Journal of Chemotherapy Vol. 15 - n. 6 (525-535) - 2003

© E.S.I.F.T. srl - Firenze ISSN 1120-009X

INTRODUCTION

The β-lactam family of antimicrobials is the cor-nerstone treatment for community-acquired infec-tions. However the increase in the incidence ofGram-negative and -positive pathogens resistant toexisting therapies, in particular Streptococcus pneu-moniae, Haemophilus influenzae, and Moraxellacatarrhalis, has a profound impact on the manage-ment of disease. Microbial resistance to β-lactamscan be expressed in four ways (Figure 1): hydrolysisof the β-lactam ring by microbial β-lactamase (BLA)production; conformational alterations of the β-lac-tam target enzymes, penicillin-binding proteins(PBPs); reduced permeability of the bacterial outermembrane to β-lactams via the down-regulation oftransmembrane pore proteins; and increased β-lac-tam efflux from the bacterial cell. In addition, theissue of cross-resistance should be heeded alongsideconsiderations related to membrane permeabilityand efflux. Of all of these mechanisms, the evolution

and spread of BLAs has been clinically the mostimportant and has driven the development of new β-lactam compounds.

b-LACTAMS - AN OVERVIEW

The β-lactams are a structurally diverse group ofantimicrobials active against Gram-positive and -neg-ative pathogens. To date, four major classes havedominated those available for clinical use: thepenams (penicillins); cephems (cephalosporins);clavams (BLA inhibitors); and carbapenems. As afamily, these classes are based on either saturated orunsaturated, 5- or 6-membered heterocyclic ringsfused to a 4-membered β-lactam ring (Figure 2). Inaddition, the atom sited at position 1 is a key char-acteristic of the class and may be carbon, oxygen orsulfur. The penams or penicillins are derivatives of6-amino-penicillanic acid and have sulfur at position1 of an unsaturated 5-membered thiazolidine ring.

Resistance to bb-Lactams - The Permutations

S.G.B. AMYES

Medical Microbiology, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, UK. Tel: +44 (0)131 650 3163; Fax: +44 (0)131 651 1385

SummaryThe bb- lactam family of antimicrobials, in particular penicil l ins and

cephalosporins, is the mainstay of treatment for community-acquired infections.However, the emergence of resistant isolates to these agents has raised concernsregarding the continued efficacy of existing therapies. Resistance to bb-lactams ismost commonly expressed by the microbial production of bb-lactamases thathydrolyze the bb-lactam ring. Three further resistance mechanisms include conforma-tional changes in penicillin-binding proteins (PBPs); permeability changes in theouter membrane; and active efflux of the antimicrobial. In addition to the pre-requi-site efficacy and tolerability profiles, new bb-lactams should address these four resis-tance mechanisms. Overcoming resistance may be a serendipitous event or arrivedat by design. A unique synthetic bb-lactam class, which demonstrates promise interms of its activity against the range of bacteria responsible for community-acquired infections and its inherent stability to hydrolysis by bb-lactamases, is thepenems. This discrete class of hybrid molecules combines properties from the peni-cillin (penam) and cephalosporin (cephem) bb-lactam classes. Faropenem is an exam-ple of a penem with a broad spectrum of activity designed to address these resis-tance issues.

Key words: Antimicrobial resistance, bb-lactam, penems.

REPRINT

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526 S.G.B. AMYES

FIGURE 1 - Mechanisms ofresistance to β-lactams (afterEliopoulos29).

FIGURE 2 - Representativemembers from the major β-lactam classes.

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RESISTANCE TO b-LACTAMS - THE PERMUTATIONS 527

Members of this class can be categorized as naturalpenicillins, isoxazolylpenicillins (penicillinase-resistantpenicillins), amidinopenicillins, or the extended-spec-trum penicillins (aminopenicillins, ureidopenicillins)1,2. The latter group is of particular interest since itsmembers possess potent activity against a widerange of pathogens as either individual agents orwhen administered in combination with BLAinhibitors as described. Group members includeampicillin, amoxicillin (aminopenicillin), ticarcillin(carboxypenicillin), mezlocillin and piperacillin (urei-dopenicillin).

The cephalosporins are derivatives of 7-aminocephalosporinic acid, with a sulfur atom atposition 1 of the 6-membered, dihydrothiazine hete-rocyclic ring 3. Cephaloridine, cefuroxime, cef-tazidime and cefpirome (cefepime) are examples off irst, second, third and fourth generationcephalosporins. Later generation cephalosporinspossess increasingly complex modifications of theside chain at C7 or substituents at C3 of the mainring.

Clavams such as clavulanic acid are inhibitors ofβ-lactamases with negligible antimicrobial activity,and they have an oxygen atom located at position 1of the heterocyclic ring. Monobactams representedby aztreonam are based upon the monocyclic natu-ral ly occurring 4-membered β- lactam ring 4.Although aztreonam has negligible activity againstGram-positive bacteria, it expresses good activityagainst Gram-negative aerobic bacteria includingPseudomonas aeruginosa.

The carbapenems, such as imipenem, meropen-em, ertapenem and panipenem, have unsaturatedheterocyclic 5-membered rings with a carbon atomlocated at position 1. They demonstrate potent

activity against a wide spectrum of Gram-negativeand -positive, aerobic and anaerobic bacterialpathogens. They maintain activity against Gram-neg-ative bacteria that produce extended-spectrum β-lac-tamases (ESBLs).

The structural variations defining each class ofthe β-lactam family are possibly driven by the envi-ronmental evolution of these naturally-occurringcompounds 5. The β-lactam ring itself providesantibacterial activity and may sterically protect theantimicrobial from hydrolysis by BLA. Modificationof the β-lactam ring via the addition of side chainscan extend the spectrum of antimicrobial activityand/or increase the penetration properties of theantimicrobial, or its binding affinities to PBPs.Despite these structural permutations designed tooutmaneuver bacterial drug resistance mechanisms,resistant isolates continue to be identified.

EXPRESSION OF ANTIMICROBIAL RESISTANCETO CURRENT THERAPIES

Antimicrobial resistance may arise spontaneouslyvia genetic mutation and/or gene transfer. β-lactamantimicrobials are agents that interfere with the syn-thesis of the bacterial cell wall as a result of theirinteraction with PBPs. The structure of the cell walldiffers in Gram-positive and -negative bacteria.Gram-positive bacteria possess a thick peptidoglycanlayer but Gram-negative bacteria have a relativelythin peptidoglycan layer within an outer membrane.β-lactams penetrating the outer membrane via chan-nels (porins) can be inactivated before reaching tar-get PBPs by BLAs released into the periplasmicspace surrounding the inner membrane, as well asextracellularly (Figure 3) 6. Of all the resistance

FIGURE 3 - β- lactamaseinduction model (modifiedafter Wiedemann 6).

528 S.G.B. AMYES

mechanisms arising in response to treatment withantimicrobials, the most common is BLA produc-tion.

b-lactamases (BLAs)BLAs can be grouped on the basis of either their

molecular structure or function. Despite the largeand still growing number of BLAs they can begrouped into only four molecular classes A, B, Cand D. Class A BLAs, including Staphylococcusaureus penicillinase, have a serine residue at theiractive site (serine-based enzymes), whereas class BBLAs are metalloenzymes (e.g. Bacillus cereusBLA). Class C BLAs are cephalosporinases andclass D are oxacillin hydrolyzing enzymes and bothare serine-based enzymes.

The functional classification scheme proposed byBush et al. also comprises four major groups (Table1) 7. Group 1 comprises the cephalosporinases thatare not inhibited by the BLA inhibitor clavulanate;group 2, the penicillinases that are inhibited byclavulanate; group 3, the metallo-β-lactamases (mol-ecular class B) that hydrolyze members from everyknown class of β-lactams with the exception ofmonobactams; and group 4, the penicillinases thatare not inhibited by clavulanate. As reported byBush, the number of unique BLAs in the publishedliterature had risen from approximately 178enzymes in 1995 to approximately 340 enzymes in2000 (Figure 4) 8. Of the four groups of enzymesidentified, the greatest increase was observed in thegroup 2 serine-based BLAs.

Group 1 BLAs are produced by most species ofEnterobacteriaceae and P. aeruginosa 7,8. Theseenzymes are chromosomally mediated cephalospori-nases and are insensitive to inhibition by clavulanicacid; representatives include the chromosomalAmpC BLAs. Genes from these chromosomalAmpC loci may have escaped to plasmids that havereached some species of enterobacteria 9. AmpCBLAs slowly hydrolyze the β-lactam ring and, cou-pled with reduced uptake or up-regulated efflux,contribute to resistance expressed by pathogens 10.

Usually, group 1 BLAs are made at trace levels;elevated BLA production arises transiently, viainduction, but hyperproducers arise by mutation 8.Group 1 BLAs hydrolyze first-, second-, third-gener-ation cephalosporins and monobactams. However,only the first-generation cephalosporins are stronginducers of group 1 BLAs so that they are rapidlyhydrolyzed and, consequently their MICs are highand clinical treatment fails. In contrast, second- andthird- generation cephalosporins and ureidopeni-cillins are poor inducers - unless a secondary inducerlike imipenem or cefoxitin is present - so that theirBLA lability is not reflected in resistance. However,these agents are highly selective for stably dere-pressed mutants overproducing AmpC BLAs.

Group 2 BLAs that include the common TEM-1BLAs (group 2b) are produced by Escherichia coli

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RESISTANCE TO b-LACTAMS - THE PERMUTATIONS 529

or H. influenzae. New types of group 2 BLAs suchas extended spectrum β-lactamases (ESBLs) havearisen. Most ESBLs are mutants of the classicalgroup 2b BLAs like TEM-1, -2 and SHV-1 enzymes.These mutations confer resistance to expandedspectrum cephalosporins and aztreonam. In contrastto group 1 AmpC BLAs, most group 2 ESBLs areinhibited by clavulanate (e.g. TEM-3 to TEM-26,belonging to group 2be) while TEM-30 to TEM-36(group 2br) do not bind clavulanate with high affini-ty, so that enzyme inhibition is only moderate 7,8.

FIGURE 4 - Number of unique BLAs in the publishedliterature.8

Group 2d β-lactamases, which include the OXAenzymes (so-called because of their ability tohydrolyze oxazolyl penicillins), can also be defined asESBLs. These enzymes confer resistance to a widerange of β-lactams, in particular to ceftazidime, withall OXA β-lactamases except OXA-17 hydrolyzingthis molecule. With the exception of OXA-18,OXAs are generally insensitive to inhibition withclavulanate 11. In a recent study, the penem faropen-em was found to be insensitive to hydrolysis withthese enzymes 12-15.

Although to date ESBL producers are predomi-nantly isolated from hospitalized patients, the wide-spread prevalence of the progenitor enzyme TEM-1in community pathogens may lead to a wider spreadof ESBL producers in the community.

Group 3 BLAs, molecular class B zinc metal-loenzymes, can hydrolyze carbapenems and they arefound in Stenotrophomonas maltophilia, P. aerug-

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inosa, Bacteroides spp. and Actinobacter bauman-nii 10,16. All metallo-BLAs utilize carbapenems assubstrates but there is much variability among indi-vidual profiles. A number of plasmid-mediated vari-ants of carbapenemases have been identified fromJapan 17 but, as pointed out by Bush, these enzymesare likely to have arisen in response to thienamycin,a natural product of Streptomyces cattelya, beforethe discovery of imipenem 18. One imipenem-hydrolyzing BLA, carried on a plasmid with multipleantibiotic resistance genes for gentamicin and sulfon-amides, has been identified in P. aeruginosa 19.Group 4 penicillinases are rare.

Efflux mechanismsActive efflux of toxic compounds from cells is a

general mechanism that bacteria have developed toprotect themselves against the adverse effects oftheir environment and the chemicals and antimicro-bials used in clinical settings are among these toxiccompounds. This protection mechanism occurs inboth bacteria and the eukaryotes. In bacteria, antibi-otics are expelled from the cells by membrane trans-porter proteins, the so-called drug-efflux pumps. Ofparticular interest are efflux pumps capable ofextruding a large variety of structurally unrelatedcompounds with different antibacterial modes ofaction. Thus, efflux pumps may mediate multi-drugresistance, which may affect different drug classes.On the other hand, exposure of a bacterium to agiven antibacterial agent may result in resistance toa structurally unrelated compound to which the bac-terium has never been exposed before.

Most of the genes encoding these multidrugresistance (MDR) pumps are normal constituents ofbacterial chromosomes. Some of these genes have arelatively high level of constitutive expression andconfer so-called intrinsic resistance to antibiotics.Other genes conferring an efflux capability are notdetected in wild-type cells, but such genes canbecome expressed after the acquisition of regulatorymutations.

At least four classes of MDR pumps have beenidentified on the basis of sequence comparisons:• the small multi-drug resistance (SMR) family, ini-

tially observed in staphylococci• the resistance-nodulation division (RND) family• the major facilitator superfamily (MFS)• the multi-drug adenosine triphosphate binding cas-

sette (ABC) transporters.

In Gram-negative bacteria, most of the effluxpumps that contribute to both intrinsic and acquiredresistance to clinically useful antibiotics are three-component structures that traverse both inner mem-branes and outer membranes. Each pump containsa transporter located in the cytoplasmic membrane,an outer membrane channel, and a periplasmic link-er protein, which is thought to bring the other two

530 S.G.B. AMYES

components into contact. This structural organiza-tion allows extrusion of substrates directly into theexternal medium, bypassing the periplasm.

P. aeruginosa is an important opportunisticpathogen that is highly resistant to antimicrobialtherapy, although fluoroquinolones, β-lactams, andaminoglycosides are among the primary agentsavailable for the treatment of infections caused bythis pathogen.

Four multicomponent MDR RND efflux pumpshave been identified in P. aeruginosa:• MexAB-OprM (Opr, outer membrane porin) has

the broadest substrate-specificity of all bacterialefflux pumps described so far and extrudes β-lac-tams, BLA inhibitors, quinolones, chlorampheni-col, tetracycline, sulfamethoxazole, and novobiocin

• MexCD-OprJ extrudes quinolones, and fourth gen-eration cephems but not conventional β-lactams(or carbacephems), and macrolides

• MexEFOprN does not contribute to β-lactam effluxbut its overexpression concomitantly reduces thecarbapenem-specific OprD porin through which,for example, imipenem is transported.Furthermore, i t extrudes chloramphenicol,trimethoprim, and quinolones.

• MexXY is responsible for the efflux of quinolones,tetracycline, and erythromycin, and is involved inthe intrinsic resistance to aminoglycosides.

Although the first three efflux systems extrudequinolones in general, the manner in which theiroverexpression affects quinolones varies. Conversely,different quinolones may select resistant subpopula-tions overexpressing these systems in different ways.Quinolones with a fluorine at position 6 and a posi-tively charged substituent at position 7 tend to selectMexCDOprJ mutants, whereas quinolones lacking apositively charged substituent at position 7 selectMexABOprM mutants. Consequently, resistance pat-terns to chemically unrelated drug classes are notthe same. These examples demonstrate that con-geners of a given drug class differ in the extent towhich they are affected by each system based ontheir chemical structure.

Analogous conclusions can be drawn for otherdrug classes. For example, overexpression of theMexABOprM efflux system in nal B mutantsincreases resistance to meropenem, but not toimipenem, which is not a substrate for theMexABOprM pump.

Permeability barriers Transmembrane pores, created by the outer

membrane proteins of Gram-negative bacteria, pro-vide the means by which many β-lactams penetratethe bacterial cell. Mutant permeability isolates of theEnterobacteriaceae appear to have reduced quanti-ties of the outer membrane protein OmpF; otherproteins include OmpC. The accompanying resis-

tance to other antimicrobials such as aminoglyco-sides, trimethoprim and tetracyclines may possiblyreflect pleiotropic mutations, not a deficiency inporin alone, demonstrating the interplay between allpossible resistance mechanisms 20,21. This resistanceroute is employed by pathogens such as P. aerugi-nosa against the carbapenems, in particular imipen-em. Imipenem is prevented from entering the cellbecause of the loss or down-regulation of the pro-tein OprD 22,23. Although the overexpression of theMexEF-OprN efflux pump is associated with adecrease in OprD expression, imipenem is taken up10 times faster than the carbapenem meropenem.

Target proteinsThe primary targets of β-lactams are PBPs,

which vary in number and size according to bacterialspecies. PBPs, located on bacterial membranes, areinvolved in the cross-linking of the cell wall as it issynthesized and they catalyze the transpeptidationreaction between two peptidoglycan strands 24,25. β-lactams bind to PBPs with varying degrees of affinityand, since the molecular backbone of a β-lactammimics the topology of the acyl-D-alanyl-D-alaninesection of a bacterial peptidoglycan strand, the β-lac-tam sterically hinders the approach of a secondstrand 26. Transpeptidation is stalled, cell growth isinhibited culminating in cell death and lysis; theremay be accompanying morphologic changes 27. β-lactam-resistant isolates of S. pneumoniae are nowmore prevalent as PBPs have evolved with alteredtarget binding sites as revealed by changes in elec-trophoretic patterns and reduced binding affinities.These changes may have arisen through the acquisi-tion of novel PBPs resulting from the formation ofmosaic genes incorporating DNA from other bacter-ial species 27. Similarly, Gram-negative bacteria suchas H. influenzae possess modified PBPs, althoughin this instance β-lactam resistance may also resultfrom co-existing permeability barriers and BLA pro-duction 24,28,29.

PBPs are responsible for the assembly, mainte-nance and regulation of the cell wall structure. Theyare multifunctional and act as space makers for theinsertion of new peptidoglycan material, as “wallzippers” during cell division, and as catalysts in therecycling of old peptidoglycan. A β-lactam mustinhibit low molecular mass PBPs (carboxypeptidases)as well as the essential high molecular mass PBPs inorder to act as a strong BLA inducer. Thus, an idealβ-lactam should have a high affinity for high molecu-lar mass PBPs but low affinity for low molecularmass PBPs.

Resistance in community-acquired infectionsThe scale of the problem associated with antimi-

crobial resistance among key pathogens responsiblefor community-acquired infections is alarming sincethe clinical utility of current therapies is compro-

RESISTANCE TO b-LACTAMS - THE PERMUTATIONS 531

mised by the emergence of resistant pathogens. S.pneumoniae causes community-acquired meningitisand pneumonia and is also the major cause of otitismedia and sinusitis. In Europe, resistance in thisorganism to penams, such as penicillin, occurs atboth intermediate (0.1−1.0 mg/L) and high levels(≥2.0 mg/L) 30. In countries worldwide with lowresistance rates to penicillin, molecular studies haveshown the importation of penicillin-resistant clones31. S. pneumoniae isolates that were formerly sus-ceptible to penicillins and cephalosporins bind peni-cillin with lower affinity upon acquiring DNA fromother bacteria. The altered PBP, resulting from theexpression of foreign DNA, synthesizes peptidogly-can with an alternative structure 32,33. Cross-resis-tance to cephalosporins, cefotaxime and ceftriaxonecan arise in S. pneumoniae with intermediate resis-tance to penicillin as shown in surveillance studiesfrom the United States 34. Despite unrelated resis-tance mechanisms,35 resistance to penicillin may beaccompanied by macrolide resistance 36. This mayarise because mutations affecting certain gene locimay alter the levels of resistance expressed againstother antimicrobials as a consequence of the inacti-vation of efflux systems and/or the down-regulationof porins. Alternatively, a pathogen may acquire anumber of plasmids that encode resistance to multi-ple antimicrobials or a single plasmid conferringmulti-drug resistance 29.

H. influenzae is an important pathogen con-tributing to the acute bacterial exacerbations ofchronic bronchitis in patients with chronic pul-monary disease. Lamentably, resistance to ampicillinand amoxicillin among H. influenzae isolates - mostof them producing a TEM-1 enzyme − hasincreased, with levels in some countries in excess of40%; and, although rare, isolates resistant tocephalosporins do occur. Current therapies includethe administration of β- lactam + β- lactamaseinhibitors, that is amoxicillin or ampicillin in combi-nation with clavulanate or sulbactam, respectively.However, there are shortcomings associated withthe dispensation of a dual compound, which irre-versibly inactivates BLAs that are not chromosomal-ly-mediated enzymes or are not being produced athigh concentrations attributed to high copy-numberplasmids 37. High level production of these BLAscan induce resistance to the β-lactam + β-lactamaseinhibitor as there may be an insufficient concentra-tion of inhibitor to inactivate the increased quantitiesof enzyme. Although resistance has taken longer toemerge with β-lactam + β-lactamase inhibitors thanwith a single β-lactam agent, and affects fewer iso-lates,18 inhibitor-resistant TEM-1 derivatives havebeen identified and appear to be increasing in fre-quency, as have other novel clavulanic acid resistantenzymes such as VAT-1 38-40.

As with H. influenzae, M. catarrhalis mostcommonly has BLA-mediated ampicillin and amoxi-cillin resistance, occurring in 97% of isolates. The

constitutively-produced BLAs, BRO-1 and BRO-2,found in M. catarrhalis, are transposon-mediated,which accounts for the rapid emergence of resis-tance. BRO-1 is more prevalent than BRO-2, occur-ring in 90% and 10% of BLA-positive isolates,respectively, as revealed by restriction enzyme analy-sis for the Bcgl restriction site present only in theBRO-2 gene 41,42.

OVERCOMING RESISTANCE - NEWAPPROACHES TO THE TREATMENT

OF RESISTANT STRAINS

The impetus directing the search for new agentsarises from both the problems related to emergentantimicrobial resistance and the greater understand-ing of resistance mechanisms. Different strategiescan be employed to address the issues surroundingresistance, involving either the design of new antimi-crobials or the improvement of existing classes sincethe likelihood of discovering a new type of β-lactamwith an, as yet, unknown structure is a remote andprobably serendipitous event. No new β-lactam hasbeen developed via this route in the past decade.

A more promising strategy is the improvementof existing antimicrobial agents, extending theirspectrum of activity and overcoming resistance.Such approaches have included modification of side-chains attached to the β-lactam core molecule (seenin the development of later generationcephalosporins), or the co-administration of existingβ-lactams with β-lactamase inhibitors (e.g. amoxy-cillin plus clavulanic acid). As with all new antimicro-bials, these agents, derived from the optimization ofcurrent β-lactams, offered a long-term but imperma-nent solution to the problem of emerging drug resis-tance. Extrapolating from Bush, there may be littleactual evolution of resistance genes to new antimi-crobials within the clinical bacterial population 18.Since new antimicrobials are derived from olderantimicrobials or new natural products, it is morelikely that an existing resistance mechanism isimported from the original antimicrobial-producingmicrobe.

There are only a few new β-lactam antimicro-bials available and most belong to existing classes.The parenteral cephalosporin RWJ-54428 (OrthoMcNeil, Johnson & Johnson) 43 reportedly circum-vents BLA cleavage and cephalosporin-targeted PBPmutations but how this is achieved is unclear. Thisantimicrobial is currently undergoing phase I trialsagainst BLA- and vancomycin-resistant infections.An injectable carbapenem, ertapenem sodium(Merck & Co) has been launched for the followingindications: community-acquired pneumonia (peni-cillin-susceptible S. pneumoniae, BLA-negative H.influenzae, and M catarrhalis strains only); skin andskin structure infections (methicillin-susceptible S.aureus); genito-urinary tract, complicated intra-

532 S.G.B. AMYES

abdominal, acute pelvic, and post-surgical gynaeco-logical infections 44. FDA regulatory approval forthis product was obtained in November 2001, andEuropean Union centralized marketing authorizationfollowed in April 2002. A randomized, double-blindstudy (016) involving 540 patients with complicatedskin and skin structure infections showed that 1 g ofertapenem sodium administered once daily was aseffective as piperacillin+tazobactam, 3.375 g q 6 h.

The IMP-1 metallo-β-lactamase inhibitor J-114870 (Banyu, Merck & Co) is in preclinical devel-opment for use as monotherapy against polymicro-bial infections associated with methicillin-resistantStaphylococcus aureus and penicillin-resistant P.aeruginosa 45.

The penems, a new class of β-lactams, representthe implementation of the function-by-design strate-gy since these synthetic, hybrid molecules arederived from penams and cephalosporins. As a classthey show promise as in vitro studies to date indi-cate that they fulfil the key requirements of any newβ-lactam antimicrobial: stability to BLAs and goodactivity against community pathogens including S.pneumoniae, H. influenzae, M. catarrhalis,Streptococcus pyogenes, S. aureus, and Klebsiellapneumoniae 46−50. Penems also appear to be moreactive than the third generation cephalosporins cefo-taxime and ceftriaxone and demonstrate eitherequivalent or more activity than the carbapenemsmeropenem and imipenem 46. One exception hasbeen observed − the penems naturally express littleactivity against P. aeruginosa compared with thecarbapenems. This may be a function of the inter-play between AmpC- BLAs and efflux systems viaeither porins OprD, OmpF or the efflux transporterproteins MexAB-OprM+, MexCD-OprJ+, MexEF-OprN+, MexXY-OprM+.

Penems in development include MEN 10700(Menarini) (Figure 5).51 While being active againstGram-positive and -negative bacteria, aerobes andanaerobes, MEN 10700 also demonstrated activityagainst penicillin-resistant S. pneumoniae and somecephalosporin-resistant Gram-negative bacteria. Inaddition, this penem exhibits in vitro stability tomost BLAs and to renal dehydropeptidase. Anotheroral penem undergoing clinical development isritipenem acoxyl, FCE 22891 (Farmitalia CarloErba, Pharmacia; Figure 5) 52. This penem wasactive in patients for the treatment of acute exacer-bations of chronic bronchitis and as active as thecephalosporin cefotiam hexetil in patients with bac-terial pneumonia.

The penem that has reached the most advancedstages of development to date is faropenem, and theproperties of this molecule that address the mecha-nistic issues related to resistance are discussed here.Faropenem sodium is licensed and marketed inJapan, and faropenem daloxate (faropenem; Bayerlicensed from Suntory), which has improved bioavail-ability, was until recently under development for the

treatment of a range of community-acquired infec-tions (Figure 5) 53.

FIGURE 5 - Chemical structures of penems under clini-cal development.

Faropenem is stable to hydrolysis by BLAs thatcurrently inactivate penici l l ins and othercephalosporins, has high binding affinities for PBPsfrom both Gram-negative and -positive bacteria andappears to have low potential for cross-resistance tocarbapenems. The in vitro investigations have com-pared the activities of faropenem with other β-lac-tams, and have shown that faropenem was highlystable to both penicillinase derived from S. aureus

a) Faropenem daloxate

N

CH3 S

OO

OO

CH3

O

O

Ob) MEN 10700

N

CH3 S

OOH

O

OH

N

CH3

O

NH2

c) Ritipenem

N

CH3 S

OOH

O

OH

O

O

NH2

RESISTANCE TO b-LACTAMS - THE PERMUTATIONS 533

and E. coli and cephalosporinase (group 2 enzymes)derived from E. coli and P. vulgaris. In addition,faropenem was highly stable against hydrolysis byvarious BLAs prepared from four B. fragilis strainsand the rate of faropenem hydrolysis by metallo-BLA was 5 times lower than that for imipenem(Dalhoff A. Personal communication). Furthermore,faropenem was also shown to be stable againsthydrolysis by ESBLs 54,57,13−15. Among the agentsstudied, faropenem was the most active against awide range of Enterobacteriaceae producing morethan 50 different ESBLs or AmpC BLAs; faropen-em exhibited a 4- to 64-fold greater antibacterialactivity than co-amoxiclav.

As proposed earlier concerning the lack ofinduction of BLA, an ideal β-lactam would expresshigh affinity for high molecular mass PBPs but lowaffinity to low molecular mass PBPs. Faropenemexhibits highest affinity to the high molecular massessential PBPs whereas its affinity to the low molec-ular mass PBPs is low. In consequence, faropeneminhibits the essential enzymes for the biosynthesis ofthe cell wall, cell division and septation, but minimal-ly affects, if at all, the sensors for BLA induction(Dalhoff A. Personal communication).

An in vitro study compared faropenem and thecephalosporins cefixime, cefaclor and cefazolin forthe acquisition of resistance by S. aureus 209P FC-1 and E. coli NIHJ JC-2 measuring MICs by the liq-uid broth dilution method 56. The faropenem MICrose 8-fold for S. aureus 209P JC-1 and 16-, 31-and 512-fold for cefixime, cefazolin and cefaclor,respectively. For E. coli, changes in MICs weremoderate for all β-lactams tested. The outcome indi-cates that faropenem had a lower propensity todevelop resistance than comparator cephalosporins.

Penem resistance depends structure-specificallyon both the up-regulation of Mex-ABOprM and theloss of OprD affecting significantly the clinical effica-cy of carbapenems used to treat difficult infections.This raises the question of whether the use of pen-ems for the treatment of community-acquired infec-tions may select for imipenem and/or meropenemresistance. This question was addressed by usingfaropenem to select for resistance to carbapenemsin P. aeruginosa. As faropenem does not penetratethe OprD porin, it does not interact with imipenemuptake through this porin. Although faropenem iseffluxed by the MexAB-OprM system, it does notinteract with other MexAB-OprM substrates e.g.meropenem, beta-lactams or quinolones etc.Consequently, faropenem may have another affinitysite.

CONCLUSIONS

The increase in antimicrobial resistance compro-mises the choice of therapies available to prescribersin the treatment of patients with community infec-tions. As noted by Jones and Pfaller, most microbes

responsible for antimicrobial-resistant infections werepredominantly Gram-positive cocci 57. Today, how-ever, there has been a shift back towards Gram-neg-ative bacilli. The most common resistance mecha-nism to β-lactams is the hydrolysis of the antimicro-bial by BLAs but this route co-exists alongside theproduction of PBPs with conformational changes,the reduction in membrane permeability and, in theinstance of P. aeruginosa, the removal of the drugby active efflux pump systems.

The prerequisite criteria for any new β-lactamare that it should have good efficacy, be well tolerat-ed and maintain stability in the presence of ever-evolving mechanisms of resistance. In comparisonwith current oral therapies with diminishing applica-tion, penems offer good BLA stability, with highaffinities for a range of PBPs. They therefore havethe potential to address prevailing resistance issues.The example of faropenem illustrates that theseagents may fulfil the needs of a new β-lactam.Coupled with good in vitro activity against commu-nity pathogens, and low propensity for resistancedevelopment, penems have a promising profile forthe treatment of community-acquired infections.

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