOUNG, MD,
College of Physicians and Surgeons, Columbia University
UCLA School of Medicine
U N D E R S T A N D I N G PENICILLINBINDING PROTEINS EDITOR'S NOTE
47
H E N R Y F. C H A M B E R S
D. AMSTERDAM
Understanding PenicillinBinding Proteins 47 H.F. CHAMBERS E D I T O R I A L COMMENT
53
H.C. NEU
Editor's Note
It has been 45 years since the therapeutic efficacy of penicillin has been documented. Yet, we are still trying to understand the mode of action of this original betalactam agent. In the attempt, the role of the penicillin-binding proteins (PBPs) has been closely followed. Dr. Chambers reports on the actions of these proteins as they relate to cell division, maintenance of the organism,
ELSEVIER
University of California, San Francisco, San Francisco General Hospital, San Francisco, California
Penicillin-binding proteins (PBPs) are enzymes that catalyze the final reactions in bacterial cell wall synthesis. With few exceptions (eg, mycoplasma, some halophilic bacteria) PBPs are present in all bacteria. Penicillin-binding proteins covalently bind penicillin and related beta-lactam antibiotics at the enzymatic site. This reaction, not readily reversible under physiologic conditions, results in inhibition of wall synthesis. Cell lysis and death typically follow. Lysis is not a passive process general physiologic function, and antimicrobial resistance. Antimicrobic chemists closely study the PBPs, for it is the elucidation of their functions that can lead to the development of new drugs that will bypass problems associated with betalactamase resistance. It is of interest to note that bacterial proteins that dissociate penicillin, turn neutralizing its lethal effects, evolved from PBPs that are part of the cell wall. This
but results from cell wall degradation by an enzyme or enzymes, termed autolysis. 42 Penicillin-binding proteins themselves directly or indirectly may trigger autolytic processes. 19 Defective autolysis is responsible for tolerance, 14 a phenomenon in which growth is inhibited at low concentrations of drug, without accompanying lysis or killing. BACTERIAL CELL WALL SYNTHESIS AND STRUCTURE Several articles reviewing the synthesis and structure of bacterial cell walls have been published. 29,40,41,44 This article will limit itself to the most essential features of the cell wall assembly, particularly as they
evolutionary step---from a surface protein to a secreted protein--was a significant adaptation of bacteria in response to a lethal compound. A corollary activity of the PBPs is the effect of lysis and killing versus tolerance. The concept of tolerance has not yet been discussed in these pages. It represents the absence of killin,g by beta-lactam drugs and is more clearly associated clinically with gram-positive than gram-negative orgamsms.
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1t {E A N T I M I C R O B I C NEWSLETTER, V O L U M E 3, NUMB[¢R 7, IULY 1986
relate to PBPs. The interested reader should refer to more comprehensive reviews for further information. The bacterial cell wall lies outside the cell membrane. In grampositive bacteria the cell wall is several layers thick and is the outermost structure of the cell (if the capsule is excluded). In gram-negative bacteria the cell wall forms a single layer located between the cell membrane and the outer membrane. Although other macromolecular polymers (eg, teichoic acids) and proteins (eg, staphylococcal protein A) may sometimes be major constituents of the cell wall, the component common to all bacterial cell walls is peptidoglycan. Peptidoglycan is composed of alternating N-acetyl-glucosamine and N-acetyl-muramic acid residues (Fig. 1). A five amino acid stem peptide is present on the lactyl group of muramic acid residues. Amino acids in the stem peptide alternate between D and L stereoisomers. The amino acid composition of the stem peptide is highly conserved among all bacteria: Typically, only the amino acid at the third position varies from
N-acetylmuramic acid
HCCH3 I co I
Niacetylglucosamine
NHCOCH3
NHCOCH3
I bala Stem l peptide
I o-glu I
LiR3-x o-ala I o-ala
Figure 1. Disaccharide monomer
subunit of peptidoglycan. Ala = alanine, glu = glutamic acid, R = the variable third amino acid residue, X = the position of the amino group or peptide where crosslinkage occurs.
-- M-G I
--
-- M-G I
L-ala
I o-g lu I
L-R3--X
--
-- M-G -I
L-ala
+
I D-ala I
I o-glu 1
L-R3--X I o-alo I D-ala
O-aJa
-- M-G I
t-ala
+
PBP~ -I~
-- M-G I
L-ala
I D-glu I
~
L-R3I X
D-ala
I D-ala I PBP
@
--
+
-- M-G I
L-ala
I D-g lU I
L-R3--X I o-ala ~
®
--
k-ala
I O-g lu ~. I
L-R3--X I o-ala I o-aJa
--
/
/
I o-glu I --R3
/tl X
D-alo I D-aJa
®
Figure 2. The transpeptidase reaction catalyzed by PBPs. (~ PBP attacks the
D-alanyl-D-alanine bond to cleave off the terminal D-alanine. (~) A short-lived PBP-peptide intermediate is formed with D-a|anine at fourth position. (~) The terminal amino group at the crosslinking position (X) is donated from a second pentapeptide chain, displaces the PBP, and crosslinks the peptidoglycan. species to species. The last two amino acids of the stem peptide are always D-alanine. If present, a branch peptide of variable composition, is located at the third amino acid residue. Penicillin-binding proteins act at the D-alanyl-D-alanine bond. Penicillin-binding proteins can hydrolyze this bond (carboxypeptidase reaction), crosslink two stem peptides (transpeptidase reaction), or cleave peptide crosslinks (endopeptidase reaction). The physiologic roles of carboxypeptidase and endopeptidase functions are not well defined. These reactions may produce structural modifications that signal specialized wall sites, such as where septation and cell division occur. The transpeptidase reaction crosslinks peptidoglycan subunits to form a network imparting integrity and strength to the cell wall. The PBP replaces the terminal alanine residue from the stem peptide (Fig. 2). The penultimate alanyl group acts as an acceptor to a donor amino acid of the branch peptide, thus crosslinking the peptidoglycan subunits. The PBP regenerated is free to repeat this reaction. Penicillin inhibits this crosshnkage step of cell wall synthesis by binding covalently to the catalytic site of PBPs. Penicillin and other
beta-lactam antibiotics possess affinity for this catalytic site probably because of stereochemical similarity between the drug and the acyl-D-alanyl-D-alanine structure in the pentapeptide chain (Fig. 3). The amide bond in the beta-lactam ring is recognized by the PBP as similar to the D-alanyl-D-alanine bond. The PBP-penicillin interaction generally is irreversible, but some PBPs (eg, PBP 4 in Staphylococcus aureus), like beta-lactamases, can hydrolyze this bond. Penicillin-binding proteins m a y catalyze more than one reaction and, to an extent, function interO
Figure 3. Stereochemical representations
of penicillin (left) and acyl-D-alanyl-Dalanine (right). Arrows indicate the betalactam bond of penicillin that is broken to acylate a PBP and the D-alanyl-Dalanine bond that is cleaved by a PBP. (Figure adapted from the original, refer ence 41, page 363, and reproduced here by permission of the Academic Press.)
The Anthnicrobic Newlsetter (ISSN 0738 1751) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY 10017. Printed in USA at (25 Sand Creek Rd., Albany, N.Y. 12205). Subscription price per year: $55.00. For air mail to Europe, add $21.00; for air mail elsewhere, add $24.00. Second-class postage pending at New York, NY, and at additional mailing offices. Postmaster: Send address changes to The AIxtimicrobic Newsletter, Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY
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changeably; some PBPs, however, perform unique, essential functions. 3°'31'45 "Essential" PBPs are required for normal cell growth and are often important targets for the lethal effects of beta-lactam antibiotics. The susceptibility of bacteria to beta-lactam antibiotics roughly correlates with the affinity of PBPs for these drugs.12 Drugs that bind to essential PBPs or PBPs that trigger autolysis at therapeutically achievable concentrations are potentially effective antimicrobial agents. DETECTION OF PENICILLINBINDING PROTEINS Assay systems to detect PBPs are based upon the specificity and stability of the covalent bond between PBPs and beta-lactam antibiotics. Radiolabelled antibiotics used in these assays bind to the PBPs and render them detectable by autoradiography, fluorography (Fig. 4), or scintillation counting. Detection of a PBP in an assay depends both upon its affinity for the radiolabelled drug and upon the number of molecules present in the cell membrane preparation. The total number of PBP molecules per cell has been estimated at 1000-2000, although there may be as few as 20 copies of a particular PBP. 31 Penicillin-binding proteins present in very small amounts, especially if of low affinity, may not be detectable in unenriched membrane preparations. Penicillin-binding proteins possessing high affinity for the label are bound and saturated at relatively low concentrations (eg, 0.011 p~g/mL) of penicillin. Low affinity PBPs may be detected less readily than high affinity PBPs unless the concentration of radiolabel used in the assay is sufficient to bind lowaffinity sites. At these higher concentrations detection still may be difficult because nonspecific binding to non-PBP proteins at times may not be easily differentiated from specific binding. Besides affinity and copy num-
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ber, several other parameters can influence the sensitivity and specificity of the PBP assay. Some PBPs rapidly hydrolyze the covalent PBP-drug bond and may not be detected unless special steps are taken to stabilize the bond (eg, lower assay temperature). Degradation, turnover, or loss of PBP from the membrane may also affect the assay, especially in whole cell systems. Membrane-associated beta-lactamases can hydrolyze the radiolabelled drug and interfere with detection of PBPs. In gram-negative organisms, the outer membrane acts as a barrier to penetration of the radiolabelled drug to PBPs, which are located at the cell membrane. If the radiolabel diffuses poorly across the outer membrane, or is inactivated by beta-lactamase located in the periplasmic space between the cell and outer membranes, PBPs may not be detected. These problems can be circumvented if the outer membrane is disrupted before the label is added, or if the label diffuses easily across the membrane and is beta-lactamase stable. Useful information such as relative affinities of PBPs for several antibiotics, morphologic effects of drug-PBP interactions, functions of PBPs, etc., can be obtained if assays are conducted in a competitive manner (Fig. 4, lane B). In competition assays samples of the cells or membranes are preincubated with "cold" (unlabelled) antibiotic at one of several concentrations. After preincubation, radiolabelled "hot" drug at a concentration that saturates PBP binding sites is added to the samples. The PBP sites not saturated at the concentration of cold drug will remain free to bind the hot drug. As the concentration of cold drug approaches saturation, the amount of hot drug binding would correspondingly diminish. This would be detected by attenuation of the protein band density in fluorographs or less counts-per-minute in the scintillation counter. Rela-
tire affinities of several drugs for a particular PBP, or several PBPs for one drug, can be compared by determination of cold drug concentrations that block uptake of hot label by the PBP, so that the PBP "disappears" (ie, is saturated by cold drug) from the fluorograph, or its band density is diminished by 50%, for example.
Figure 4. Flurograph of PBPs in a
strain of intrinsically resistant S. Lane A is the direct PBP assay. 20 ~g/mL of 3H-penicillin was added to the membrane preparation. Lane B is the competition assay of a sample from the same membrane preparation. This sample was preincubated with 10 ~g/mL of nonradioactive nafcillin before addition of the 3H-penicillin. In the direct assay PBP 2a, although present, is not seen because :adiolabelled PBP 2 runs in nearly the ~ame position. In the competition assay, the high affinity PBPs 1,2,3,4, have been saturated (and are not available to bind the 3H-penicillin) during nafcillin incubation. The lowaffinity PBP 2a sites have remained free to bind the 3H-penicillin (added at a saturating concentration) and PBP 2a now is readily visible. aureus.
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THE ANTIMICROB1C NEWSLETTER, VOI,UME 3, NUMBER 7, JULY 1986
these often are not essential for cell growth or survival. The physiologic functions of PBPs have been characterized best for E. coli. Penicillin-binding proteins are important for cell division and maintenance of cell shape. Penicillin-binding protein 1 has transpeptidase activity 7'3° ~'~" and probably is the lethal target of betaqactam antibiotics in E. coli and most gram-negative rods. Penicillin-binding protein 1 has 2 components, la and lb. Penicillinbinding protein lb, in turn, can be resolved into 2 or 3 components. 3~" Although cell lysis is clearly triggered by binding of beta-lactam antibiotics to PBP 1, which component mediates lysis is unresolved. Initiation of lysis has been attributed to binding of drug to PBP la. 7 However, PBP la apparently is not essential to cell growth, 33 and drugs that preferentially bind the lb component have a marked lytic effect compared with those that bind la. Other studies 3~"examining thermosensitive mutants of E. coli defective in PBP lb suggest that this PBP is the lytic target. Penicillin-binding protein 2 is not a lethal target of beta-lactam antibiotics. However, this PBP is important for maintenance of the characteristic rod shape in gramnegative bacteria. 3°'3~'37 Cells exposed to drugs (eg, mecillinam) that specifically bind to PBP 2 are not killed but form round rather than rod shapes. Penicillin-binding protein 3 is important for cell division and is a
preferential target of m a n y betalactam antibiotics. 73''':~j Inhibition Crosslinkage and.assembly of the of this PBP by drugs causes the cell wall is a coordinated and orformation of long filamentous dered process. As a general rule forms. This is not a lethal effect the more complicated the cell because once the drug is removed, shape (eg, rods versus cocci), the cell division and growth resumes more different PBPs are present in (ie, no postantibiotic effect). the cell (Table 1). As few as six Penicillin-binding proteins 4, 5, and as m a n y as nine PBPs have and 6 are not essential PBPs, that been found in strains of Escherichia is, mutant cells lacking one or coil, 30"32'36 although seven PBPs is more of these PBPs appear to the number most often detected. grow normally. 2' Penicillin-binding Differences in numbers are beprotein 4 has both carboxypepticause minor PBPs or PBPs of simidase 24 and transpeptidase funclar molecular weight may be detions and may be important for tectable under some assay, maturation of the cell wall. Penicilgrowth, or electrophoretic condilin-binding proteins 5 and 6 are tions and not others. By convencarboxypeptidases and their physition PBPs are labelled by number ologic role in peptidoglycan synaccording to molecular weight as thesis is not well characterized. determined by sodium dodecyl Other PBPs (PBP 7, PBP 8) are ocsulfate (SDS)-polyacrylamide gel casionally identified but their funcelectrophoresis with PBPs of hightions are not known. er molecular weights being asPenicillin-binding proteins probsigned lower numbers. Related ably also have specific physiologic PBPs of similar molecular weights functions in gram-positive COCCi. 12 are assigned a letter. For example, In S. aureus, PBPs 1 and 4 are not PBP 1 in E. colJ can be resolved by essential for cell growth. 45 HowSDS-polyacrylamide gel electroever, PBP 4 has an important role phoresis into two distinct bands, in the higher degrees of crosslinkPBP la and PBP l b . 33 age characteristic of the cell wall in Penicillin-binding proteins of staphylococci. Cells lacking this gram-negative rods are similar in PBP or grown in the presence of number, molecular weight, and cefoxitin, which has high affinity function.~2 Usually there are seven for this PBP, have 60% crosslinkage of their walls, compared with to nine PBPs ranging in molecular weights from 30 to 100 kilodaltons. the typical 90%. The essential PBPs and those with Penicillin-binding proteins 2 and 3 are essential for cell growth and specialized functions tend to be of higher molecular weight. Although survival. The exact physiologic function of these PBPs are not well lower molecular weight PBPs comdefined but PBP 2 has been impliprise the majority of total PBPs, cated as an important target mediating cell lysis. 12 TABLE 1. Penicillin-binding Proteins in Some Clinically Important Although unique functions Bacterial Species sometimes can be attributed to Range of molecular hnportant PBP targets specific PBPs, the effects of betaNo. of PBPs weights (kilodaltons) of beta-lactams lactam antibiotics in vivo are mediated through binding not to just Gram-negative rods one, but to several PBPs. "NonesEscherichia coli 7-9 40-96 1, 2, 3 sential" PBPs may be able to subPs. aeruginosa 8 35-118 1, 2, 3 stitute for the functions of physioGram-negative cocci logically more important PBPs. Neisseria gonorrheae 3 48-90 2 Gram-positive cocci Because the lethal effects of a drug Streptococcus pneumoniae 5 52-100 1, 2 may be the cumulative result of inStaphylococcus aureus 4-5 42-92 2, 3 hibition of two or more PBPs, terms such as "lethal target," and PBPs = Penicillin-binding proteins. FUNCTION OF PENICILLINBINDING PROTEINS
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"essential" PBP, can be misleading. Nevertheless, certain PBPs play a dominant role in peptidoglycan synthesis. The more active, bactericidal agents tend to have these PBPs as their targets. Discrepancies between the minimum inhibitory concentration (MIC) and concentrations that bind to these PBPs probably are due to effects on multiple PBP targets, some of which may be "nonessential." PENICILLIN-BINDING PROTEINS AS MEDIATORS OF ANTIBIOTIC RESISTANCE
Resistance to beta-lactam antibiotics is one of two types: beta-lactamase-mediated or intrinsic (Table 2). Beta-lactamase-mediated resistance is the type most often encountered clinically. This mechanism involves drug inactivation either by enzymatic hydrolysis of the amide bond in the beta-lactam ring or, possibly, by a "trapping" mechanism 38 whereby drug is irreversibly complexed with beta-lactamase in the periplasmic space of gram-negative organisms. In intrinsic resistance, drug is not inactivated. Rather, the active drug fails to reach and/or bind to the target PBPs. Intrinsic resistance due to impermeability of outer membrane potion proteins results from inadequate diffusion of drug into the periplasmic space so that not enough drug reaches the target PBPs. Intrinsic resistance also may be due to a PBP alteration that prevents bindings of drug to the active site. Alerations in target PBPs have been associated with antibiotic resistance in several pathogenic bacT A B L E 2. Types of Bacterial
Resistance to BetaLactam Antibiotics Beta-lactamase mediated Enzymatic hydrolysis "Trapping"? Intrinsic Permeability barrier Alteration in PBPs PBPs = P e n i c i l l i n - b i n d i n g p r o t e i n s . 0738-1751/86/$0.00 + 2.20
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terial species: Streptococcus pneumoniae, 46 viridans streptococci and enterococci, Neisseria gonorrhoeae, s Staphylococcus aureus, 2'16 and Haemophilus influenzae. 23 Non-beta-lactamase-mediated resistance in the pneumococcus and gonococcus is an emerging clinical problem as the prevalence of these strains is increasing, ls18 Methicillin-resistant strains of S. aureus are an important cause of serious infection in hospitals and some communities. 13,28
Neisseria gonorrhoeae The exquisite susceptibility of gonococcal isolates to penicillin, when this drug was first introduced, has been steadily declining ever since. This general trend toward penicillin resistance is reflected by an increasing percentage of strains that have MICs of 1-2 p,g/mL, values 100-fold greater than those of fully susceptible strains. This resistance, which is not beta-lactamase mediated, has required the use of higher and higher doses of penicillin to avoid treatment failure. Unlike beta-lactamase-mediated resistance, which can be overcome by use of beta-lactamase-stable drugs, high level intrinsic resistance may render the usual beta-lactam alternatives ineffective.34 Intrinsic resistance is mediated by alterations in penicillin-binding affinity of essential PBPs. s'9 Gonococci have three PBPs. Penicillinbinding protein 3 is not the lethal target of penicillin because cells continue to grow at concentrations well above those that saturate this PBP. Penicillin-binding protein 3 is not altered in resistant strains. In susceptible strains PBP 2 is an important target for the lethal action of penicillin. Intermediate levels of resistance (MICs of 0.06 to 0.5 ~g/mL) are accompanied by a stepwise decrease of affinity of PBP 2 for penicillin. 8"9 However, this lower affinity PBP 2 is saturated by some beta-lactam antibiotics at concentrations below the MIC. This suggests that a second alteration--a shift of lethal target from
PBP 2 to PBP 1--also may be important for expression of intermediate levels of resistance. Strains exhibiting the highest level of intrinsic resistance have MICs greater than 1 ~g/mL. This level of resistance is accompanied by reduced affinity of both PBP 1 and PBP 2 for penicillin. Other beta-lactam antibiotics show similar changes in MICs and PBP affinity patterns. Alterations of PBPs 1 and 2 in resistant gonococci result in alterations in cell wall product. 1° These alterations include changes in the degree of o-acetylation at the 6 position in muramic acid and alterations in crosslinkage. These changes may affect the autolytic processes in cells exposed to betalactam drugs and may play a role in mediation of resistance. Gonococcal infections not responding to appropriate doses of penicillin, particularly if reinfection is unlikely, suggest the occurrence of intrinsically resistant strains. Such strains first should be evaluated for penicillinase production. Once this has been excluded, measurement of the MIC by agar dilution method or disk diffusion test 1 is recommended to test for intrinsic resistance. Beta-lactamase negative strains with MICs of/>0.1 ~g/ mL (or a zone of inhibition of <20 mm around the penicillin disk) should be submitted to a reference laboratory for confirmation so that epidemiologic investigations may be initiated, if indicated.
Streptococcus pneumoniae As for the gonococcus, low level resistance of the pneumococcus to penicillin has become more prevalent since its introduction into clinical medicine. In 1977 high level resistance in multiply antibiotic-resistant strains of pneumococci was first reported.iS Zigelboim and Tomasz 46 showed that intrinsic resistance is mediated through alterations in PBPs. The pnuemococcus has three groups of PBPs. Groups 1 and 2 can each be resolved into two
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components, a and b. Like resistance in gonococci, intrinsic resistance in pnuemococci is associated with alterations in PBP groups 1 and 2 and not in PBP 3. H o w e v e r , the p n e u m o c o c c u s has five PBPs, compared with three in the gonococcus, so PBP alterations are more complex in the case of the pneumococcus. Zigelboim and Tomasz 4" investigated intrinsic resistance in the p n e u m o c o c c u s by first transforming a c o m p e t e n t laboratory strain with DNA from a highly resistant South African strain and then selecting for penicillin resistance in recipients. T h e y f o u n d that acquisition of resistance was orderly, stepwise, and occurred at several discrete levels. It was not possible to produce highly resistant transformants of susceptible cells in a single step. Rather, the first level transformants expressed a m o d e s t increase in MIC from 0.006 ~g/mL to 0.012-0.025 i~g/mL but no higher. Transformation to the next higher level of resistance (eg, 0.05 to 0.1 ~g/mL MICs) could only be accomplished in c o m p e t e n t transformants already expressing the next lower level of resistance (eg, MIC of 0.025 ~g/mL), and so on. Each incremental increase in the MIC was accompanied by discrete changes in PBPs. The lowest level of resistance (penicillin MIC of 0.012 ~g/mL) was accompanied only by a decrease in affinity of PBP 2a. At the next level (MIC 0.025 ~,g/mL) a new, higher molecular weight PBP, designated PBP 2a', was detected. Penicillin-binding protein 2a' had been reported in wild-type resistance p n e u m o • 25 COCCI.
At the third level of resistance (MIC = 0.05 p,g/mL) PBP 2b could no longer be detected. At an MIC = 0.4 bLg/mL, PBPs la and l b disappeared and a new, s o m e w h a t lower molecular weight PBP, PBP lc, a p p e a r e d instead. At the highest levels of resistance, PBP 2a' f o u n d in the intermediately resistant transformants also disappeared and only three
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PBPs remained; a novel PBP lc, PBP 2a (of markedly reduced affinity), and PBP 3 (unchanged). The PBP pattern of these transformants was the same as that of the highly resistant DNA d o n o r strain. Changes in PBP profiles also have been f o u n d in genetically unrelated resistant clinical isolates. H a r d w e r g e r and Tomasz 1~ studied laboratory strains selected for lowlevel penicillin resistance and found alterations in PBPs that corr e s p o n d e d to PBPs of clinical isolates expressing low-level resistance. This strongly suggests that accumulation of sequential PBP alterations acquired u n d e r pressure of penicillin selection is responsible for resistance. Thus, higher levels of resistance can be anticipated in pneumococci as a result of the continued selective pressure exerted by penicillins and other beta-lactam antibiotics. Intrinsic resistance in p n e u m o cocci can be readily screened for by a modified Kirby-Bauer method.~ A 1 ~g oxacillin disk is placed on a blood agar surface. A zone of <20 m m after incubation in 5% CO2, 37°C overnight indicates intrinsic resistance. A broth or agar dilution MIC of ~>0.1 btg/mL also indicates resistance. Staphylococcus aureus
Intrinsic resistance, also termed methicillin resistance, in strains of S. aureus also has been associated with an alteration in PBPs. Susceptible strains possess four PBPs. Resistant strains possess an additional PBP, PBP 2', or PBP 2a 16 that has a molecular weight very nearly the same as PBP 2. Penicillin-binding proteins 2a has a very low affinity for beta-lactam antibiotics and may not be saturated at drug concentrations of ~>1000 ~g/mL or greater. Intrinsic resistance in strains of S. aureus differs from that found in the gonococcus and pneumococcus. In the latter two species, the MIC correlates closely with a concentration that binds the altered PBPs and the expression of resis-
© 1986 BY ELSEVIER SCIENCE PUBLISHING CO., INC.
tance is uniform t h r o u g h o u t the population of ceils, ie, the population of cells is h o m o g e n e o u s l y resistant. Some strains of S. a u r e , s are h o m o g e n e o u s , but the more c o m m o n pattern is a heterogeneous expression of resistance• 17,22 In h e t e r o g e n e o u s strains only a small fraction of the population (typically 1 cell in 10(') actually expresses high levels of resistance. The vast majority of ceils remain susceptible to beta-lactam antibiotics and are inhibited or killed at concentrations of 1-5 ~g/mL of nafcillin, for example. In addition, these h e t e r o g e n e o u s strains m a y appear susceptible in vitro to some beta-lactam antibiotics (most notably, the cephalosporins) and not others (eg, the penicillinase-resistant penicillins). Despite this apparent susceptibility, the balance of clinical and experimental data indicate that these strains are crossresistant to all beta-lactam antibiotics. H e t e r o g e n e o u s resistance poses an important practical problem for the clincial microbiology laboratory because, unless special precautions are taken, these strains may appear falsely susceptible. Various c o n d i t i o n s - - p r i o r selection in the presence of beta-lactam antibiotics, incubation at 30°C, and/or for 48 hours s u p p l e m e n t a t i o n of the media with sodium chloride or suc r o s e - e n h a n c e expression of resistance. For Kirby-Bauer m e t h o d of susceptibility testing, best results are obtained if a 4 ~g oxacillin disk is used*. 2° For microtiter method, supplementation of the media with 2 to 2.5% NaC1 is r e c o m m e n d e d . B° Either m e t h o d is associated with a low percentage of both false-resistant and false-susceptible results. One feature that distinguishes heterogeneously resistant from susceptible strains is that the resistant subpopulation in the former, once selected out by exposure to drug in agar at or slightly above *A 1.0 p,g d i s k also m a y be u s e d w i t h confidence.
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the MIC, produces progeny that are uniformly resistant and are able thereafter to grow at higher concentrations of drug. The ability of any colonies to grow, after an inoculum of 108 CFU/mL onto tryptic soy agar containing 5-10 ~g/mL of nafcillin incubated for 48 hours at 35°C, is very suggestive of intrinsic resistance. If such a colony produces progeny that can survive higher concentrations of nafcillin (eg, 10-50 p,g/mL in agar), then the strain is intrinsically resistant. Heterogeneous expression of resistance in S. aureus raises questions about the role of PBP 2a in the mediation of resistance. Penicillin-binding proteins 2a or a similar PBP has been found only in resistant strains, and there are no reports of intrinsic resistance in strains that do not produce this PBP. This is true whether the strain is homogeneous or heterogeneous in its expression of resistance. If PBP 2a mediates the resist-" ance in both homogeneous and heterogenous strains, why is it that in heterogeneous strains it fails to prevent inhibition and killing of the vast majority of cells? This discrepancy between the presence of PBP 2a and the expression of resistance suggests that some other factor is present that influences expressions of resis-
53
tance. One possibility is that PBP 2a is an inducible protein 6"27"43and that susceptible cells are those that are killed or inhibited before sufficient, protective amounts can be produced. Another possibility, and the one that seems increasingly more likely, is that some other alteration or factor is required before resistance is fully expressed. The nature of this factor is speculative at present, but good evidence suggests that the autolytic pathway is involved. 3,26 Vancomycin is the drug of choice for treatment of infections caused by intrinsically resistant staphylococci. Beta-lactam antibiotics should not be used because, despite apparent susceptibility to these drugs, treatment failures are likely to occur as the highly resistant subpopulation emerges during therapy. 4 Although the combination of an aminoglycoside and a beta-lactam antibiotic may be synergistic and bactericidal in vitro, this cannot be recommended for definitive therapy because resistance can still emerge. Other agents, such as trimethoprim-sulfamethoxazole (TMP-SMX) and rifampin are active against these strains. Trimethoprim-sulfamethoxazole has been successfully used as a single agent to treat serious infections caused by these strains. Rifampin must be used in combi-
nation with another effective drug because resistance emerges if it is used alone. Newer agents, such a teicoplanin and the quinolones are active in vitro and appear efficacious in some animal models, 5"3s but these drugs are investigational at present and their role for treatment of human infections is undefined.
1970s and in the early part of this decade, methicillin-resistant staphylococci again became a significant problem in many hospitals. As Dr. Chambers reviews, this resistance can be either heterogenous or homogenous and now appears to be due to induction of a new PBP 2'. Altered PBPs are an important mechanism of resistance in Streptococcus pneumoniae as illustrated by the South African outbreak. Penicilin-binding proteins with poor affinity for penicillin also are seen in some Neisseria gonorrhoeae. Does this fortell an end to beta-lactam
antibiotics? No, this form of resistance is important, but clearly of a much lesser magnitude than that due to beta-lactamases. It may be possible with a better understanding of and ability to isolate PBPs to construct compounds with affinity for the altered PBPs. Already some penems and carbapenems have been synthesized that can inhibit some organisms with altered PBPs. Hopefully the medicinal chemists will tackle this problem as they did the problem of beta-lactamases. H. C. NEU
CONCLUSIONS Intrinsic resistance due to alterations in the target PBPs is a clinically important cause of treatment failures. Its occurrence so far has been limited to a few bacterial species. Not surprisingly, the species that have developed this type of resistance are those for which penicillins are almost always used therapeutically. Intrinsic resistance is almost certainly a consequence of the selective pressures exerted by the use of these drugs. Further studies to delineate better the complex relationships among PBP, autolysis, and cell wall product are important to help in the search for newer, perhaps more specifically targeted, agents. Until usage patterns of beta-lactam antibiotics change, intrinsic resistance caused by alterations in PBPs will occur in more and more species and will continue to be an important clinical problem. References on back cover
Editorial C o m m e n t
The concept of altered targets as a mechanism of bacterial resistance has been well known. The altered ribosomes of gram-positive species render organisms resistant to macrolides, and altered DNA-directed RNA polymerase makes rifampin ineffective. The mechanism of resistance of Staphylococcus aureus to the beta-lactamase-stable penicillins such as methicillin and oxacillin was not well understood when the problem first appeared in the early 1960s. Methicillin-resistant staphylococci as a problem seemed to fade, but by the end of the 0738-1751/86/$0.00 + 2.20
© 1986 BY ELSEVIER SCIENCE PUBLISHING CO., INC.
ISSN 0738-1751
VOLUME 3, NUMBER 7, JULY 1986
EDITORIAL BOARD Editor
Associate Editors
DANIEL AMSTERDAM, PhD,
RONALD N. JONES, MD,
State University of New York
at Buffalo and Erie County Laboratory
CLYDE THORNSBERRY,PhD,
Kaiser-Permanente Medical Care Program
Center for Infectious Diseases, Centers for Disease Control
HAROLD C. NEU, MD,
LOWELL S. YOUNG, MD,
College of Physicians and Surgeons, Columbia University
UCLA School of Medicine
U N D E R S T A N D I N G PENICILLINBINDING PROTEINS EDITOR'S NOTE
47
H E N R Y F. C H A M B E R S
D. AMSTERDAM
Understanding PenicillinBinding Proteins 47 H.F. CHAMBERS E D I T O R I A L COMMENT
53
H.C. NEU
Editor's Note
It has been 45 years since the therapeutic efficacy of penicillin has been documented. Yet, we are still trying to understand the mode of action of this original betalactam agent. In the attempt, the role of the penicillin-binding proteins (PBPs) has been closely followed. Dr. Chambers reports on the actions of these proteins as they relate to cell division, maintenance of the organism,
ELSEVIER
University of California, San Francisco, San Francisco General Hospital, San Francisco, California
Penicillin-binding proteins (PBPs) are enzymes that catalyze the final reactions in bacterial cell wall synthesis. With few exceptions (eg, mycoplasma, some halophilic bacteria) PBPs are present in all bacteria. Penicillin-binding proteins covalently bind penicillin and related beta-lactam antibiotics at the enzymatic site. This reaction, not readily reversible under physiologic conditions, results in inhibition of wall synthesis. Cell lysis and death typically follow. Lysis is not a passive process general physiologic function, and antimicrobial resistance. Antimicrobic chemists closely study the PBPs, for it is the elucidation of their functions that can lead to the development of new drugs that will bypass problems associated with betalactamase resistance. It is of interest to note that bacterial proteins that dissociate penicillin, turn neutralizing its lethal effects, evolved from PBPs that are part of the cell wall. This
but results from cell wall degradation by an enzyme or enzymes, termed autolysis. 42 Penicillin-binding proteins themselves directly or indirectly may trigger autolytic processes. 19 Defective autolysis is responsible for tolerance, 14 a phenomenon in which growth is inhibited at low concentrations of drug, without accompanying lysis or killing. BACTERIAL CELL WALL SYNTHESIS AND STRUCTURE Several articles reviewing the synthesis and structure of bacterial cell walls have been published. 29,40,41,44 This article will limit itself to the most essential features of the cell wall assembly, particularly as they
evolutionary step---from a surface protein to a secreted protein--was a significant adaptation of bacteria in response to a lethal compound. A corollary activity of the PBPs is the effect of lysis and killing versus tolerance. The concept of tolerance has not yet been discussed in these pages. It represents the absence of killin,g by beta-lactam drugs and is more clearly associated clinically with gram-positive than gram-negative orgamsms.
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1t {E A N T I M I C R O B I C NEWSLETTER, V O L U M E 3, NUMB[¢R 7, IULY 1986
relate to PBPs. The interested reader should refer to more comprehensive reviews for further information. The bacterial cell wall lies outside the cell membrane. In grampositive bacteria the cell wall is several layers thick and is the outermost structure of the cell (if the capsule is excluded). In gram-negative bacteria the cell wall forms a single layer located between the cell membrane and the outer membrane. Although other macromolecular polymers (eg, teichoic acids) and proteins (eg, staphylococcal protein A) may sometimes be major constituents of the cell wall, the component common to all bacterial cell walls is peptidoglycan. Peptidoglycan is composed of alternating N-acetyl-glucosamine and N-acetyl-muramic acid residues (Fig. 1). A five amino acid stem peptide is present on the lactyl group of muramic acid residues. Amino acids in the stem peptide alternate between D and L stereoisomers. The amino acid composition of the stem peptide is highly conserved among all bacteria: Typically, only the amino acid at the third position varies from
N-acetylmuramic acid
HCCH3 I co I
Niacetylglucosamine
NHCOCH3
NHCOCH3
I bala Stem l peptide
I o-glu I
LiR3-x o-ala I o-ala
Figure 1. Disaccharide monomer
subunit of peptidoglycan. Ala = alanine, glu = glutamic acid, R = the variable third amino acid residue, X = the position of the amino group or peptide where crosslinkage occurs.
-- M-G I
--
-- M-G I
L-ala
I o-g lu I
L-R3--X
--
-- M-G -I
L-ala
+
I D-ala I
I o-glu 1
L-R3--X I o-alo I D-ala
O-aJa
-- M-G I
t-ala
+
PBP~ -I~
-- M-G I
L-ala
I D-glu I
~
L-R3I X
D-ala
I D-ala I PBP
@
--
+
-- M-G I
L-ala
I D-g lU I
L-R3--X I o-ala ~
®
--
k-ala
I O-g lu ~. I
L-R3--X I o-ala I o-aJa
--
/
/
I o-glu I --R3
/tl X
D-alo I D-aJa
®
Figure 2. The transpeptidase reaction catalyzed by PBPs. (~ PBP attacks the
D-alanyl-D-alanine bond to cleave off the terminal D-alanine. (~) A short-lived PBP-peptide intermediate is formed with D-a|anine at fourth position. (~) The terminal amino group at the crosslinking position (X) is donated from a second pentapeptide chain, displaces the PBP, and crosslinks the peptidoglycan. species to species. The last two amino acids of the stem peptide are always D-alanine. If present, a branch peptide of variable composition, is located at the third amino acid residue. Penicillin-binding proteins act at the D-alanyl-D-alanine bond. Penicillin-binding proteins can hydrolyze this bond (carboxypeptidase reaction), crosslink two stem peptides (transpeptidase reaction), or cleave peptide crosslinks (endopeptidase reaction). The physiologic roles of carboxypeptidase and endopeptidase functions are not well defined. These reactions may produce structural modifications that signal specialized wall sites, such as where septation and cell division occur. The transpeptidase reaction crosslinks peptidoglycan subunits to form a network imparting integrity and strength to the cell wall. The PBP replaces the terminal alanine residue from the stem peptide (Fig. 2). The penultimate alanyl group acts as an acceptor to a donor amino acid of the branch peptide, thus crosslinking the peptidoglycan subunits. The PBP regenerated is free to repeat this reaction. Penicillin inhibits this crosshnkage step of cell wall synthesis by binding covalently to the catalytic site of PBPs. Penicillin and other
beta-lactam antibiotics possess affinity for this catalytic site probably because of stereochemical similarity between the drug and the acyl-D-alanyl-D-alanine structure in the pentapeptide chain (Fig. 3). The amide bond in the beta-lactam ring is recognized by the PBP as similar to the D-alanyl-D-alanine bond. The PBP-penicillin interaction generally is irreversible, but some PBPs (eg, PBP 4 in Staphylococcus aureus), like beta-lactamases, can hydrolyze this bond. Penicillin-binding proteins m a y catalyze more than one reaction and, to an extent, function interO
Figure 3. Stereochemical representations
of penicillin (left) and acyl-D-alanyl-Dalanine (right). Arrows indicate the betalactam bond of penicillin that is broken to acylate a PBP and the D-alanyl-Dalanine bond that is cleaved by a PBP. (Figure adapted from the original, refer ence 41, page 363, and reproduced here by permission of the Academic Press.)
The Anthnicrobic Newlsetter (ISSN 0738 1751) is issued monthly in one indexed volume per year by Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY 10017. Printed in USA at (25 Sand Creek Rd., Albany, N.Y. 12205). Subscription price per year: $55.00. For air mail to Europe, add $21.00; for air mail elsewhere, add $24.00. Second-class postage pending at New York, NY, and at additional mailing offices. Postmaster: Send address changes to The AIxtimicrobic Newsletter, Elsevier Science Publishing Co., Inc., 52 Vanderbilt Avenue, New York, NY
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THE ANTIMICROBIC NEWSLETFER, VOLUME 3, NUMBER 7, JULY 1986
changeably; some PBPs, however, perform unique, essential functions. 3°'31'45 "Essential" PBPs are required for normal cell growth and are often important targets for the lethal effects of beta-lactam antibiotics. The susceptibility of bacteria to beta-lactam antibiotics roughly correlates with the affinity of PBPs for these drugs.12 Drugs that bind to essential PBPs or PBPs that trigger autolysis at therapeutically achievable concentrations are potentially effective antimicrobial agents. DETECTION OF PENICILLINBINDING PROTEINS Assay systems to detect PBPs are based upon the specificity and stability of the covalent bond between PBPs and beta-lactam antibiotics. Radiolabelled antibiotics used in these assays bind to the PBPs and render them detectable by autoradiography, fluorography (Fig. 4), or scintillation counting. Detection of a PBP in an assay depends both upon its affinity for the radiolabelled drug and upon the number of molecules present in the cell membrane preparation. The total number of PBP molecules per cell has been estimated at 1000-2000, although there may be as few as 20 copies of a particular PBP. 31 Penicillin-binding proteins present in very small amounts, especially if of low affinity, may not be detectable in unenriched membrane preparations. Penicillin-binding proteins possessing high affinity for the label are bound and saturated at relatively low concentrations (eg, 0.011 p~g/mL) of penicillin. Low affinity PBPs may be detected less readily than high affinity PBPs unless the concentration of radiolabel used in the assay is sufficient to bind lowaffinity sites. At these higher concentrations detection still may be difficult because nonspecific binding to non-PBP proteins at times may not be easily differentiated from specific binding. Besides affinity and copy num-
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ber, several other parameters can influence the sensitivity and specificity of the PBP assay. Some PBPs rapidly hydrolyze the covalent PBP-drug bond and may not be detected unless special steps are taken to stabilize the bond (eg, lower assay temperature). Degradation, turnover, or loss of PBP from the membrane may also affect the assay, especially in whole cell systems. Membrane-associated beta-lactamases can hydrolyze the radiolabelled drug and interfere with detection of PBPs. In gram-negative organisms, the outer membrane acts as a barrier to penetration of the radiolabelled drug to PBPs, which are located at the cell membrane. If the radiolabel diffuses poorly across the outer membrane, or is inactivated by beta-lactamase located in the periplasmic space between the cell and outer membranes, PBPs may not be detected. These problems can be circumvented if the outer membrane is disrupted before the label is added, or if the label diffuses easily across the membrane and is beta-lactamase stable. Useful information such as relative affinities of PBPs for several antibiotics, morphologic effects of drug-PBP interactions, functions of PBPs, etc., can be obtained if assays are conducted in a competitive manner (Fig. 4, lane B). In competition assays samples of the cells or membranes are preincubated with "cold" (unlabelled) antibiotic at one of several concentrations. After preincubation, radiolabelled "hot" drug at a concentration that saturates PBP binding sites is added to the samples. The PBP sites not saturated at the concentration of cold drug will remain free to bind the hot drug. As the concentration of cold drug approaches saturation, the amount of hot drug binding would correspondingly diminish. This would be detected by attenuation of the protein band density in fluorographs or less counts-per-minute in the scintillation counter. Rela-
tire affinities of several drugs for a particular PBP, or several PBPs for one drug, can be compared by determination of cold drug concentrations that block uptake of hot label by the PBP, so that the PBP "disappears" (ie, is saturated by cold drug) from the fluorograph, or its band density is diminished by 50%, for example.
Figure 4. Flurograph of PBPs in a
strain of intrinsically resistant S. Lane A is the direct PBP assay. 20 ~g/mL of 3H-penicillin was added to the membrane preparation. Lane B is the competition assay of a sample from the same membrane preparation. This sample was preincubated with 10 ~g/mL of nonradioactive nafcillin before addition of the 3H-penicillin. In the direct assay PBP 2a, although present, is not seen because :adiolabelled PBP 2 runs in nearly the ~ame position. In the competition assay, the high affinity PBPs 1,2,3,4, have been saturated (and are not available to bind the 3H-penicillin) during nafcillin incubation. The lowaffinity PBP 2a sites have remained free to bind the 3H-penicillin (added at a saturating concentration) and PBP 2a now is readily visible. aureus.
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THE ANTIMICROB1C NEWSLETTER, VOI,UME 3, NUMBER 7, JULY 1986
these often are not essential for cell growth or survival. The physiologic functions of PBPs have been characterized best for E. coli. Penicillin-binding proteins are important for cell division and maintenance of cell shape. Penicillin-binding protein 1 has transpeptidase activity 7'3° ~'~" and probably is the lethal target of betaqactam antibiotics in E. coli and most gram-negative rods. Penicillin-binding protein 1 has 2 components, la and lb. Penicillinbinding protein lb, in turn, can be resolved into 2 or 3 components. 3~" Although cell lysis is clearly triggered by binding of beta-lactam antibiotics to PBP 1, which component mediates lysis is unresolved. Initiation of lysis has been attributed to binding of drug to PBP la. 7 However, PBP la apparently is not essential to cell growth, 33 and drugs that preferentially bind the lb component have a marked lytic effect compared with those that bind la. Other studies 3~"examining thermosensitive mutants of E. coli defective in PBP lb suggest that this PBP is the lytic target. Penicillin-binding protein 2 is not a lethal target of beta-lactam antibiotics. However, this PBP is important for maintenance of the characteristic rod shape in gramnegative bacteria. 3°'3~'37 Cells exposed to drugs (eg, mecillinam) that specifically bind to PBP 2 are not killed but form round rather than rod shapes. Penicillin-binding protein 3 is important for cell division and is a
preferential target of m a n y betalactam antibiotics. 73''':~j Inhibition Crosslinkage and.assembly of the of this PBP by drugs causes the cell wall is a coordinated and orformation of long filamentous dered process. As a general rule forms. This is not a lethal effect the more complicated the cell because once the drug is removed, shape (eg, rods versus cocci), the cell division and growth resumes more different PBPs are present in (ie, no postantibiotic effect). the cell (Table 1). As few as six Penicillin-binding proteins 4, 5, and as m a n y as nine PBPs have and 6 are not essential PBPs, that been found in strains of Escherichia is, mutant cells lacking one or coil, 30"32'36 although seven PBPs is more of these PBPs appear to the number most often detected. grow normally. 2' Penicillin-binding Differences in numbers are beprotein 4 has both carboxypepticause minor PBPs or PBPs of simidase 24 and transpeptidase funclar molecular weight may be detions and may be important for tectable under some assay, maturation of the cell wall. Penicilgrowth, or electrophoretic condilin-binding proteins 5 and 6 are tions and not others. By convencarboxypeptidases and their physition PBPs are labelled by number ologic role in peptidoglycan synaccording to molecular weight as thesis is not well characterized. determined by sodium dodecyl Other PBPs (PBP 7, PBP 8) are ocsulfate (SDS)-polyacrylamide gel casionally identified but their funcelectrophoresis with PBPs of hightions are not known. er molecular weights being asPenicillin-binding proteins probsigned lower numbers. Related ably also have specific physiologic PBPs of similar molecular weights functions in gram-positive COCCi. 12 are assigned a letter. For example, In S. aureus, PBPs 1 and 4 are not PBP 1 in E. colJ can be resolved by essential for cell growth. 45 HowSDS-polyacrylamide gel electroever, PBP 4 has an important role phoresis into two distinct bands, in the higher degrees of crosslinkPBP la and PBP l b . 33 age characteristic of the cell wall in Penicillin-binding proteins of staphylococci. Cells lacking this gram-negative rods are similar in PBP or grown in the presence of number, molecular weight, and cefoxitin, which has high affinity function.~2 Usually there are seven for this PBP, have 60% crosslinkage of their walls, compared with to nine PBPs ranging in molecular weights from 30 to 100 kilodaltons. the typical 90%. The essential PBPs and those with Penicillin-binding proteins 2 and 3 are essential for cell growth and specialized functions tend to be of higher molecular weight. Although survival. The exact physiologic function of these PBPs are not well lower molecular weight PBPs comdefined but PBP 2 has been impliprise the majority of total PBPs, cated as an important target mediating cell lysis. 12 TABLE 1. Penicillin-binding Proteins in Some Clinically Important Although unique functions Bacterial Species sometimes can be attributed to Range of molecular hnportant PBP targets specific PBPs, the effects of betaNo. of PBPs weights (kilodaltons) of beta-lactams lactam antibiotics in vivo are mediated through binding not to just Gram-negative rods one, but to several PBPs. "NonesEscherichia coli 7-9 40-96 1, 2, 3 sential" PBPs may be able to subPs. aeruginosa 8 35-118 1, 2, 3 stitute for the functions of physioGram-negative cocci logically more important PBPs. Neisseria gonorrheae 3 48-90 2 Gram-positive cocci Because the lethal effects of a drug Streptococcus pneumoniae 5 52-100 1, 2 may be the cumulative result of inStaphylococcus aureus 4-5 42-92 2, 3 hibition of two or more PBPs, terms such as "lethal target," and PBPs = Penicillin-binding proteins. FUNCTION OF PENICILLINBINDING PROTEINS
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"essential" PBP, can be misleading. Nevertheless, certain PBPs play a dominant role in peptidoglycan synthesis. The more active, bactericidal agents tend to have these PBPs as their targets. Discrepancies between the minimum inhibitory concentration (MIC) and concentrations that bind to these PBPs probably are due to effects on multiple PBP targets, some of which may be "nonessential." PENICILLIN-BINDING PROTEINS AS MEDIATORS OF ANTIBIOTIC RESISTANCE
Resistance to beta-lactam antibiotics is one of two types: beta-lactamase-mediated or intrinsic (Table 2). Beta-lactamase-mediated resistance is the type most often encountered clinically. This mechanism involves drug inactivation either by enzymatic hydrolysis of the amide bond in the beta-lactam ring or, possibly, by a "trapping" mechanism 38 whereby drug is irreversibly complexed with beta-lactamase in the periplasmic space of gram-negative organisms. In intrinsic resistance, drug is not inactivated. Rather, the active drug fails to reach and/or bind to the target PBPs. Intrinsic resistance due to impermeability of outer membrane potion proteins results from inadequate diffusion of drug into the periplasmic space so that not enough drug reaches the target PBPs. Intrinsic resistance also may be due to a PBP alteration that prevents bindings of drug to the active site. Alerations in target PBPs have been associated with antibiotic resistance in several pathogenic bacT A B L E 2. Types of Bacterial
Resistance to BetaLactam Antibiotics Beta-lactamase mediated Enzymatic hydrolysis "Trapping"? Intrinsic Permeability barrier Alteration in PBPs PBPs = P e n i c i l l i n - b i n d i n g p r o t e i n s . 0738-1751/86/$0.00 + 2.20
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terial species: Streptococcus pneumoniae, 46 viridans streptococci and enterococci, Neisseria gonorrhoeae, s Staphylococcus aureus, 2'16 and Haemophilus influenzae. 23 Non-beta-lactamase-mediated resistance in the pneumococcus and gonococcus is an emerging clinical problem as the prevalence of these strains is increasing, ls18 Methicillin-resistant strains of S. aureus are an important cause of serious infection in hospitals and some communities. 13,28
Neisseria gonorrhoeae The exquisite susceptibility of gonococcal isolates to penicillin, when this drug was first introduced, has been steadily declining ever since. This general trend toward penicillin resistance is reflected by an increasing percentage of strains that have MICs of 1-2 p,g/mL, values 100-fold greater than those of fully susceptible strains. This resistance, which is not beta-lactamase mediated, has required the use of higher and higher doses of penicillin to avoid treatment failure. Unlike beta-lactamase-mediated resistance, which can be overcome by use of beta-lactamase-stable drugs, high level intrinsic resistance may render the usual beta-lactam alternatives ineffective.34 Intrinsic resistance is mediated by alterations in penicillin-binding affinity of essential PBPs. s'9 Gonococci have three PBPs. Penicillinbinding protein 3 is not the lethal target of penicillin because cells continue to grow at concentrations well above those that saturate this PBP. Penicillin-binding protein 3 is not altered in resistant strains. In susceptible strains PBP 2 is an important target for the lethal action of penicillin. Intermediate levels of resistance (MICs of 0.06 to 0.5 ~g/mL) are accompanied by a stepwise decrease of affinity of PBP 2 for penicillin. 8"9 However, this lower affinity PBP 2 is saturated by some beta-lactam antibiotics at concentrations below the MIC. This suggests that a second alteration--a shift of lethal target from
PBP 2 to PBP 1--also may be important for expression of intermediate levels of resistance. Strains exhibiting the highest level of intrinsic resistance have MICs greater than 1 ~g/mL. This level of resistance is accompanied by reduced affinity of both PBP 1 and PBP 2 for penicillin. Other beta-lactam antibiotics show similar changes in MICs and PBP affinity patterns. Alterations of PBPs 1 and 2 in resistant gonococci result in alterations in cell wall product. 1° These alterations include changes in the degree of o-acetylation at the 6 position in muramic acid and alterations in crosslinkage. These changes may affect the autolytic processes in cells exposed to betalactam drugs and may play a role in mediation of resistance. Gonococcal infections not responding to appropriate doses of penicillin, particularly if reinfection is unlikely, suggest the occurrence of intrinsically resistant strains. Such strains first should be evaluated for penicillinase production. Once this has been excluded, measurement of the MIC by agar dilution method or disk diffusion test 1 is recommended to test for intrinsic resistance. Beta-lactamase negative strains with MICs of/>0.1 ~g/ mL (or a zone of inhibition of <20 mm around the penicillin disk) should be submitted to a reference laboratory for confirmation so that epidemiologic investigations may be initiated, if indicated.
Streptococcus pneumoniae As for the gonococcus, low level resistance of the pneumococcus to penicillin has become more prevalent since its introduction into clinical medicine. In 1977 high level resistance in multiply antibiotic-resistant strains of pneumococci was first reported.iS Zigelboim and Tomasz 46 showed that intrinsic resistance is mediated through alterations in PBPs. The pnuemococcus has three groups of PBPs. Groups 1 and 2 can each be resolved into two
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components, a and b. Like resistance in gonococci, intrinsic resistance in pnuemococci is associated with alterations in PBP groups 1 and 2 and not in PBP 3. H o w e v e r , the p n e u m o c o c c u s has five PBPs, compared with three in the gonococcus, so PBP alterations are more complex in the case of the pneumococcus. Zigelboim and Tomasz 4" investigated intrinsic resistance in the p n e u m o c o c c u s by first transforming a c o m p e t e n t laboratory strain with DNA from a highly resistant South African strain and then selecting for penicillin resistance in recipients. T h e y f o u n d that acquisition of resistance was orderly, stepwise, and occurred at several discrete levels. It was not possible to produce highly resistant transformants of susceptible cells in a single step. Rather, the first level transformants expressed a m o d e s t increase in MIC from 0.006 ~g/mL to 0.012-0.025 i~g/mL but no higher. Transformation to the next higher level of resistance (eg, 0.05 to 0.1 ~g/mL MICs) could only be accomplished in c o m p e t e n t transformants already expressing the next lower level of resistance (eg, MIC of 0.025 ~g/mL), and so on. Each incremental increase in the MIC was accompanied by discrete changes in PBPs. The lowest level of resistance (penicillin MIC of 0.012 ~g/mL) was accompanied only by a decrease in affinity of PBP 2a. At the next level (MIC 0.025 ~,g/mL) a new, higher molecular weight PBP, designated PBP 2a', was detected. Penicillin-binding protein 2a' had been reported in wild-type resistance p n e u m o • 25 COCCI.
At the third level of resistance (MIC = 0.05 p,g/mL) PBP 2b could no longer be detected. At an MIC = 0.4 bLg/mL, PBPs la and l b disappeared and a new, s o m e w h a t lower molecular weight PBP, PBP lc, a p p e a r e d instead. At the highest levels of resistance, PBP 2a' f o u n d in the intermediately resistant transformants also disappeared and only three
THE ANTIMICROBIC NEWSLETTER, VOI,UME 3, NUMBER 7, IULY 1986
PBPs remained; a novel PBP lc, PBP 2a (of markedly reduced affinity), and PBP 3 (unchanged). The PBP pattern of these transformants was the same as that of the highly resistant DNA d o n o r strain. Changes in PBP profiles also have been f o u n d in genetically unrelated resistant clinical isolates. H a r d w e r g e r and Tomasz 1~ studied laboratory strains selected for lowlevel penicillin resistance and found alterations in PBPs that corr e s p o n d e d to PBPs of clinical isolates expressing low-level resistance. This strongly suggests that accumulation of sequential PBP alterations acquired u n d e r pressure of penicillin selection is responsible for resistance. Thus, higher levels of resistance can be anticipated in pneumococci as a result of the continued selective pressure exerted by penicillins and other beta-lactam antibiotics. Intrinsic resistance in p n e u m o cocci can be readily screened for by a modified Kirby-Bauer method.~ A 1 ~g oxacillin disk is placed on a blood agar surface. A zone of <20 m m after incubation in 5% CO2, 37°C overnight indicates intrinsic resistance. A broth or agar dilution MIC of ~>0.1 btg/mL also indicates resistance. Staphylococcus aureus
Intrinsic resistance, also termed methicillin resistance, in strains of S. aureus also has been associated with an alteration in PBPs. Susceptible strains possess four PBPs. Resistant strains possess an additional PBP, PBP 2', or PBP 2a 16 that has a molecular weight very nearly the same as PBP 2. Penicillin-binding proteins 2a has a very low affinity for beta-lactam antibiotics and may not be saturated at drug concentrations of ~>1000 ~g/mL or greater. Intrinsic resistance in strains of S. aureus differs from that found in the gonococcus and pneumococcus. In the latter two species, the MIC correlates closely with a concentration that binds the altered PBPs and the expression of resis-
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tance is uniform t h r o u g h o u t the population of ceils, ie, the population of cells is h o m o g e n e o u s l y resistant. Some strains of S. a u r e , s are h o m o g e n e o u s , but the more c o m m o n pattern is a heterogeneous expression of resistance• 17,22 In h e t e r o g e n e o u s strains only a small fraction of the population (typically 1 cell in 10(') actually expresses high levels of resistance. The vast majority of ceils remain susceptible to beta-lactam antibiotics and are inhibited or killed at concentrations of 1-5 ~g/mL of nafcillin, for example. In addition, these h e t e r o g e n e o u s strains m a y appear susceptible in vitro to some beta-lactam antibiotics (most notably, the cephalosporins) and not others (eg, the penicillinase-resistant penicillins). Despite this apparent susceptibility, the balance of clinical and experimental data indicate that these strains are crossresistant to all beta-lactam antibiotics. H e t e r o g e n e o u s resistance poses an important practical problem for the clincial microbiology laboratory because, unless special precautions are taken, these strains may appear falsely susceptible. Various c o n d i t i o n s - - p r i o r selection in the presence of beta-lactam antibiotics, incubation at 30°C, and/or for 48 hours s u p p l e m e n t a t i o n of the media with sodium chloride or suc r o s e - e n h a n c e expression of resistance. For Kirby-Bauer m e t h o d of susceptibility testing, best results are obtained if a 4 ~g oxacillin disk is used*. 2° For microtiter method, supplementation of the media with 2 to 2.5% NaC1 is r e c o m m e n d e d . B° Either m e t h o d is associated with a low percentage of both false-resistant and false-susceptible results. One feature that distinguishes heterogeneously resistant from susceptible strains is that the resistant subpopulation in the former, once selected out by exposure to drug in agar at or slightly above *A 1.0 p,g d i s k also m a y be u s e d w i t h confidence.
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THE ANTIMICROBIC NEWSLETTER, VOLUME 3, NUMBER 7, JULY 1986
the MIC, produces progeny that are uniformly resistant and are able thereafter to grow at higher concentrations of drug. The ability of any colonies to grow, after an inoculum of 108 CFU/mL onto tryptic soy agar containing 5-10 ~g/mL of nafcillin incubated for 48 hours at 35°C, is very suggestive of intrinsic resistance. If such a colony produces progeny that can survive higher concentrations of nafcillin (eg, 10-50 p,g/mL in agar), then the strain is intrinsically resistant. Heterogeneous expression of resistance in S. aureus raises questions about the role of PBP 2a in the mediation of resistance. Penicillin-binding proteins 2a or a similar PBP has been found only in resistant strains, and there are no reports of intrinsic resistance in strains that do not produce this PBP. This is true whether the strain is homogeneous or heterogeneous in its expression of resistance. If PBP 2a mediates the resist-" ance in both homogeneous and heterogenous strains, why is it that in heterogeneous strains it fails to prevent inhibition and killing of the vast majority of cells? This discrepancy between the presence of PBP 2a and the expression of resistance suggests that some other factor is present that influences expressions of resis-
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tance. One possibility is that PBP 2a is an inducible protein 6"27"43and that susceptible cells are those that are killed or inhibited before sufficient, protective amounts can be produced. Another possibility, and the one that seems increasingly more likely, is that some other alteration or factor is required before resistance is fully expressed. The nature of this factor is speculative at present, but good evidence suggests that the autolytic pathway is involved. 3,26 Vancomycin is the drug of choice for treatment of infections caused by intrinsically resistant staphylococci. Beta-lactam antibiotics should not be used because, despite apparent susceptibility to these drugs, treatment failures are likely to occur as the highly resistant subpopulation emerges during therapy. 4 Although the combination of an aminoglycoside and a beta-lactam antibiotic may be synergistic and bactericidal in vitro, this cannot be recommended for definitive therapy because resistance can still emerge. Other agents, such as trimethoprim-sulfamethoxazole (TMP-SMX) and rifampin are active against these strains. Trimethoprim-sulfamethoxazole has been successfully used as a single agent to treat serious infections caused by these strains. Rifampin must be used in combi-
nation with another effective drug because resistance emerges if it is used alone. Newer agents, such a teicoplanin and the quinolones are active in vitro and appear efficacious in some animal models, 5"3s but these drugs are investigational at present and their role for treatment of human infections is undefined.
1970s and in the early part of this decade, methicillin-resistant staphylococci again became a significant problem in many hospitals. As Dr. Chambers reviews, this resistance can be either heterogenous or homogenous and now appears to be due to induction of a new PBP 2'. Altered PBPs are an important mechanism of resistance in Streptococcus pneumoniae as illustrated by the South African outbreak. Penicilin-binding proteins with poor affinity for penicillin also are seen in some Neisseria gonorrhoeae. Does this fortell an end to beta-lactam
antibiotics? No, this form of resistance is important, but clearly of a much lesser magnitude than that due to beta-lactamases. It may be possible with a better understanding of and ability to isolate PBPs to construct compounds with affinity for the altered PBPs. Already some penems and carbapenems have been synthesized that can inhibit some organisms with altered PBPs. Hopefully the medicinal chemists will tackle this problem as they did the problem of beta-lactamases. H. C. NEU
CONCLUSIONS Intrinsic resistance due to alterations in the target PBPs is a clinically important cause of treatment failures. Its occurrence so far has been limited to a few bacterial species. Not surprisingly, the species that have developed this type of resistance are those for which penicillins are almost always used therapeutically. Intrinsic resistance is almost certainly a consequence of the selective pressures exerted by the use of these drugs. Further studies to delineate better the complex relationships among PBP, autolysis, and cell wall product are important to help in the search for newer, perhaps more specifically targeted, agents. Until usage patterns of beta-lactam antibiotics change, intrinsic resistance caused by alterations in PBPs will occur in more and more species and will continue to be an important clinical problem. References on back cover
Editorial C o m m e n t
The concept of altered targets as a mechanism of bacterial resistance has been well known. The altered ribosomes of gram-positive species render organisms resistant to macrolides, and altered DNA-directed RNA polymerase makes rifampin ineffective. The mechanism of resistance of Staphylococcus aureus to the beta-lactamase-stable penicillins such as methicillin and oxacillin was not well understood when the problem first appeared in the early 1960s. Methicillin-resistant staphylococci as a problem seemed to fade, but by the end of the 0738-1751/86/$0.00 + 2.20
© 1986 BY ELSEVIER SCIENCE PUBLISHING CO., INC.