Bacterial Antibiotic Resistance

Shahriar Mobashery, Wayne State University, Detroit, Michigan, USA Eduardo F Azucena Jr, Wayne State University, Detroit, Michigan, USA
Mechanisms of bacterial antibiotic resistance refer to the processes that enable bacteria to counter the harmful action of antibacterial agents against them.

Introductory article
Article Contents
. Selection Pressure and the Evolution of Resistance . Biochemistry of Resistance . Accumulation of Multiresistance . Human and Economic Costs of Drug Resistance . End of the Antibiotic Era?

Selection Pressure and the Evolution of Resistance

Studies of protein structures have made signicant advances within the past decade. The ndings from these eorts have led some to suggest that there may not be more than 1000 unique protein folds in nature. In light of the fact that there are more enzymes than this number in the genomes of most organisms, it indicates that nature has utilized the primordial proteins with the unique folds, diversied their sequences with retention of the folds, to acquire additional functions. This must be a common occurrence for both simple and complex forms of life. As it pertains to microorganisms, the numbers are on their side. A typical bacterial population would double its size every 30 minutes. Bacteria can reach very high population densities during uninhibited growth (107 cells mL 2 1 in infections of blood and as much as 109 cells mL 2 1 in infections of tissues). The rate of mutation in living bacteria is relatively low (10 2 10 per base). However, considering the large sizes of the genomes (e.g. that for Escherichia coli K-12 is 4.6 106 base pairs) and the high potential for bacterial population densities under favourable growth conditions, this rate is not insignicant. Simple mathematical manipulation of these numbers (population density genome size rate of mutation) indicates that approximately 105 106 mutations might occur in each millilitre of bacterial growth under favourable conditions. Many of these mutations would take place in noncoding regions; others would be silent mutations, yet a proportion would result in lethal alterations of the genome. However, a fraction of these random mutations might permit selection of new phenotypes, some of which would make organisms that possess them more t for growth and survival. With exposure to antibiotics, in the environment or in a clinical setting, some of these mutant organisms might have an antibiotic-resistant phenotype and be more selectively t. As will be discussed here, nature has met the challenge of antibiotics eectively by selecting a variety of mechanisms for drug resistance. The events of random mutation and the attendant natural selection in the presence of a given antibiotic provide ample opportunity for the advent of the resistance

phenotype. It is interesting to note that the cost to the microorganism of the evolution of drug resistance may or may not be important. One could envision that, in the case of a plasmid-borne resistance factor, the organism must synthesize the additional genetic material and the protein that it encodes. Furthermore, alterations of chromosomal genes encoding the targets for antibacterials may present the opportunity for selection of resistance to antibiotics. However, such modications may jeopardize the normal function of the gene products and make the organism that carries them less t in the absence of the antibiotic. Numerous studies indicate that genetic adaptation by secondary mutations elsewhere in the genome and natural selection reduces this cost to the organism in several generations of growth, such that the cost is either reduced or eliminated entirely. Evolution of the drug-resistance phenotype presents an ideal case study for the advent of function in biological systems. Our knowledge of many aspects of this process is still rudimentary. Undoubtedly, the critical importance of antibiotic chemotherapy to the well-being of humankind will stimulate further research in this area in the immediate future. This entry highlights important aspects of what is currently known about drug resistance.

Biochemistry of Resistance
Understanding the mechanisms of resistance to antibiotics has become a signicant biochemical issue over the past several years. Although the manner of acquisition of resistance may vary among bacterial species, drug resistance is created by only a few mechanisms. Bacteria may prevent entry of drugs into the cell by changing their cell membrane composition or by altering the drug uptake system. When the drug does penetrate into the cell, it may be modied by cellular enzymes, rendering it inactive, or it may be excreted (modied or not). If none of these mechanisms is operative, the cell may alter the target of the drug to eect weaker interaction between the target and the drug, or bacteria may resort to alternative pathways to bypass the inactivated target. Which of these mechanisms
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prevails depends on the nature of the antibiotic, its target site, the bacterial species and whether it is mediated by a resistance plasmid (R plasmid) or by a chromosomal mutation.

Impermeability and drug efflux

Some bacterial species prevent easy access of antibiotics to their cellular targets by having cell membranes with low permeability to antimicrobial agents. Gram-positive bacteria surround their cytoplasmic membrane with a porous peptidoglycan layer that oers sturdiness to the cell but has little resistance to inux of many antibiotics. Gramnegative bacteria have an additional outer membrane, the outer leaet of which consists of lipopolysaccharide molecules with six or seven all-saturated fatty acid constituents. The higher levels of saturation and the higher number of fatty acid chains (67) make lipopolysaccharides more rigid and less permeable than the more common glycerophospholipids, which usually have two unsaturated fatty acid chains. Diusion channels, which consist of proteins called porins, traverse the outer membrane. The openings of these porin channels are big enough to allow the inux of essential nutrients, but are suciently constricted that they hinder passage of antibiotics larger than 1 kDa. In addition, these openings are lined with charged amino acids that restrict the entry of lipophilic drugs. Notwithstanding the organization of the bacterial membrane, total exclusion of drugs from the bacteria is not ensured. Small hydrophilic antibacterials such as certain b-lactams or aminoglycosides can still diuse through the porin channels. To counteract this, dierent membrane-barrier schemes are adopted by certain bacteria to further reduce membrane permeability. For instance, Pseudomonas aeruginosa has various porin channels, each specializing in the uptake of specic nutrient molecules and each having about a 100-fold lower permeability to small molecules than do most diusion channels found in other organisms. In the case of mycobacteria, the outer membrane is composed of long (more than 70 carbon atoms) fatty acid chains, called mycolic acid. An arabinogalactan molecule holds hundreds of mycolic acid molecules together in one unit and links them to the peptidoglycan beneath it. Specialized porin channels present in small numbers allow the ingress of nutrients at a slow rate. This arrangement aords mycobacterial membranes even greater resistance to drug inux. Mutations that reduce production of specic channels make certain Gram-negative cells less permeable to specic agents. For example, some P. aeruginosa variants exhibit resistance to imipenem, a carbapenem antibiotic, as these strains have lost the porin channel that normally permits transit of this antibiotic. Interestingly, Escherichia coli is shown to decrease transiently its production of certain
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porins in response to exposure to salicylate produced in host plant tissues. Similarly, oxygen stress represses porin production in some pathogenic bacteria. Despite the dierent strategies adopted by bacteria, decreasing the membrane permeability alone does not elevate the minimum inhibitory concentrations for several drugs to levels of clinical signicance. This is because reduction of membrane permeability merely retards diusion of antibiotics through the channels. Nevertheless, drugs can penetrate the membrane barriers suciently rapidly to be eective as antibacterials. A more ecacious resistance mechanism is the energy-dependent extrusion of drugs from the cytoplasm into the milieu by eux pumps, which consist of proteins located in the cytoplasmic membrane. In many Gram-negative organisms, accessory proteins link the eux pumps to the outer membrane, possibly by complexing with porins. These bridges ensure that the drug is released directly into the medium, and not into the periplasm (the space between the outer and cytoplasmic membranes). The process of drug extrusion in some Gram-negative eux systems lacking these accessory proteins is not clear. In many cases, low membrane permeability is coupled with the presence of eux proteins. In fact, in some bacteria, the gene that encodes eux proteins also represses the production of porin channels, thereby creating the dual eects of low permeability and eux. In some instances, biosynthesis of an eux pump (usually accompanied by reduced porin production) can be induced by the antibiotic it euxes. Many drug eux genes have been identied: some are plasmid-borne; others are chromosomal. Most eux systems can extrude a wide range of drugs and thus engender multidrug resistance in many bacteria. For example, the pseudomonal multidrug eux pump (MexB), which belongs to one of the four families of eux systems, extrudes quinolones, tetracyclines, chloramphenicol, pyoverdine and several b-lactam antibiotics.

Drug inactivation by structure modification

Bacterial species may become insensitive to certain antimicrobial agents because of their possession of enzymes that inactivate these antibiotics. This is an important resistance mechanism against b-lactams, aminoglycosides, macrolides and chloramphenicol. b-Lactam antibiotics irreversibly acylate the active sites of enzymes called penicillin-binding proteins (PBPs), which catalyse the transpeptidation reaction in cell wall biosynthesis. Once acylated, the PBP can no longer perform its normal function, and death of the bacterium ensues. To oset the detrimental eects of b-lactams, most bacteria synthesize b-lactamases, enzymes that hydrolyse the b-lactam moieties of these antibiotics, resulting in products that can no longer inhibit PBPs. These b-lactam-inactivating enzymes

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are ubiquitous in Gram-negative bacteria and are present in Gram-positive bacteria such as staphylococci and enterococci. They are a diverse group of proteins categorized into four classes according to substrate prole, inhibition behaviour, molecular weight and catalytic parameters. Some b-lactamases are inducible (i.e. their biosynthesis can be increased by the presence of certain compounds), while others are expressed constitutively (i.e. they are biosynthesized at constant levels). There are certain b-lactamases that possess such a broad substrate prole that they turn over virtually all existing b-lactam antibiotics. The b-lactamase genes are found in chromosomes, in plasmids or in transposons. The plasmid-borne b-lactamase genes are responsible for most of the b-lactam resistance found in the majority of pathogens. Since plasmids are more readily transferable from one cell to another, the presence of these genes in plasmids assures dissemination of b-lactam resistance among bacteria. Of all drug-resistance enzymes, the fastest evolving are blactamases. This is in part because of the extensive use of blactam antibiotics in the clinic. Furthermore, since many of the genes for these enzymes are on multicopy plasmids (i.e. multiple copies of the same gene in one cell), this presents more opportunity for the gene to acquire mutations. Yet another reason for the facile mutability of substrate prole among b-lactamases is the fact that they do not interact with any cofactors (second substrates). Hence, even a single amino acid substitution in b-lactamases can alter their substrate preference range. This enables b-lactamases to change their substrate specicity and inactivate newer blactams. For instance, within a few years of the introduction of cefotaxime into clinical use, a b-lactamase mutant was found capable of hydrolysing it. Aminoglycosides exert bactericidal action by interacting with the bacterial ribosomal ribonucleic acid (rRNA). The exact sequence of events that leads to bacterial cell death after aminoglycoside action is still elusive. It has been proposed that, because of this aminoglycosiderRNA interaction, messenger RNA (mRNA) is mistranslated and nonfunctional proteins are produced, which in turn produces nonviable cells. Resistance to aminoglycosides is mostly the result of their inactivation by aminoglycosidemodifying enzymes. There are three types of aminoglycoside-modifying enzymes, each of which transfers a functional group on to the aminoglycoside structure to render the antibiotic ineective: (1) aminoglycoside nucleotidyltransferases (ANTs) transfer a nucleotide from nucleotide triphosphates; (2) aminoglycoside acetyltransferases (AACs) transfer the acetyl group from acetyl coenzyme A (CoA) and (3) aminoglycoside phosphotransferases (APHs) transfer the phosphoryl group from adenosine triphosphate (ATP). Each enzyme group has dierent isozymic forms that dier in substrate regiospecicity for their reactions. At least 30 dierent genes for aminoglycoside-modifying enzymes are known in bacteria. In contrast

to the b-lactamase genes, these genes do not undergo easy mutational changes in their substrate specicities. Many Gram-positive pathogens harbour a unique bifunctional enzyme that has both AAC and APH activities. This dual activity appears to be due to the merger of two independent resistance genes, a process that might have taken place during insertion into a transposon. Chloramphenicol is generally a bacteriostatic agent that binds to bacterial ribosomes. Chloramphenicol acetyltransferase (CAT) inactivates chloramphenicol by acetylating it using acetyl CoA as the acetyl group donor. At least a dozen dierent cat genes exist that encode dierent chloramphenicol acetyltransferases. In Gram-negative bacteria, CAT is biosynthesized constitutively, while in Gram-positive bacteria CAT biosynthesis is inducible in the presence of chloramphenicol. The host toxicity of chloramphenicol has led to a decline in the use of this drug; hence, chloramphenicol resistance has become of minor importance from a therapeutic standpoint. A few other less common antibiotic-modifying enzymes are known. Fosfomycin, which interferes with bacterial cell wall biosynthesis, is inactivated by a plasmid-determined glutathione-S-transferase that catalyses the formation of the glutathione-fosfomycin adduct. In some Gram-positive bacteria, the lincosamide antibiotics are inactivated by nucleotidyltransferases. Enzymatic phosphorylation as well as hydrolytic modication of the drug erythromycin have been observed in a number of bacterial strains.

Target modification
Some bacteria that lack b-lactamases counter the pernicious eects of b-lactams by altering their chromosomal PBP genes such that the resultant PBPs have markedly reduced anity for the drugs. Bacteria possess multiple PBPs, each performing a specic role in cell wall biosynthesis during the cell cycle. Studies have established conserved sequence motifs around the active sites of the PBP transpeptidase domains, the sites of binding of blactam antibiotics. Not surprisingly, mutations that result in reduced anity of PBP for b-lactams are found within, or near, these motifs. It is speculated that instead of sequential amino acid substitution, genetic exchange (by transformation) among bacteria might have given rise to resistant PBPs. Consistent with this hypothesis is the fact that the best-studied examples of this type of resistance occur among naturally transformable species. This is exemplied by Streptococcus pneumoniae, which is normally transformable and whose altered PBPs are entirely responsible for high resistance to penicillins and cephalosporins, including expanded-spectrum cephalosporins. An interesting case is observed in methicillin-resistant Staphylococcus aureus (MRSA), a serious problem in nosocomial (i.e. hospital-acquired) infections. MRSA does not possess altered forms of its four
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wild-type staphylococcal PBPs. Instead, it has an additional PBP that is immune to inactivation by essentially all available b-lactam antibiotics. It is remarkable that, when challenged by b-lactam antibiotics, this unusual PBP can take over the tasks of the normal PBPs, as cell wall biosynthesis is believed to require multiple PBPs. Unlike most bacteria that exhibit PBP-mediated resistance, MRSA is not naturally transformable; hence, the genesis of resistance in MRSA remains obscure. PBP-mediated resistance to b-lactams is rare because, in general, b-lactam antibiotics kill bacteria by inactivating more than one PBP. Thus, in order to cause signicant resistance to b-lactams, the anity of each of the physiologically important target PBPs should be reduced, and achieving this would be dicult. Another limitation to PBP-mediated resistance stems from the fact that blactams are structural analogues of the normal PBP substrates, namely the peptidoglycans. Consequently, altered PBPs must be able to dierentiate between blactams and their physiological substrates. It is dicult to have such a PBP variant by replacement of individual amino acids. A few PBP amino acid substitutions can confer increased resistance in certain Gram-negative bacteria only in combination with reduced membrane permeability. Trimethoprim, sulfonamides, rifamycins and quinolones are drugs for which inactivating enzymes have not been found. Resistance to these drugs normally takes place by genetic mutations of a target enzyme, resulting in mutant variant proteins with poor anities for their inhibitors, the antibiotics. Since each of these groups of drugs inhibits a single target enzyme, resistance to these drugs via target alteration occurs more readily and more commonly than PBP-mediated resistance. Trimethoprim and sulfonamides are competitive inhibitors of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), respectively. Both enzymes are essential for biosynthesis of tetrahydrofolate a critical source for single carbon in the biosynthesis of various metabolites. Chromosomal mutations that produce altered DHFR or altered DHPS in certain clinical isolates are responsible for resistance to trimethoprim or sulfonamides, respectively. In some strains, alteration of the target enzyme, in combination with its overproduction, provides high-level resistance. In the case of rifamycins (RNA polymerase inhibitors) and quinolones (deoxyribonucleic acid (DNA) gyrase inhibitors), drug resistance is attainable by single amino acid substitutions in their respective targets, because neither drug is a substrate analogue. Thus, the mutant variants of the target proteins can discriminate between the drugs and their substrates. An alternative resistance mechanism of this genre involves genes encoding enzymes that catalyse modication of drug targets. An example of this is the enzymatic methylation of rRNA, which desensitizes certain microorganisms to rRNA-interacting antibiotics, such as
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aminoglycosides, macrolides, lincosamides, synergimycins and thiostrepton, all of which disrupt the protein biosynthetic machinery of bacteria. In some bacterial strains, resistance to this group of antibiotics is a consequence of gene mutations of the target ribosomal proteins or the target rRNA.

Target bypass
Some bacteria become refractory to specic antibiotics by possessing an alternative pathway that bypasses the lethal eects of inactivation of a given enzyme. This mode of resistance is observed in many trimethoprim-and sulfonamide-resistant bacteria. In bacteria, major biochemical pathways are rarely duplicated, as this would pose a heavy burden on the cells resources. Accordingly, inhibition of a step in a critical metabolic pathway generally brings cellular metabolism to a standstill. Duplication of the synthetic pathway for tetrahydrofolate, however, is benecial to bacteria, in that it provides a means for drug resistance. As mentioned above, DHFR and DHPS are enzymes involved in tetrahydrofolate biosynthesis, and are inhibited by trimethoprim and sulfonamides, respectively. In several trimethoprim-and sulfonamide-resistant strains, a second enzyme that is plasmid-determined, and that has low anity for the inhibitors, is produced. The plasmidencoded enzyme and its chromosomal counterpart usually dier in molecular weight, mechanism of drug interaction and specic activity. The cell that carries both chromosomal and plasmid enzymes is able to synthesize tetrahydrofolate even in the presence of inhibitors because, despite inhibition of the chromosomal enzyme, the plasmidencoded drug-resistant enzyme remains unharmed and functional. The genes for target bypass are found mostly in R plasmids, often as components of transposons. The genes of some nonchromosomal, drug-resistant DHFRs may be transposed temporarily on the chromosome, but are still referred to as transferable or plasmid-borne DHFRs. A well-documented transposon that confers trimethoprim resistance is transposon 7 (Tn7), which also confers resistance to streptomycin and spectinomycin. In bacteria that parasitize animals, in which trimethoprim resistance is more common than those in humans, plasmids conferring trimethoprim resistance were also found to contain Tn7. Thus, transposition may have been the mechanism responsible for the inter-and intraspecies dissemination of trimethoprim resistance from strains in animals to those in humans. The origin of trimethoprim resistance genes in bacteria is a subject of speculation. The molecular weights of plasmid-borne DHFRs in trimethoprim-resistant E. coli indicate that these genes could not have evolved from an ancestral trimethoprim-sensitive E. coli enzyme. Instead, the source of the genes could have been a bacteriophage

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Accumulation of Multiresistance
Multidrug resistance can be dened as resistance to two or more classes of antibiotics. Resistance to multiple drugs may arise from acquisition of plasmids or transposons that carry multiple resistance genes against dierent classes of antibiotics. For example, the plasmid pSa carries a gene for chloramphenicol resistance, a gene for streptomycin and spectinomycin resistance, and another gene for kanamycin and gentamicin resistance. It is believed that the genes in these multiresistance plasmids and transposons come from unrelated species, as suggested by the varied codon usage in the constituent resistance genes. Studies conducted in recent years have shown that these multiresistance plasmids and transposons uniformly contain two conserved DNA sequences, called the 5- and 3-conserved segments. Between these segments is a recombination site in which one or more genes, called cassettes, can be inserted. These DNA elements, with or without the inserted cassettes, are collectively called integrons. The vast majority of cassettes found in integrons are resistance genes, although inserts of genes for other functions are also likely to be found. Resistance gene cassettes have been found for each class of known antibiotic, and the gene products are involved in various mechanisms of resistance, such as eux, target bypass and drug inactivation. The 5conserved segment contains a gene encoding an enzyme called integrase that catalyses site-specic recombinational insertion of one or more cassettes into the recombination site in a specic orientation. This gene orientation is such that the genes can be transcribed from a single promoter located at the 5-conserved segment. Gene cassettes are mobile and can exist freely as circular DNA structures possessing their own recombination site that is recognized by the integrase. Freely existing gene cassettes cannot be transcribed unless picked up by an integron because they lack transcription promoters. The integron-promoted cassette transcripts contain a ribosome-binding site required for translation to protein products. Thus, integrons serve not only as cloning vectors but also as ecient expression vectors for resistance gene cassettes. The integrase-catalysed recombination events also serve other functions, such as excision of cassettes from integrons, repositioning of inserted genes and transfer of resistance genes from one integron-bearing plasmid to another. Thus, a plasmid with a preexisting resistance gene cassette can acquire additional resistance gene cassettes from donor plasmids. The combination of dierent resistance genes, their rearrangement and transfer between integrons all promote the emergence of novel multiresistance integrons. It has been demonstrated that the integrase can transfer a gene cassette from an integron to a secondary plasmid site where the gene becomes more stable, i.e. less prone to excision. It has also been proposed that integrons themselves are mobile, but the modus

operandi by which mobility of integrons occurs has not yet been elucidated.

Human and Economic Costs of Drug Resistance

The World Health Organization reported that one-third of the 52.2 million human deaths in the world in 1997 were caused by communicable parasitic diseases. Of these infectious diseases, the three leading killers were acute respiratory infections (3.7 million), tuberculosis (2.9 million) and diarrhoea (2.5 million). In the United States alone, an estimated two million hospitalizations a year result in nosocomial infections. Thus, infectious diseases carry an urgent and consequential public healthcare concern, both at a national level and on a global scale. Owing to a lack of systematic surveillance for antibiotic resistance worldwide, the contribution of drug resistance to this global problem is dicult to assess quantitatively. It is estimated that 60% of nosocomial infections in certain developed nations are due to resistant bacteria, while in most developing countries it is unknown. Resistance to antibiotics contributes signicantly to the already increasing healthcare cost, which in the United States is estimated at $4.5 billion per annum on nosocomial infections alone. Failure of an antimicrobial agent in treatment of an infection due to microbial resistance results in prolonged illness, or worse. Consequently, resistance to antibiotics incurs additional direct healthcare costs in the form of more laboratory tests, need for further treatment and extended hospitalization, as well as indirect costs due to lost productivity. Furthermore, prolonged illness incurs the risk of dissemination of resistance to other patients. Bacterial resistance to the older, and relatively cheaper, rst-line antibiotics (mainly given orally) usually compels physicians to prescribe the newer, second-line antibiotics. Many of the second-line antibiotics are more expensive, and are likely to have further indirect costs associated with them, such as trained nurses, syringes, needles, intravenous lines, etc. Taking into account all these factors, the annual societal costs attributable to antibiotic resistance in the United States is estimated to range from $150 million (without deaths) to $3 billion (with deaths). A study conducted in 1995 on hospitals in metropolitan New York compared the costs of treating infections caused by MRSA versus methicillin-sensitive S. aureus (MSSA) the leading causes of nosocomial infections in the United States in the 1990s. This study reported that infections due to MRSA had a higher per-patient cost and average death rate than MSSA: $34 000 versus $31 500 (1995 US dollars), and 21% versus 8%, respectively. This dierence reects the higher cost of vancomycin, which is the drug of choice for treatment of MRSA infection, as well as isolation procedures. The economic factors and mortality rate
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associated with S. aureus infections would increase, if the new S. aureus strain (discovered in Japan in 1996) that has intermediate resistance to vancomycin spreads, or if a vancomycin-resistant strain emerges. This situation is reminiscent of the twofold increase in mortality rate due to bacteraemia after the penicillin-resistant S. aureus emerged in the 1950s.

End of the Antibiotic Era?

Eective antibacterial treatment options became available essentially during the 1940s. The early successes in antibacterial development were so spectacular that they stimulated search for novel classes of antibiotics. These eorts led to a period which has come to be known as the golden age of antibiotics, spanning the 1960s and 1970s. A perception of a glut in antibiotics by practitioners, both in the clinic and in the pharmaceutical industry, existed. This view was so pervasive by the late 1980s that many leading pharmaceutical companies stopped further research on antibacterials, and simply continued producing the existing successful antibiotics for clinical use. These views and perceptions have changed in a most dramatic fashion. Now, at the threshold of the new millennium, one nds bacterial populations that have become resistant to multiple antibacterial classes. To compound the problem, no less than 30 new infectious agents have been discovered since 1973. Furthermore, there are organisms that cannot be treated with any of the existing antibacterials, or can only be treated with a single antibiotic, as discussed earlier. We live in a time of an epidemic of microbial resistance. Mitchell Cohen of the US Centers for Disease Control and Prevention has stated recently that: unless currently eective antimicrobial agents can be successfully preserved and the transmission of drug-resistance organisms curtailed_ postantimicrobial era may be rapidly approaching in which infectious disease

wards housing untreatable infections will again be seen. This is a sobering thought at the end of the twentieth century. As of the past few years, there has been a renewed interest in research on identifying essential targets in bacteria for development of novel antibiotics. These eorts are stimulated by the recent signicant advances in genomics of bacteria, which provide the entire genetic make-up for each organism. Only time will tell how many of the new targets that are being identied will prove ecacious in the development of new antibiotics. These asyet-unknown antibiotics are needed in the near future to preserve our therapeutic options in the face of challenges by disease-causing organisms that have proven dicult to treat.

Further Reading
bile-Cuevas CF (ed.) (1996) Antibiotic Resistance: From Molecular Ama Basics to Therapeutic Options. New York: Chapman and Hall. Bryan LE (ed.) (1989) Antimicrobial Resistance. Handbook of Experimental Pharmacology, vol. 91. Berlin: Springer. Chadwick DJ and Goode J (eds) (1997) Antibiotic Resistance: Origins, Evolution, Selection and Spread. New York: Wiley. Ewald PW (1994) Evolution of Infectious Disease. New York: Oxford University Press. Fisher JA (1994) The Plague Makers. New York: Simon and Schuster. Garrett L (1994) The Coming Plague: Newly Emerging Diseases in a World out of Balance. New York: Farrar, Straus and Giroux. Harrison PF and Lederberg J (eds) (1998) Antimicrobial Resistance: Issues and Options. Washington DC: National Academy Press. Jungkind DL, Mortensen JE, Fraimow HS and Calandra GB (eds) (1995) Antimicrobial Resistance: A Crisis in Health Care. New York: Plenum Press. Levy SB (1992) The Antibiotic Paradox: How Miracle Drugs Are Destroying the Miracle. New York: Plenum Press. Rosen BP and Mobashery S (eds) (1998) Resolving the Antibiotics Paradox. New York: Plenum Press. Wilson ME, Levins R and Spielman A (1994) Disease in evolution: global changes and emergence of infectious diseases. Annals of the New York Academy of Sciences 740.

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