penicillin‐binding protein‐mediated resistance in pneumococci and staphylococci

S353

Penicillin-Binding Protein–Mediated Resistance in Pneumococci andStaphylococci

Henry F. Chambers Medical Service, San Francisco General Hospital, and Department ofMedicine, University of California, San Francisco

Target alteration underlies resistance to b-lactam antibiotics in both Staphylococcus species andStreptococcus pneumoniae. The penicillin-binding protein (PBP) targets in penicillin-resistant strainsof S. pneumoniae are modified, low-binding-affinity versions of the native PBPs. Multiple PBPtargets may be modified by transformation and homologous recombination with DNA from PBPgenes of viridans streptococci. The level of resistance is determined by how many and to what extenttargets are modified. In contrast, methicillin resistance in staphylococci is due to expression of PBP2a, a novel, low-affinity PBP for which there is no homologue in methicillin-susceptible strains. PBP2a is encoded by mecA, a highly conserved gene most likely acquired by a rare transposition fromStaphylococcus sciuri or a closely related ancestor. Expression of resistance can be highly variable,but this seems not to be determined by PBP modifications. Several non-PBP factors are requiredfor high-level resistance.

Resistance to b-lactam antibiotics in gram-positive cocci is tivity is mechanistically similar [1]. These enzymes catalyzethe transpeptidation reaction that cross-links the peptidoglycanmediated by production either of a b-lactamase that hydrolyzes

and inactivates drug or of an altered target or targets, that is, of the bacterial cell wall. b-lactam antibiotics, which are sub-strate analogues, covalently bind to the PBP active site serinepenicillin-binding proteins (PBPs). b-lactamase as a clinically

important resistance mechanism is almost exclusively found in and inactivate PBPs at concentrations that are about the sameas the MICs. The high-molecular-weight PBPs (mass ú60staphylococci. Staphylococcal b-lactamases are narrow-spec-

trum penicillinases with relatively poor activity against semi- kDa) are the critical antibiotic targets. In staphylococci, PBPs1, 2, and 3, which have high affinity for most b-lactam antibiot-synthetic antistaphylococcal penicillins, cephalosporins, and

carbapenems. Clinically significant methicillin resistance, this ics, are essential for cell growth and survival of susceptiblestrains. Binding of b-lactams by these PBPs is lethal [2, 3].term being most commonly used for resistance to b-lactamase–

stable antibiotics in staphylococci, is due to target alteration. Low-molecular-weight PBP 4, although it may be importantin normal cell wall synthesis and participate to a limited extentb-lactamases are not known to occur in Streptococcus spe-

cies. Staphylococcal b-lactamase has been acquired by entero- in resistance, is not considered a critical target and is dispens-able [4]. The high-molecular-weight PBPs in Streptococcuscocci, but b-lactamase–producing clinical isolates are rare. As

with methicillin resistance in staphylococci, penicillin resis- pneumoniae are PBP 1 (which can be resolved into PBPs 1aand 1b, which appear functionally interchangeable) and PBPtance in enterococci, in viridans strains of streptococci, and in

pneumococci is due to alteration of target PBPs. Whether in 2 (actually three proteins, 2a, 2b, and 2x). These are essentialand have high penicillin-binding affinity. PBP 3 is the low-methicillin-resistant staphylococci or penicillin-resistant pneu-

mococci, the new PBP target has a lower affinity for binding molecular-weight PBP in pneumococci.penicillin than does that in wild type strains. The molecularbasis for PBP alterations, however, differs between pneumo-

Penicillin Resistance in Pneumococcicocci and staphylococci.

Pneumococci are normally exquisitely sensitive to penicillin,PBPs which is lethal at concentrations õ0.1 mg/mL. Typical penicil-

lin MICs for highly resistant strains are 4–8 mg/mL. ThePBPs are membrane-bound D,D-peptidases that havethreshold MIC at which treatment failures are likely to occurevolved from serine proteases, to which their biochemical ac-is unclear because of a paucity of clinical data. Serum concen-trations averaging 10–15 mg/mL are readily achievable withhigh-dose intravenous penicillin regimens, which should pro-In part adapted with permission from Clin Microbiol Rev 1997;10:781–91.

Grant support: NIH (AI-32496). vide a comfortable margin above the MIC for all but the mostReprints or correspondence: Dr. Henry F. Chambers, Box 0811, Division resistant strains. However, at lower doses or with less activeof Infectious Diseases, San Francisco General Hospital, University of Califor-

b-lactams (table 1), the margins would be less, increasing thenia San Francisco School of Medicine, 3rd and Parnassus Aves., San Francisco,CA 94143 (chipc likelihood of treatment failure. Treatment failure can and doesitsa.ucsf.edu). occur with meningitis caused by a penicillin-resistant strain,The Journal of Infectious Diseases 1999;179(Suppl 2):S353–9 because achievable concentrations of penicillin and other b-q 1999 by the Infectious Diseases Society of America. All rights reserved.0022–1899/99/79S2–0011$02.00 lactam antibiotics in cerebrospinal fluid average only a fraction,

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S354 Chambers JID 1999;179 (Suppl 2)

Table 1. Comparison of MIC90 of several b-lactam antibiotics for tion in the normal binding without alterations of PBPs 1 or 2b.penicillin-resistant strains of Streptococcus pneumoniae. Strains with MICs ú1 mg/mL typically have more marked

reductions in binding affinity affecting all PBPs. The low-b-lactam MIC90 (mg/mL)

molecular-weight PBP 3 has been thought to play no role inresistance, because its affinity is unchanged in highly resistantPenicillin 4

Ampicillin 4 strains. However, reduced expression of PBP 3 lowers resis-Amoxicillin 4 tance, suggesting that it may play a supportive function [7].Piperacillin 4 The reason for the widely differing MICs among the variousTicarcillin 128

b-lactam antibiotics (table 1) is that specific compounds willCeftriaxone 2differ in the degree to which binding is affected by a structuralCefuroxime 8

Cefaclor ú32 change or changes in one or more PBPs.Ceftazidime ú32 From the organism’s point of view, a multiple-step pointCeftizoxime ú32 mutation approach to resistance is a laborious process that runsCefixime ú32

the risk of introducing a lethal mutation, a silent mutation,Imipenem 1or one that only marginally improves survival. A meaningfulMeropenem 2

survival advantage would require the simultaneous presence ofmultiple mutations in multiple targets. The likelihood of thisoccurring through a series of single-step mutations seems re-mote. Sequence analysis of altered PBP 2b genes from resistant5%–15%, of those in serum [5]. On the basis of animal models,

anecdotal cases, and pharmacokinetic considerations, MICs clinical isolates reveals that an alternative strategy has beenused [8]. Extensive modification of the PBP-coding frame hasú0.25 mg/mL are likely to be associated with penicillin treat-

ment failure. Since Ç25% of S. pneumoniae isolates in the occurred in a way that cannot be explained by serial accumula-tion of point mutations (figure 2). For example, PBP 2b fromUnited States exhibit intermediate (0.1 mg/mL £ MIC £ 1

mg/mL) or high-level (MIC ú1 mg/mL) penicillin resistance, South African strains of penicillin-resistant S. pneumoniae hassubstitutions with blocks of mutant nucleotide sequence dis-the impact on clinical practice, particularly the choice of empir-

ical therapy for infections likely to be caused by S. pneumoniae, persed throughout the transpeptidase domain (figures 2, 3) [9].These mosaic genes are assembled by homologous recombina-is significant.

Penicillin resistance in pneumococci is the result of the ac- tion between pneumococcal PBPs and PBP genes from closelyrelated species of viridans streptococci (figure 4) [9, 10]. DNAquisition of series of stepwise mutations in PBP genes. Muta-

tions that reduce the amount of drug binding are selected for is introduced into the cell by transformation, a highly efficientmechanism for genetic exchange used by S. pneumoniae. Veryby b-lactam antibiotics. In the laboratory, very low-level resis-

tance to penicillin can result from a point mutation leading to large amounts of DNA and multiple loci can be exchangedquickly and efficiently. Moreover, since the gene pool for pneu-amino acid substitution within the transpeptidase domain of

the PBP. Mutations described in the PBP 2x gene of penicillin- mococci is potentially immense, including not only S. pneu-moniae but also other viridans streptococci, the possibilitiesresistant mutants of S. pneumoniae R6 are instructive [6]. Muta-

tions occurred within the penicillin-binding domain of the PBP for variability are great.2x molecule (figure 1), often in regions near one of the threepenicillin-binding motifs, SXXK (which contains the activesite serine), S(Y)XN, and K(H)T(S)G. One point mutation re-sulted in a modest increase in MIC from 0.02 to 0.16 mg/mL (table 2). A second mutation doubled that MIC to 0.32.Additional mutations in PBP 2x were associated with higherlevels of resistance, with MICs of 1.28 mg/mL. However, trans-formants with a PBP 2x double, triple, or quadruple mutant allhad MICs of 0.32 mg/mL. It has been shown that higher levelsof resistance require mutations within other PBP genes (andperhaps non-PBP genes, as well) in addition to the PBP 2xmutations. The more extensively PBPs are altered, both instructure and in number of distinct protein species, the higherthe MIC (table 3). Penicillin MICs are õ0.1 mg/mL in strains

Figure 1. PBP 2x transpeptidase domains from cefotaxime-selectedexpressing wild type PBPs 1 and 2, which have high-affinitymutants of Streptococcus pneumoniae R6. Locations of penicillin-

penicillin binding. Strains with intermediate MICs show low- binding motifs STMK, SSN, and KSG and amino acid residues thataffinity binding of some but not all PBPs. Such a strain, for are altered in low- and zero-binding-affinity mutants are shown (see

[6]).example, might have PBP 2a and 2x showing a modest reduc-


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S355JID 1999;179 (Suppl 2) PBPs and Resistance

Table 2. Point mutations and corresponding MICs in PBP 2x mutants selected for penicillin resistanceby serial passage of Streptococcus pneumoniae R6 in cefotaxime.

Amino acid residue (nucleotide point mutation)No. of MIC, mg/mLmutations 289 422 597 601 (MIC of transformant)

0 Met Gly Gly Gly 0.021 Met Gly Gly Val 0.16 (0.16)

(GGA r GTA)2 Met Gly Asp Val 0.32 (0.32)

(GGT r GAT)3 Met Gly Asp Val 0.64 (0.32)4 Met Gly Asp Asp 0.64 (0.32)5 Thr Gly Asp Val 1.28 (0.32)

(ATG r ACG)6 Thr Asp Asp Val 1.28 (0.32)

(GGT r GAT)

NOTE. See [6].

Resistance comes at a price biochemically [11]. PBP modi- Much has been learned concerning the genetics of methicillinresistance. Although a single gene determines the trait, numer-fications probably require a supportive biochemical infrastruc-

ture [12]. If resistance alone were sufficient for clinical success, ous other elements are also involved in expression of the methi-cillin-resistance phenotype. The important elements are de-one would have difficulty explaining the remarkable clonality

displayed by penicillin-resistant clinical isolates. Other viru- scribed below (for a more comprehensive review, see [17]).mec DNA. The methicillin-resistance determinant, mecA,lence or compensatory mutations are almost certainly required

to account for the clonality of the penicillin-resistant strains of encodes PBP 2a, a novel low-affinity PBP. mecA is locatedwithin Ç30–50 kb of additional chromosomal DNA, mec, notpneumococci that have spread worldwide.present in susceptible strains [18]. mec is always found nearthe pur-nov-his gene cluster on the S. aureus chromosomeMethicillin Resistance in Staphylococci[19] (figure 5). Two regulatory elements, mecI and mecR1,

Initially regarded as a laboratory curiosity when first de- controlling mecA transcription are upstream from mecA. Therescribed in 1961 [13], methicillin-resistant strains of Staphylo- is no mecA homologue in susceptible strains. The gene product,coccus aureus quickly proved to be able pathogens [14]. During PBP 2a (also termed PBP 2*), is an inducible 76-kDa penicillin-the last 30 years, the epidemiology of methicillin-resistant binding protein (PBP). PBP 2a has low affinity for binding b-staphylococci has shifted from that of a primarily nosocomial lactam antibiotics [20] and apparently can substitute for theorganism acquired in tertiary care centers. Methicillin-resistant essential functions of high-affinity PBPs at otherwise lethalstrains have made their way into the community and are found concentrations of antibiotic. mecA is highly conserved amongthroughout hospitals in the United States and Europe [15, 16]. staphylococcal species [21–24]. The mecA promoter region,About one-quarter of S. aureus strains isolated in clinical labo- the first 300 nucleotides of mecA, and its regulatory genes areratories of hospitals in the United States are resistant to methi- similar in sequence to the analogous regions of the staphylococ-cillin. cal b-lactamase [25, 26]. A homologue with 88% amino acid

similarity to mecA of methicillin-resistant staphylococci has

Table 3. Relationship between penicillin MIC (mg/mL) and bindingaffinity of pneumococcal PBPs.

PBP

MIC 1a/1b 2a/2b 2x

õ0.1 / / /0.1–1.0 { { {ú1.0 0 0 0

Figure 2. Extensive nucleotide and amino acid residue changes forsame coding region of PBP 2b in susceptible strain of StreptococcusNOTE. /, high-affinity-binding PBP;{, slight decrease in binding affinitypneumoniae (top sequences) compared with resistant strain (bottom)for some but not all PBP species; 0, low-affinity binding for most or all PBP

species. (see [8]).


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pressure of b-lactam antibiotics for mutants lacking strongrepressor activity, so that a sufficient amount of PBP 2a isproduced to offer a survival advantage.

mec-associated DNA. mec consists of 25–50 kb of addi-tional DNA that may contain up to 100 open-reading frames[18, 41]. Transposons and insertion sequences are present.There can be considerable variability within mec due to dele-tion, rearrangement, and recombination events. IS431, an ex-tremely common insertion sequence in staphylococcal chromo-some and plasmids, is present within mec. It has been associatedwith numerous resistance determinants, including mercury,cadmium, and tetracycline [31, 42]. The aadD gene, for exam-ple, which encodes an enzyme for tobramycin resistance, isFigure 3. Comparison of mosaic PBP 2b genes from 2 types (Alocated on a plasmid integrated into mec-associated DNAand B) of penicillin-resistant pneumococci to wild type PBP 2b se-

quence (top). Transpeptidase domain of protein is indicated by hori- within IS431 (figure 5) [43]. IS431 elements probably servezontal arrow. Vertical arrow indicates location of SXXK motif. Open as a trap for resistance determinants with similar IS elements,regions indicate wild type sequence. Shaded boxes indicate regions thereby accounting for the multiple drug resistance phenotypecontaining sequences altered from wild type, but derived from related

typical of methicillin-resistant staphylococci.donor. Solid region indicates non–wild type sequence from anotherHeterogeneous resistance. Methicillin resistance is hetero-donor (see [9]).

geneous [44, 45]. The level of resistance varies with cultureconditions and the b-lactam antibiotic being used. The majorityof cells (i.e., §99.9%) are susceptible to b-lactam antibioticbeen identified in Staphylococcus sciuri [27]. mecA probably

originated in a coagulase-negative Staphylococcus species, per- concentrations in the range of 1–10 mg/mL. A small subpopu-lation (e.g., 1 cell in 106) can grow at concentrations §50 mg/haps a close evolutionary relative of S. sciuri [21, 28]. All

methicillin-resistant strains of S. aureus are clonal descendants mL. Heterogeneous strains can, however, appear homogeneous(i.e.,§1% of cells grow at 50 mg/mL methicillin) in hypertonicfrom the few ancestral strains that acquired mecA [29]. How

mecA was acquired by methicillin-resistant staphylococci is not culture medium supplemented with NaCl or sucrose, or onincubation at 307C [46]. Addition of EDTA, pH 5.2, or incuba-known, but transposition is a plausible mechanism [30, 31].

mecI and mecR1. mecI and mecR1 are divergently tran- tion at 437C may suppress resistance entirely. These changesare phenotypic. Passage of a heterogeneous strain in a b-lactamscribed regulatory genes located immediately upstream from

the mecA promoter [32, 33]. They resemble the staphylococcal antibiotic also alters the resistance phenotype by selecting forhighly resistant mutant clones. The phenotype tends to be un-b-lactamase regulatory elements, blaI and blaR1 [34]. mecI

and blaI encode repressors of mecA and b-lactamase gene stable, although some isolates, the COL strain of S. aureusbeing most extensively studied, and laboratory-derived mutantstranscription. mecR1 and blaR1 encode signal-transducing

PBPs that in the presence of b-lactam antibiotic lead to mecA remain homogeneous despite repeated subculture.Numerous genes affect the methicillin-resistance phenotype.and b-lactamase gene transcription, although the precise mech-

anism by which this occurs is not known. Strains isolated before b-lactamase regulatory genes may affect expression of resis-tance [47, 48] by exerting control over mecA transcription.1970 generally have deletions of the penicillin-binding domain

of mecR1 and the complete downstream mecI [21, 35]. PBP The best-characterized of the factors are the so-called fem (forfactors essential for methicillin resistance) or aux (auxiliary)2a production in these isolates is constitutive, unless the strain

contains an inducible b-lactamase, the regulatory genes for factors. These are chromosomal loci physically distinct fromwhich will also regulate mecA expression [36, 37].

Strains isolated since 1980 tend not to have regulatory genedeletions but have mecI polymorphisms and mecA promotermutations [18]. It has been hypothesized that intact mec regula-tory genes may overly strongly repress production of PBP 2a[38, 39] and render the cell susceptible to methicillin. PBP2a in such strains is poorly inducible, and they can appearmethicillin-susceptible. Elements other than mecI-mecR1 alsodetermine resistance phenotype [40]. Provided that some PBP2a is produced, amounts of PBP 2a and whether its productionis constitutive or inducible do not correlate with the level of Figure 4. Scheme whereby mosaic genes are generated by sequen-resistance expressed. Nevertheless, the mecI polymorphisms tial cross-over and recombination events occurring between donor

and wild type DNA (shaded and open regions, respectively).and mecA promoter mutations probably reflect the selective


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Figure 5. Organization of Ç50 kb mec region andits chromosomal location relative to fem factors andpur-nov-his. IS indicates insertion element IS431(same as IS257) flanking tobramycin resistance plas-mid pUB110. Tn554 is transposon with ermA encod-ing inducible erythromycin resistance (reprinted from[17] with permission).

mec that are required for full expression of resistance (figure Inactivation of llm, which encodes a 38-kDa hydrophobicprotein of unknown function, in a homogeneous methicillin-5) [49–51]. fem factors are present in both susceptible and

resistant strains. Six fem genes, femA, femB, femC, femD, femE, resistant strain produced a heterogeneous pattern of resistance[56]. agr (accessory gene regulator) and sar (staphylococcaland femF, which map to sites throughout the staphylococcal

genome, have been characterized. accessory regulator) are involved in controlling expression offem mutants have altered peptidoglycan metabolism (figure

6). The femAB operon encodes two proteins required for forma-tion of the pentaglycine interpeptide bridge that serves as thecross-link of peptidoglycan [52]. femA adds the second andpossibly third glycine, and femB adds the fourth and perhapsfifth glycine to the cross-bridge. Disruption of these genesreduces the level of resistance to nearly susceptible levels.femC has been mapped to the open-reading frame that encodesa glutamine synthetase repressor gene, glnR [53]. Disruption ofglnR has a polar effect on the adjacent downstream glutaminesynthetase gene, glnA, resulting in a block in glutamine produc-tion. Glutamine is the NH4

/ donor for amidation of glutamate,the third amino acid residue of peptidoglycan pentapeptide.The cell wall product has reduced peptidoglycan cross-linkingand a heterogeneous pattern of resistance. femD inactivationresults in disappearance of unsubstituted disaccharide penta-peptide monomer from the cell wall [54]. Inactivation of femF

Figure 6. Sites of peptidoglycan precursor synthesis at whichcauses a block in peptidoglycan precursor synthesis at the ly- blocks occur in fem mutants. UDP-Mur, uridine-diphospho-muramyl-sine addition step and heterogeneous expression of resistance peptide precursor; NAG-NAM, N-acetylglucosamine-N-acetylmu-

ramic acid disaccharide (reprinted from [17] with permission).[55].


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