In the last quarter of this century, the emer-gence of completely new pathogenic microbeshas occurred somewhat rarely. Neverthelesssometimes the phenomenon is spectacular,no more so perhaps than with the appearanceof the human immunodeficiency virus andthe characterization of new hepatitis viruses.A number of bacterial species, too, have beenidentified as pathogens since 1975: Legionellagormanii, Borrelia burgdorferi, Helicobacterpylori, Bartonella henselae and Chlamydiapneumoniae. The relative rarity of these dis-coveries warrants neither naive optimism norcomplacency. For bacterial infectious dis-eases, the remarkable genetic variability andadaptability of microorganisms constitutes arecurrent source of problems for clinicians.Mutations and exchanges of genetic materialresult in strains that resist the usual chemo-therapeutic agents. They will be selected forand thrive when the agents are used.

Other factors also serve to dull optimismin those combating bacterial disease. In devel-oped countries, relatively innocuous infec-tious agents can represent a lethal threat forthe elderly. Prevailing socioeconomic condi-tions, rather than microbial evolution, havebeen responsible for a resurgence in tubercu-losis among the poor. Both the poor and theelderly represent increasing proportions ofthe populations of industrialized nations.

The resistance problem is exacerbated inintensive care units, where many people aretreated with high doses of antibiotics to com-bat actual or potential acute infections.Furthermore, immunodepressed AIDSpatients or those receiving transplants mustreceive large doses of antibiotics for extendedperiods and may, in consequence, becomereservoirs of potentially dangerous strains.

As we enter the antibiotic era’s secondcentury, the need is as pressing as ever tounderstand how bacteria acquire resistance toantibiotics and reemerge as pathogens.

The history of b-lactamsb-lactam antibiotics (penicillins, cephalo-sporins, and related compounds; Fig. 1) arethe most popular and widely used antibacter-ial compounds. Their history supplies a strik-ing example of the evolution of distinct resis-

tance mechanisms in the bacterial world.Benzylpenicillin was the first compoundintroduced into clinical practice. At the outsetof the antibiotic era, it transpired that manyspecies were “intrinsically” resistant.Subsequent indiscriminate use led to therapid selection of resistant strains of previ-ously sensitive species such as Staphylococcusaureus. To circumvent these shortcomings,researchers and companies developed newcompounds by making minor modificationsof the initial molecules: The R and R´ groupsshown in Figure 1 were altered.Simultaneously, they discovered or synthe-sized new families of antibiotics (e.g.,

cephalosporins) that exhibited different andsometimes vastly enlarged activity spectra1.

In every case, despite humankind’s mostinventive efforts, some bacteria alwaysdevised efficient mechanisms to circumventthe lethal activity of the new antibiotics. Tobetter understand the mechanisms of resis-tance, we need to understand what, at a fun-damental biochemical level, makes a goodantibiotic.

Good antibioticsAlmost by definition, the prime requirementof any antibiotic is that it disables, or inter-feres with, a function of the target microor-ganism, the loss of which either kills theorganism or stops its growth. A second fun-damental requirement, if the antibiotic isgoing to be of any medical or veterinary use, is

NATURE BIOTECHNOLOGY VOL 17 SUPPLEMENT 1999 http://biotech.nature.com BV 17

that its direct effects on humans or animalsshould be mild or harmless. Good antibioticswill also satisfy several other requirements.

First, they should readily overcome the“passive” resistance of the target microorgan-isms by passing through the physical andchemical permeability barriers represented bythe various components of the bacterial cellenvelope. Thus they can reach their biochem-ical targets. Second, they should exhibit highaffinity for their biochemical targets by bind-ing specifically and tightly to their biochemi-cal targets. From the microbe’s point of view,it will have “intrinsic” resistance to an antibi-otic if that antibiotic has low affinity for itstarget. As well as avoiding “passive” and“intrinsic” resistance mechanisms, a goodantibiotic should also avoid “active” resis-tance mechanisms. These include enzymesthat destroy the antibiotics or that eject themfrom the cell.

The ideal antibiotic will have other prop-erties of great practical importance such asbiodegradability, lack of allergenicity, andcost. But consideration of these will not con-tribute much to a discussion of resistancemechanisms.

How do b-lactam antibiotics measureup against these yardsticks?The targets of b-lactam antibiotics are DD-transpeptidases (also known as penicillinbinding proteins or PBPs). These are essentialenzymes in the synthesis of a functional pep-tidoglycan, the most important componentof the bacterial cell wall2,3. Mammalian cellsdo not have cell walls; consequently, humansand animals do not possess the enzymes thatare the antibiotic’s target. PBPs are located onthe outer face of the bacterial cytoplasmicmembrane; thus, the antibiotic does not haveto permeate this barrier. However, the outermembrane of Gram-negative genera4 and themycolic acid layer of mycobacteria (a groupthat includes the causative organisms oftuberculosis and leprosy) can considerablyslow down the diffusion of the antibiotic.

The b-lactams inactivate the DD-transpeptidases by acylating their essentialactive-site serines. The sensitivity of the targetto the antibiotic is characterized by a second-order acylation rate constant. This rate con-stant can vary widely—by a factor of 1000 ormore—depending both on the type of bac-terium (and its enzymes) and the type ofantibiotic. This variability of the reaction rateexplains, at least in part, the “intrinsic” sensi-tivity of a species to a given b-lactam.

HUMAN HEALTH

Pathogens old, new, and revivedJean-Marie Frère, Alain Dubus, and Eveline Fonzé

Jean-Marie Frère, Alain Dubus, and EvelineFonzé are at Centre d’Ingénierie des Protéines,Université de Liège, Institut de Chimie, B6,Sart-Tilman, B4000 Liège, Belgium(jmfrere@ulg.ac.be).

Figure 1. b-lactam antibiotic family members;(A) penicillins or penams; (B) cephalosporinsor cephems; (C) cephamycins or 7-b-methoxy-cephems; (D) carbapenems; (E)monobactams. New variants of thesecompounds were developed by changing theR and R´ groups.

A B

C

DE

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HUMAN HEALTH

Although active efflux systems that canexpel b-lactams from the periplasm5 appearto be mainly restricted to Pseudomonas aerug-inosa, many bacteria produce b-lactamases,enzymes that hydrolyze the amide bond in theb-lactam ring.

Development of resistanceResistance in a bacterial population resultsfrom mutations or the acquisition of newgenes (from mobile genetic elements such asplasmids and transposons), followed by selec-tion. For b-lactam antibiotics, resistancemechanisms that are “passive,” “intrinsic,”and “active” have all been identified.

One passive mechanism operating inGram-negative bacteria, for instance, is theloss or alteration of porins, proteins thatallow the diffusion of hydrophilic molecules(like some antibiotics) through the outer bac-terial membrane. This mechanism reducesthe rate at which the antibiotic can get intothe periplasm, the region between the outerand inner bacterial membrane. However,unless there is also b-lactamase activity in theperiplasm, this and other passive resistancemechanisms are only marginally importantfor b-lactam antibiotics4.

“Intrinsic” resistance to b-lactams is cor-related with the appearance of variants of theDD-transpeptidase enzymes that have verypoor affinity for the antibiotics. Their sec-ond-order acylation rate constants arearound 1–10 M–1s–1, much lower than the100,000–300,000 M–1s–1 seen in enzymes fromsensitive bacteria. In some cases, the genesencoding these “resistant” proteins are whollyacquired: PBP2a of the methicillin-resistantStaphylococcus aureus, the origins of whichremain undetermined, is an example here.Alternatively, the genes will arise throughextensive modifications of the bacterium’soriginal genes, probably by recombinationwith the corresponding PBP gene from aresistant, related species. Where a bacterialstrain possesses more than one variant of thetarget enzyme, it may indulge in metabolicswitching. Thus the gene for a minor resistantPBP can be amplified, so that the correspond-ing enzyme can take over the function of itsmore sensitive counterparts6.

A cycle of selectionWhile both “passive” and “intrinsic” resis-tance mechanisms are known for b-lactamantibiotics, it is the “active” mechanisms ofresistance—notably the actions of b-lacta-mases—that represent the major sources ofclinical problems.

The genes encoding b-lactamases areoften plasmid- or transposon-borne. Bacteriacan thus readily acquire one or more of them.Moreover, point mutations can alter the catal-ysis of the enzymes or deregulate their controlmechanism. One consequence can be that the

amount of b-lactamase accumulating in theperiplasm becomes so high that it confersresistance even to compounds against whichthe enzyme is only weakly active7,8.

These phenomena propel a three-step b-lactamase cycle. First, the widespread use ofb-lactams selects microbial strains that pro-duce b-lactamases; second, new antibiotics—such as third-generation cephalosporins orcarbapenems—are designed to escape theactivity of these enzymes; then, finally, new b-lactamases emerge that hydrolyze these newcompounds. The most recent example is thespread of metallo-b-lactamases. Theseenzymes hydrolyze carbapenems, com-pounds that are resistant to the activity ofnearly all the more abundant active-site ser-ine b-lactamases.

Future strategiesAn efficient approach to resistance phenome-na should rest on a balance between an opti-mization of compounds directed at “old tar-gets” and the search for potential “new tar-gets.” Both types of targets must be specificbacterial structures or metabolic processes.The biosynthesis of peptidoglycan is a meta-bolic pathway unique to the bacterial worldand involving D-amino acids making it par-ticularly attractive for future research.

The intracellular steps of peptidoglycansynthesis have been well-characterized9 andthe discovery of specific inhibitors of the cor-responding enzymes certainly represents aninteresting goal. However, these compoundswill have to cross the cytoplasmic membrane.

The two synthetic steps that occur outsidethe cell are catalyzed respectively by a transg-lycosylase and the penicillin-sensitive (orresistant) transpeptidase. The substrates ofthe former might not be very different frommetabolites found in the cells of higherorganisms, but it might not be impossible tofind relatively specific inhibitors. Because ofthe D-alanyl-D-alanine structure of its sub-strate, the transpeptidases are likely to remainthe targets of choice. Even the penicillin-resis-tant transpeptidases must process precursorsthat contain the D-alanyl-D-alanine structure(since they act by a mechanism similar to thatof their sensitive counterparts). This opensthe door to the design of inhibitors with alarge activity spectrum.

Avoiding the “active” resistance conferredby bacterial b-lactamases remains the majorproblem, the more so because DD-transpep-tidases and many b-lactamases share struc-tural and functional similarities6,7. In thisrespect, an alternative strategy for combatingresistance has been successfully used in thepast: A classical b-lactam is administeredtogether with an inactivator of b-lactamases.Here again though, the microbial worldseems to be one step ahead of human endeav-ors: Some b-lactamases are intrinsically resis-

tant to the inhibitors and the use of combina-tion treatments has led to the emergence ofresistant bacterial populations.

The efforts of researchers thus far havebeen inspired by the metabolic capability ofmicrobes. b-lactams, even when they are syn-thesized chemically, are based on designs per-fected by bacteria or fungi. With this in mind,an alternative strategy would be to developinactivators of the DD-transpeptidases or ofthe b-lactamases that are not b-lactams.These would be fundamentally different fromthe natural compounds. We could hope thatthere is no enzyme in the microbial world thatwould transform them, no gene in the micro-bial pool that could be mobilized to counter-act them.

There are other reasons to hope that ourfight against microbial pathogens is not ahopeless one. The complete nucleotidesequences of several bacterial genomes havealready been determined. Analysis of these isexpected to identify new potential targets.The proteins encoded by a large number ofopen reading frames have never been isolatedor characterized. However, we can safely pre-dict that the properties of these “new targets”will vary according to the species. We can alsopredict that the difficulties encountered withb-lactams will not disappear when inhibitorsof newly discovered enzymes are studied.

Although the rate and efficiency of newdrug discovery will be increased by computer-aided analysis of vast compound libraries andthe automated study of their inhibitory prop-erties, a better understanding of bacterialphysiology and biochemistry remains thecornerstone of the development of new com-pounds. When new antibiotics are developed,doctors will also need new methods that per-mit rapid and simple identification ofpathogen strains to sensibly and rationallyadminister specific antibacterial agents.

AcknowledgmentsThe work of our laboratory is supported by theBelgian Government through the PAI Program(P4/03). The authors thank Professor P. De Mol forhis interesting suggestions.

1. Burton, G., et al. The b-lactam antibiotics In:Burgers’s Medicinal Chemistry and Drug Discovery,5th Ed. Vol. 4: Therapeutic Agents, (ed. Wolff, M.E.)(John Wiley and Sons, New York, 1997).

2. Frère, J.M. and Joris, B. CRC Crit. Rev. in Microbiol.11, 299–396 (1985).

3. Ghuysen J.M. Annu. Rev. Microbiol. 45, 37–67(1991).

4. Nikaido, H. and Normark, S. Mol. Microbiol. 1, 29–36(1987).

5. Nikaido, H. J. Bacteriol. 178, 5853–5859 (1996).6. Frère, J.M., et al. Mode of action: interaction with

penicillin-binding proteins. In: The Chemistry of b-lactams (ed. Page, M.I.) (Blackie Academic andProfessional, London, 1992).

7. Matagne, A. et al. Nat. Products Rep. 16, In press(1999).

8. Lakaye, B., et al. Mol. Microbiol. 30, In press (19990). 9. Van Heijenoort, J. Cell. Mol. Life Sci. 4, 300–304

(1998).

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