Biosci. Biotechnol. Biochem.

, 70 (5), 1060–1075, 2006

Review
Antibiotic Resistance in Bacteria and Its Future
for Novel Antibiotic Development
Hiroshi YONEYAMAy and Ryoichi K ATSUMATA
Laboratory of Animal Microbiology, Department of Microbial Biotechnology,
Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Since the first introduction of the sulfa drugs and
penicillin into clinical use, large numbers of antibiotics
have been developed and hence contributed to human
health. But extensive use of antibiotics has raised a
serious public health problem due to multiantibiotic
resistant bacterial pathogens that inevitably develop
resistance to every new drug launched in the clinic.
Consequently, there is a pressing need to develop new
antibiotics to keep pace with bacterial resistance. Recent
advances in microbial genomics and X-ray crystallog-
raphy provide opportunities to identify novel antibacte-
rial targets for the development of new classes of
antibiotics and to design more potent antimicrobial
compounds derived from existing antibiotics respective-
ly. To prevent and control infectious diseases caused by
multiantibiotic resistant bacteria, we need to under-
stand more about the molecular aspects of the patho-
gens’ physiology and to pursue ways to prolong the life
of precious antibiotics.
Key words: antibiotics; multiantibiotic resistance; bac-
teria; chemotherapy
The history of humankind can be regarded from a
medical point of view as a struggle against infectious
diseases. Although infectious diseases were the leading
cause of death worldwide at the beginning of the
twentieth century, the introduction of sulfa drugs and
penicillin into clinical use in the 1930s and 1940s
respectively had a striking impact on the treatment of
infectious diseases and decreased mortality dramatical-
ly.1) After the clinical success of penicillin, intensive
screening for antibacterial agents led to the discovery
and successful clinical development of streptomycin,
chloramphenicol, tetracycline, erythromycin, rifamycin,
vancomycin, and cephalosporin between 1940 and 1960.
‘‘The era of antibiotics’’ led to optimism that infectious
diseases can be controlled and prevented, and confi-
dence that modern medicine would prevail against
infectious diseases.1) However, contrary to this opti-
mism, infectious diseases are still the second-leading
cause of death worldwide and the third-leading cause of
death in developed countries.2,3) The global public
health problem has been caused by emerging infections
not yet recognized, re-emerging infections experienced
previously that have reappeared in more virulent forms,
and antimicrobial-resistant bacterial infections.2) Anti-
microbial resistance was recognized soon after the
deployment of sulfonamides and penicillins, and it
now appears that the emergence of antibiotic-resistant
bacteria is inevitable to most every new drug.1) To
prevent the emergence and dissemination of resistant
bacteria, continuing efforts to develop new antibacterial
agents are needed.4,5)
In this review, we focus on antibacterial agents,
resistance to them, and their future development.
I. Definition of Antibiotics
Antibiotics, literally ‘‘against life,’’ are chemical
compounds produced by actinomycetes, fungi, or bac-
teria that interfere with some essential bacterial structure
or process with no effects on the eukaryotic host having
the infectious agents.6) Antibiotics exert two effects on
bacteria: bacteriostatic and bactericidal. Bacteriostatic
agents prevent the bacterial cells from growing, as
exemplified by the drug chloramphenicol, and bacter-
icidal agents kill bacteria, as exemplified by penicillin.6)
Although the term ‘‘antibiotic’’ was first introduced
by Waksman in 1942 to denote chemical substances
produced by a microorganism, semisynthetic modifica-
tions of natural products have produced a large variety
of antibacterial agents, such as
-lactam antibiotics and
macrolides, that are also called antibiotics.7) This
definition distinguishes antibiotics from antibacterial
agents totally synthesized by the chemist. At present,

y
To whom correspondence should be addressed. Tel: +81-22-717-8915; Fax: +81-22-717-8708; E-mail: yoneyama@bios.tohoku.ac.jp
Abbreviations: PBP, penicillin binding protein; MRSA, methicillin resistant Staphylococcus aureus; VRE, vancomycin resistant Enterococci; MFS,
major facilitator superfamily; RND, resistance-nodulation-division; SMR, small multidrug resistance; MATE, multidrug and toxic compounds
extrusion; ABC, ATP-binding cassette; MLSB , macrolides, lincosamides, and streptogramin B; ORF, open reading frame; IVET, in vitro expression
technology; DFI, differential fluorescence induction; STM, signature-tagged mutagenesis; PKS, polyketide synthase; PDF, peptide deformylase; FAS,
fatty acid synthase; DHP, dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; MCIPC, cloxacillin; DOC, deoxycholate; AC, acriflavine; TC,
tetracycline; Erm, erythromycin ribosome methylation; EM, erythyromycin; CAT, chloramphenicol acetyltransferase
 Antibiotic Resistance in Bacteria
Cytoplasmic membrane
Outer membrane
Cell wall
(peptidoglycan)
DNA/RNA biosynthesis
DNA gyrase
DNA
RNA
polymerase
ribosome
mRNA
Protein biosynthesis
Gram-negative
Fig. 1. Major Targets of Antibitoics.
PBP, penicillin binding protein; DHP, dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate.
there are three classes of chemically synthesized anti-
bacterial agents in clinical use: the sulfa drugs intro-
duced in the 1930s, the quinolones introduced in the
1960s, and an oxazolidinone approved in the United
States in 2000.4,7) These purely synthesized antibacterial
agents are also treated as antibiotics in this review.
II. Targets of Antibiotics
Although a large number of antibiotics are used
clinically, the variety of targets that they inhibit is
limited. Antibiotics are usually classified on the basis of
their chemical structure and mode of action. To under-
stand how antibiotics work and why bacteria become
resistance to them, a brief description of the targets of
the main classes of antibiotics is required. The main
classes of antibiotics inhibit four classical targets
(Fig. 1): (i) cell wall biosynthesis, (ii) protein biosyn-
thesis, (iii) DNA and RNA biosynthesis, and (iv) folate
biosynthesis.8)
1. Inhibition of bacterial cell wall biosynthesis
Since bacteria have high intracellular pressure, they
must protect themselves from osmolysis with a rigid
peptidoglycan, a meshwork of strands of glycan and
peptide covalently crosslinked (Fig. 1). Gram-negative
bacteria, such as Escherichia coli and Pseudomonas
aeruginosa, and Gram-positive bacteria, such as Staph-
ylococcus aureus and Streptococcus pneumoniae, both
1061
PBP
Cell wall biosynthesis
DNA GTP
dTTP DHP
mRNA
dTMP DHF
dUMP THF
Folic acid
metabolism
Gram-positive
have a peptidoglycan layer as part of their cell-wall
structure. The peptidoglycan layer of Gram-positive
bacteria is generally much thicker than that of Gram-
negative bacteria, and constitutes the outermost layer.9)
The peptidoglycan undergoes crosslinking of the glycan
strands by the action of transglycosidase, and of the
peptide strands by the action of transpeptidase.6) Bifunc-
tional enzymes possessing both transglycosidase and
transpeptidase domains are the target sites of penicillins
and cephaolosporins, collectively called
-lactams.10)
-
lactams attack and acylate the active site of the
transpeptidase, leading to inactivation of the enzymes,
hence they are called penicillin-binding proteins or
PBPs.10)
In addition to
-lactams, vancomycin, considered a
last resort among antibiotics, also targets peptidoglycan
to block cell-wall biosynthesis.11) It binds to the D-Ala4 -
D-Ala5 moiety of pentapeptides and thereby keeps the
substrates away from transpeptidase and transglycosi-
dase.11) This substrate sequestration leads to the failure
of peptidoglycan crosslinks, making the cell wall
susceptible to osmolysis.
2. Inhibition of protein biosynthesis
Protein biosynthesis, a central reaction for cellular
function, is catalyzed by ribosomes, composed of two
nucleoprotein subunits, 30S and 50S subunits with about
two-thirds RNA and one-thirds protein, in prokaryotes
(Fig. 1).12) This machinery of prokaryotes differs sub-
1062 H. YONEYAMA and R. KATSUMATA
stantially from the eukaryotic counterpart, which ex-
plains the effectiveness and selective toxicity of many
clinically important antibiotics. The X-ray structure of
30S and 50S subunits12–14) and of 70S ribosome15)
reveals the architecture of this giant protein biosynthesis
machinery in detail, and the peptidyltransferase activity
deriving from the catalytic ribozyme domain of the 23S
rRNA with no obvious assistance from protein sub-
units.12) Due to the large number of steps involved in
protein biosynthesis, initiation, elongation, and termi-
nation, it is not surprising that there are many steps to be
blocked, or that many clinically useful antibiotics that
interact with the conserved sequences of the 16S rRNA
of the 30S subunit (aminoglycosides and tetracyclines)
and the 23S rRNA of the 50S subunit (macrolides,
chloramphenicol, lincosamides, and quinupristin-dalfo-
pristin) have been found.16–18)
3. Inhibition of DNA and RNA biosynthesis
DNA replication is an essential process for all
organisms. Bacterial chromosomal DNA is packed in a
highly twisted (supercoiling) state in the cell, and
dynamically changes its structure during the replication
process.19) The enzymes involved in interconversion of a
topologically different form of the DNA are called
topoisomerases. The DNA topoisomerases are classified
into type I and type II. Type I topoisomerase transiently
breaks either one strand at a time (also called a nick),
while type II topoisomerase breaks both strands at the
same time.20) Due to the essential character of DNA
replication, it is natural that microorganisms such as
streptomycetes produce metabolites such as novobiocin
and coumermycin that kill their neighbors by inhibiting
the DNA replication process, the target of which has
been found to be type II topoisomerase, specifically
DNA gyrase.20) Nalidixic acid, the prototype quinolone,
was discovered to possess antibacterial activity in
196221) and was approved for urinary tract infections
in 1964.22) This finding led to more potent quinolone
antibiotics, such as norfloxacin, ciprofloxacin, ofloxacin,
and gatifloxacin, also called fluoroquinolone due to the
presence of a fluorine substituent. These quinolones
target the DNA gyrase and affect the double-strand
cleavage/double-strand religation equilibrium in the
gyrase catalytic reaction, in such a way that the cleaved
complex accumulates by stabilization of the complex in
the presence of quinolones.20) A second type II top-
oisomerase, known as topoisomerase IV, is also impli-
cated in DNA replication and decatenation of knotted
daughter chromosomes at the end of bacterial DNA
replication,23) and thus is an important target, probably
the primary one in Staphylococcus aureus.24)
Transcription is an essential process for decoding
genetic information from DNA to RNA in all organisms.
RNA polymerase of bacteria composed of different
subunits with a stoichiometry of 2

0 ! to form the core
enzyme catalyzes transcription.25) Rifampicin (known in
some countries as rifampin) is a semisynthetic version of
rifamycin B, isolated from a strain of Amycolatopsis
mediterranea (formerly known as Streptomyces medi-
terranea or Nocardia mediterranea). It is used princi-
pally in the treatment of tuberculosis and leprosy.22)
Rifampicin inhibits RNA polymerase by binding to the

subunit of the enzyme at an allosteric site, not at the
active site, as revealed by resistant mutations in clinical
isolates of Mycobacterium tuberculosis and M. leprae26)
and X-ray structural analysis of the Thermus aquaticus
core RNA polymerase complexed with rifampicin.27)
4. Other targets
Folic acid metabolism. The longest-used antibacterial
agents are the sulfa drugs, or sulfonamides, first
introduced in the 1930s.6,8) The current generation of
sulfa drugs is sulfamethoxazole, which is used in
combination with trimethoprim.22) Each antibacterial
agent is bacteriostatic when used individually, but they
are bactericidal when used together. This effect makes
the combination recipe remains clinically relevant in the
treatment of many bacterial infections, such as urinary
and respiratory tract infections.22) Each of the drugs
inhibits distinct steps in folic acid metabolism (Fig. 1).
Sulfamethoxazole inhibits dihydropteroate synthase in
the biosynthetic pathway of folate in a competitive
manner with higher affinity for the enzyme than a
natural substrate, p-aminobenzoic acid.22) The resulting
dead-end product cannot be a substrate for the next
enzyme, dihydrofolate synthase. Since humans do not
have dihydropteroate synthase, sulfa drugs achieve high
selectivity against bacteria. Trimethoprim inhibits dihy-
drofolate reductase, a key enzyme providing a one-
carbon carrier, tetrahydrofolate, for the synthesis of
deoxythymidylate, purine nucleotides, and the amino
acids glycine and methionine.28) In contrast to dihy-
dropteroate synthase, dihydrofolate reductase is essen-
tial for all organisms. Trimethoprim is a competitive
inhibitor of dihydrofolate reductase, and is 50,000 to
100,000 times more active against bacterial enzymes
than against the human counterparts, achieving high
selectivity.22) A combination of trimethoprim and sulfa
drugs acting at distinct steps on the same biosynthetic
pathway shows synergy and a reduced mutation rate for
resistance as compared to the use of each drug alone,
providing validation of combination chemotherapy.
Cell membranes. The integrity of cell membranes,
including the outer membrane of Gram-negative bac-
teria, is essential to all organisms. Cationic peptides
exemplified by the polymyxins, a group of cyclic
peptides with a fatty acid chain attached to the peptide,
attack the cytoplasmic membrane of Gram-positive and
Gram-negative bacteria, and the outer membrane of
Gram-negative bacteria.29) The interaction of the cat-
ionic peptide and the membrane disrupts membrane
organization and causes increased permeability of cell
components, which eventually leads to cell-killing.29)
The toxicity of polymyxin B due to its membrane-active
nature restricts its use in topical applications.29)
 Antibiotic Resistance in Bacteria
A second set of antibacterial peptides is bacteriocins
produced by both Gram-positive and Gram-negative
bacteria to compete with neighboring bacterial cells.30)
With some exceptions, most of these bacteriocins, such
as nisin, permeabilize target cell membranes.29,30)
Studies on the mechanism of action of nisin show that
it binds with high affinity to a docking molecule, lipid II,
a precursor of cell-wall biosynthesis, leading to the
hypothesis of the dual functionality of nisin.31,32) Recent
structural analysis of the nisin-lipid II complex reveals
that a novel lipid II recognition motif of nisin and related
lantibiotics (lanthionine-containing antibiotic peptides)
binds to the pyrophosphate moiety of lipid II. This
provides a basis for rational design of future anti-
biotics.33)
III. Resistance to Antibiotics
Since sulfonamide and penicillin were introduced into
clinical use in the 1930s and 1940s respectively, people
were captured by the illusion that infectious diseases
were totally controlled by antibiotics. The widespread
use of antibiotics, however, imposes strong selection
pressure for the development of antibiotic resistance, a
presentday major public health problem.4,34) We must
now accept that the occurrence of antibiotic resistance is
inevitable, that is, it is only a question of time.35) As the
frequency of antibiotic use increases, the speed of
resistance development also increases, thereby reducing
the effectiveness of the antibiotics. Clinically significant
resistance appears after months to years whenever a new
antibiotic is introduced into medical use.36,37) For
example, penicillin resistance was detected a few years
after its clinical debut in 1942,38) and streptomycin, a
year after its discovery in 1944.35,39) In the exceptional
case of vancomycin, its resistance appeared almost 30
years (in 1987) after its clinical introduction.40) The long
delay was likely due in part to limited use of
vancomycin in the first 25 years, since other effective
antibiotics were available during the ‘‘antibiotic era’’
of the 1950s through the 1960s. But then indiscriminate
use of antibiotics resulted in the emergence of methi-
cillin-resistant Straphylococcus aureus (MRSA), which
exhibits multiantibiotic resistance against many struc-
turally unrelated antibiotics.41) In turn, vancomycin-
resistant Enterococci (VRE) appeared in 1986 due to
widespread use of vancomycin, which was regarded as
the antibiotic of last resort.11,42) Then the discovery of
partially vancomycin-resistant strains of S. aureus in
1997 lead to the first case of fully vancomycin-resisitant
S. aureus.43)
More to the point, what makes the problem more
complicated is that about 50% of antibiotics are used for
prophylaxis, chemotherapy, and growth promotion in
animals.42,44–46) There is now evidence that antibiotic
use in animals plays a role in the development of
antibiotic-resistance in human pathogens.47) A case of
causal relationship of the glycopeptides, vancomycin
1063
and avoparicin, pushed the European Union to ban the
use of avoparcin in 1997 for growth promotion on the
basis of the ‘‘Precautionary Principle.’’48)
To combat these life-threatening problems, continu-
ing efforts to develop new antibiotics are obviously
needed. Since the introduction of nalidixic acid in 1962,
a totally new class of antibiotic oxazolidinones (line-
zolid) and cyclic lipopeptides (daptomycin) were ap-
proved in 2000 and 2003 respectively for clinical use in
the United States.4,8) Although these new antibiotics are
effective against vancomycin-resistant MRSA and En-
terococci, it is clear that the problematic pathogens will
eventually develop resistance to these compounds too,
and that the therapeutic lifetime of the new antibiotics
will be shortened if we use them indiscriminately.
Indeed, clinical isolates of S. aureus and Enterococcus
faecium showing linezolid resistance were first reported
in 2001, a year after launch.49,50)
Antibiotic resistance can be either intrinsic (natural)
or acquired. An example of intrinsic resistance is
Pseudomonas aeruginosa, whose low membrane per-
meability is likely to be a main reason for its innate
resistance to many antibiotics.9,51) Acquired resistance is
caused by acquisition of a plasmid and/or transposon
that harbors determinants for resistance, or by chromo-
some mutation.35,42) The major mechanisms of antibiotic
resistance are (i) prevention of accumulation of anti-
biotics either by decreasing uptake or increasing efflux,
(ii) inactivation of antibiotics either by hydrolysis or by
modification, and (iii) qualitative alteration of the target
that reduces the affinity for antibiotics either by mutation
or by target modification, or quantitatively by over-
production of the target (Fig. 2).9,26,35,36)
A
C
B
or
P
Fig. 2. Principle Mechanisms of Antibiotic Resistance.
A, Prevention of accumulation of antibiotics by decreasing uptake
or increasing efflux. B, Inactivation of antibiotics by hydrolysis or
modification, in this case, phosphorylation. C, Alteration of targets
qualitatively to reduce the affinity for antibiotics or quantitatively by
overproduction of the target.
1064 H. YONEYAMA and R. KATSUMATA

1. Efflux pumps
Prevention of antibiotic access to their targets results A
from either decreasing the influx or increasing the efflux
of antibiotics across biological membranes (Fig. 2).9,52)
Pseudomonas aeruginosa, an opportunistic pathogen,
exemplifies this mechanism of antibiotic resistance.
Mutants lacking an outer membrane porin channel,
OprD2, decrease the uptake of carbapenem, a
-lactam
antibiotic, and increase resistance to this antibiotic due
to low accessibility of the drug to its target, penicillin-
binding protein.9,53) A mutation resulting in overexpres-
sion of a multidrug efflux pump, MexAB-OprM, renders
this organism resistant to a wide variety of structurally
unrelated antibiotics.54,55) Since a loss-of-function mu-
tation in the MexAB-OprM efflux pump causes hyper-
sensitivity toward various antibiotics, the intrinsic B Side view Vertical view
resistance of this organism appears to be due mainly
to an active efflux of antibiotics.54,56)
OprM
From the first indication that plasmid-coded tetracy- trimer
cline resistance in E. coli is due to energy-dependent
efflux of the drug,52) this mechanism of antibiotic
resistance, namely transmembrane efflux of antibiotics, MexA
attracted scientists’ attention, and led to the recognition
of the widespread presence of multidrug efflux pumps in
bacteria that create resistance to clinically important
drugs.9,51) The efflux pumps are grouped into five
families based on their mechanisms and primary
sequence homologies: (i) the major facilitator super- MexB
trimer
family (MFS), (ii) the resistance-nodulation-division
(RND) family, (iii) the small multidrug resistance
(SMR) family, (iv) the multidrug and toxic compounds
extrusion (MATE) family, and (v) the ATP-binding
cassette (ABC) superfamily.57) Each member of the first Fig. 3. 3D Structure of Multidrug Exporter.
A, Cutaway view displaying the solvent-accessible surface of
four groups is a secondary transporter that uses proton
AcrB. The front protomer is removed. Broken lines indicate the
motive force (or sodium ion motive force for the MATE putative membrane boundaries and the framework of the funnel and
family) to expel antibiotics from cells.57) Members of cavity. Broken arrows indicate the putative substrate translocation
the ABC superfamily are primary transporters that use pathway. Pore helices and transmembrane helices comprising a
energy liberated by ATP hydrolysis such as LmrA of groove are depicted in yellow and green respectively. MCIPC,
cloxacillin; DOC, deoxycholate; AC, acriflavine; TC, tetracycline.
Lactococcus lactis.58)
Adapted with permission from (67). B, Assembly model of the
The ubiquitous multidrug efflux pumps raised the MexAB-OprM effulx pump. The figure is based on the 3-fold
question how they recognize and transport multiple MexA-dimer model. Adapted with permission from (61).
structurally unrelated antibiotics, and the question what
their natural physiological role is. Recently, there has
been tremendous progress in structural analysis of stoichiometry of the pump is predicted to be 1:1:1, 1:1:2,
multidrug efflux pumps and their cognate regulators or 1:1:3 for MexB:OprM:MexA.61,62,65) Quantitative
that has provided significant insight into these ques- analysis of each subunit in intact cells supports the
tions.57,59) Efflux pumps of the RND family are 3-fold MexA-dimer model in which the functional pump
composed of an inner membrane proton antiporter, a is composed of MexB trimer, OprM trimer, and three
periplasmic membrane fusion protein, and an outer dimers of MexA (Fig. 3B).61,66) The mechanism of
membrane exit channel.9,51,57) The crystal structures multidrug efflux by the tripartite pump has been
of an inner membrane component, AcrB of E. coli suggested by the striking features of the AcrB struc-
(Fig. 3A),60) a periplasmic protein, MexA of P. aerugi- ture.60) Substrates are collected from the inner and outer
nosa (Fig. 3B),61,62) and outer membrane channels, TolC leaflet of the plasma membrane through vestibules,
of E. coli63) and OprM of P. aeruginosa (Fig. 3B),64) which are formed between AcrB protomers, captured on
have been solved. It has been suggested that the the wall of the central cavity, and then actively trans-
functional tripartite efflux pump of P. aeruginosa ported through the pore into the TolC channel
comprises MexB trimer, a homolog of AcrB, OprM (Fig. 3A).60,67,68) The wide substrate specificity of the
trimer, and three to nine protomers of MexA. Hence the multidrug efflux pump can be explained by the large
 Antibiotic Resistance in Bacteria
central cavity that interacts with different substrates,
mainly through hydrophobic interaction, as well as
electrostatic interaction.69)
2. Inactivation of antibiotics
Enzymatic inactivation of antibiotics either by hy-
drolysis or by modification is a major mechanism of
resistance in pathogenic bacteria to natural antibiotics
such as
-lactams (penicillins and cephalosporins),
aminoglycosides, and chloramphenicol.35,36) The classic
case is the hydrolysis of the four-membered
-lactam
ring in penicillins and cephalosporins by hydrolytic
enzymes,
-lactamases, resulting in products that cannot
inhibit their targets, penicillin binding proteins (PBPs)
(Fig. 4A).35,36) A group of
-lactamases, which have
serine residue at their active sites, form penicilloyl-O-
Ser
-lactamase covalent intermediates as in the
catalytic cycle of PBPs, which also form penicilloyl-
O-Ser PBPs.70) The different lifetimes of each penicil-
loyl-O-Ser enzyme intermediate result in totally differ-
ent outcomes (Fig. 4A). The long lifetime of penicilloyl-
O-Ser PBPs results in inactivation of PBP transpeptidase
activity, leading to prevention of cross-linking.70,71) By
contrast, the deacylation rate of penicilloyl-O-Ser
-
lactamase is fast, resulting in hydrolyzed, ring-opened
penicilloic acid that is deactivated and nonfunctional as
a PBP pseudosubstrate.36) The structural and mechanical
similarities of the serine
-lactamases to the PBPs
suggest evolution of the
-lactamase from PBPs.71,72)
In contrast to
-lactams, the aminoglycoside anti-
biotics do not possess hydrolytically fragile groups.
Thus, the enzymatic inactivation strategy for amino-
glycoside-resistant bacteria is covalently to modify
the OH and NH2 groups of the aminoglycosides
(Fig. 4B).73,74) There are three types of aminoglyco-
side-modifying enzymes: aminoglycoside acetyltrans-
ferases, which transfer the acetyl group from acetyl
CoA, aminoglycoside phosphotransferases, which trans-
fer the phosphoryl group from ATP, and aminoglycoside
adenylyltransferases, which transfer the AMP group
from ATP.73,74) Aminoglycosides bind specifically to
regions near the A site for aminoacyl-tRNA binding of
16S rRNA on the 30S ribosome subunit by hydrogen
bond network through the various hydroxyl and amino
substitutions of the antibiotics.16,17) This high affinity
binding of aminoglycosides with rRNA inhibits protein
biosynthesis.16,17) Enzymatic modification of aminogly-
cosides disrupts the hydrogen bond network, resulting in
the considerably lower affinity of the drugs for the
rRNA.
Chloramphenicol has been found to inhibit protein
biosynthesis by preventing binding of tRNA to the A site
of the ribosome.75,76) Recent structural analysis of the
bacterial 50S ribosome subunit complexed with chlo-
ramphenicol has revealed that the antibiotic interacts
specifically with various nucleotides of the peptidyl
transferase cavity of the 23S rRNA through hydrogen
bonds without any interactions with ribosomal pro-
1065
18)
teins. Chloramphenicol acetyltransferases, widely dis-
tributed among bacterial pathogens of all genera,
inactivate chloramphenicol by transferring the acetyl
group from acetyl CoA (Fig. 4C).77) This modification
of chloramphenicol again results in the considerably
lower affinity of the antibiotic for the rRNA.
Enzymatic inactivation of antibiotics is one of the
most common mechanisms of antibiotic resistance.35)
The antibiotic-resistant clinical isolates in most cases
inherit the antibiotic-resistance genes on R (resistance)
plasmids.35) Although definitive evidence for the origin
of the antibiotic-resistant determinants remains to be
found, such resistance determinants most probably are
acquired by pathogenic bacteria from a pool of resist-
ance genes in other microbial genera, including anti-
biotic-producing organisms.35)
-lactamase was identi-
fied in 1940, several years before the introduction of
penicillin into clinical use, indicating that there has been
a pool of antibiotic resistant genes in nature.78)
3. Alteration of the target
In contrast to enzymatic inactivation of natural
antibiotics, no enzymes that hydrolyze or modify man-
made antibiotics such as sulfonamides, trimethoprim,
and quinolones have been found.26) The mechanism of
antibiotic resistance for these synthetic antibiotics and
those natural products such as rifampicin, where
inactivating enzymes have not been found, is commonly
achieved by target alterations.26)
The most common mechanism of target alteration is
the acquisition of new genes, carried on plasmids or
transposons, that result in enzymatic modification of the
normal target so that it does not bind the antibiotics. The
erythromycin family of macrolides and the streptogra-
min B family exemplify this mechanism of antibiotic
resistance.36,79) In erythromycin-resistant bacteria, the
exocyclic amino group of a specific adenine residue,
A2058, in 23S rRNA is mono- or dimethylated by an
Erm (erythromycin ribosome methylation) methyltrans-
ferase, resulting in cross-resistance to macrolides,
lincosamides, and streptogramin B (MLSB phenotype)
(Fig. 5A).80,81) This modification reduces the affinity of
the rRNA for the antibiotics but does not interfere with
protein biosynthesis. The recent crystal structure of the
50S subunit of the ribosome complexed with erythro-
mycin revealed the importance of hydrogen bonds
between erythromycin and A2058 of 23S rRNA.18)
Erm is the major resistance mechanism in antibiotic-
resistant Staphylococcus aureus and is present in
erythomycin-producing organisms as a self-protection
mechanism.79,80)
Another example of target alteration can be seen in
VRE. Five clinical phenotypes of vancomycin resistance
have been identified so far, VanA, VanB, VanC, VanD,
and VanE.82) VanA, VanB, VanD, and VanE are
acquired resistance phenotypes, and VanC is an intrinsic
resistance phenotype.83) The enzymes in VanA, VanB,
and VanD resistance are involved in reprogramming of
1066 H. YONEYAMA and R. KATSUMATA

A
R R S
S
H2 O
+ β−lactamase
O N O N
H H
O COOH HO COOH
rapid
Ser deacylation
β−lactamase
R β−lactamase
S
N
O
COOH PBP H2 O
R S
O N
H very slow
O deacylation
COOH
Ser

PBP

B O

HN HO
HO O O OH
N−Acetylation HO NH2
OH HO
HO
O O
H2 N NH 2

NH 2 HO NH 2 HO
HO O O OH O HO O O OH
HO NH2 O−Phosphorylation O NH2
OH HO P OH HO
HO HO HO
O O OH O O
H2 N NH2 H2 N NH2

NH2 HO
AMP O O O OH
O−Adenylation HO NH2
OH HO
HO
O O
H2 N NH2

C
O
C CH3
nonenzymatic
OH OH O reaction O
CAT
OH O C CH3 OH
HN CHCl2 HN CHCl2 HN CHCl2
O2N O2N O2 N
O Acetyl−CoA O O

Acetyl−CoA
CAT

O
C CH3
O O
O C CH3
HN CHCl2
O2N
O

Fig. 4. Inactivation of Antibiotics Either by Hydrolysis or Modification.

A, Inactivation of penicillin by
-lactamases. During the inactivation of PBPs by penicillins, hydrolysis of the penicilloyl-O-Ser PBP
intermediate is very slow due to the inaccessibility of the active site to water. By contrast, free access of water to the penicilloyl-O-Ser
-
lactamases hydrolyses the intermediate very rapidly, leading to the inactivation of penicillins. B, Inactivation of aminoglycosides by acetylation,
phosphorylation, and adenylation. Adapted with permission from (6). C, Inactivation of chloramphenicol by chloramphenicol acetyltransferase
(CAT). CAT catalyzes acetylation of chloramphenicol at C-3 to yield 3-acetylchloramphenicol. Next, nonenzymatic intramolecular transfer of
the acetyl group from C-3 to C-1 occurs to produce a mixture of 1- and 3-acetylchloramphenicol. Finally, the 1-acetylchloramphenicol is
acetylated again by CAT to yield 1,3-diacetylchloramphenicol. Both mono- and di-acetylchloramphenicol are inactive.
 Antibiotic Resistance in Bacteria 1067

A
EM EM

CH3 CH3
NH2 N
Erm
N methyltransferase N
N N

N N N N

A2058 A2058

B
HO HO
NH2 NH2
O O
OH OH
O OH O OH
O O OH O O OH
Cl Cl
O O O O
OH OH
HO Cl O HO Cl O
O O H O O H
H N H N
O N N N O N N N
N N H H N N H H
H H H H
NH O O NH O O
HO O HO O
NH2 NH2
O OH O OH
OH OH
HO HO

O H O O O
N O
R N O R N O
H O H O
N−Acyl−D−Ala−D−Ala N−Acyl−D−Ala−D−Lactate

Fig. 5. Antibiotic Resistance Caused by Target Alterations.

A, Enzymatic dimethylation of the amino group of a specific adenine residue, A2058, in 23S rRNA disrupts interaction of the reactive groups
of erythromycin (EM) with the nucleotides of the peptidyl transferase cavity. B, Reprograming of the peptidoglycan termini from D-Ala-D-Ala
to D-Ala-D-lactate causes loss of one hydrogen bond between vancomycin and D-Ala-D-lactate moiety, leading to a 1,000-fold decrease in
binding affinity. Adapted with permission from (6).

the peptidoglycan termini from D-Ala-D-Ala to D-Ala-
D-lactate, which decreases the binding affinity of
vancomycin to its target (D-Ala-D-Ala) by 1,000-fold,
leading to high vancomycin resistance (Fig. 5B).84) On
the other hand, VanC and VanE replace the terminal D-
Ala with D-Ser, leading to low-level resistance.85)

-lactam antibiotic resistance can be caused not only
by
-lactamase, but also by alteration (or mutation) of
PBPs that have low affinity for the
-lactams, as well as
by acquisition of new PBPs with low affinity for the
antibiotic.26,36) Unlike many other pathogens, Strepto-
coccus pneumoniae does not employ
-lactamases as the
major mechanism of resistance to
-lactams. Its resist-
ance to
-lactams is due to alteration of PBPs.26) The
most highly
-lactam-resistant clinical isolates produce
altered forms of high-molecular weight (Mr) PBPs (1A,
1B, 2A, 2B, and 2X) that have reduced affinity for
-
lactams.86–89) It is now known that low-affinity forms of
high-Mr pneumococcal PBPs have arisen by interspecies
homologous recombination,26,90,91) suggesting the oc-
currence of gene shuffling in nature. MRSA acquires the
mecA gene, which encodes a new PBP, termed PBP20
(also PBP2A), with low affinity for all
-lactams.26,36)
Although the source of the PBP20 gene is not known,
horizontal transmission of the mecA gene from other
Staphylococcus species has been postulated.26,92–94)
IV. New Approaches to Development of
Novel Antibiotics
There is a pressing need for new antibiotics due to the
inevitable development of resistance that follows the
introduction of antibiotics to the clinic. In the past 10–15
years, there has been a dramatic increase in the
emergence of antibiotic-resistant bacteria, including
multiantibiotic resistant strains in both community and
hospital settings.95,96) The main strategy of the pharma-
ceutical industry for development of new therapeutics
has been modification of existing antibiotics, such as the
second- and third-generation molecules of the best-
selling antibiotic groups of
-lactams, macrolides, and
fluoroquinolones.8) Although this approach is effective,
it has turned out to be increasingly difficult to launch
new drugs to meet the needs of the community.96)
1068 H. YONEYAMA and R. KATSUMATA
Almost 40 years were required for the development of a
new structural class of synthetic antibiotics of broad
spectrum, the oxazolidinone linezolid, since the intro-
duction of nalidixic acid in 1962.8) Since the main
classes of antibiotic targets are cell wall, protein, DNA/
RNA, and folate biosynthetic pathways, expanding the
number of potential targets is of great importance for
discovery and development of new antibiotics to keep
pace with the emergence of antibiotic resistance.
The explosion in microbial genome sequencing in the
past decade has revealed over 200 complete bacterial
genome sequences, including important pathogens (270
as of January 8, 2006; http://gib.genes.nig.ac.jp/), which
provides more opportunities to identify and validate new
antibacterial targets.8,96) The bioinformatics approach is
based on searches of open reading frames (ORFs)
conserved among potential pathogenic bacteria, as well
as ORFs found only in prokaryotes, not in eukar-
yotes.8,96) An example of this approach compared the
4,289 E. coli genes against the genomes of Haemophilus
influenzae, Mycoplasma, S. pneumoniae, Streptococci,
Chlamydia pneumoniae, Klebsiella, and P. aeruginosa.
The process identified 246 conserved genes in all these
bacteria, of which 68 genes are not found in humans.97)
Next, loss-of-function tests showed that 18 genes were
essential, 16 genes nonessential, and 34 genes unknown
as to function. In this bioinformatic analysis, three genes
were finally chosen as targets for new respiratory-tract
antibiotics.97)
The key aspect of the potential targets is that they are
essential for bacterial growth or viability and are
expressed under the conditions of the infection proc-
ess.96,98) There is now good evidence to suggest that the
expression pattern of pathogens during infection can be
quite different from that in culture medium.3,96,99) In
fact, traditional whole-cell screenings focus on targets
that are important for in vitro growth in rich media and
thus might not identify inhibitors of target systems that
are induced specifically in vivo and are relevant to the
infection process.
Several new techniques detect and monitor gene
expression during infection. In vivo expression technol-
ogy (IVET) allows identification of genes specifically
induced in the host. It has been applied successfully to
several pathogens.100,101) A similar technique, differ-
ential fluorescence induction (DFI), utilizes promoter
fusions with green fluorescent protein to identify active
promoters in vivo.101,102) Signature-tagged mutagenesis
(STM) is another technique that identifies the genes
required for the infection process and viability in
vivo.101,103)
Combinatorial chemistry has a significant impact on
the discovery of new antibiotics.104) This new approach
to high-throughput screening requires large, diverse
synthetic compounds and natural products. In nature,
biosynthetic assembly lines of polyketides and non-
ribosomal peptides furnish an example of combinatorial
libraries.104) The structures of complex polyketide
natural products, such as erythromycin, are assembled
by multifunctional polyketide synthases (PKSs) that
contain modular arrangements of functional domains.104)
A library of >50 macrolides that are not produced by
chemical methods have been generated by simultaneous
manipulation of multiple catalytic centers of erythro-
mycin PKS genes.105)
The high-resolution X-ray crystal structures of
the 30S and 50S ribosomal subunits as well as whole
70S ribosome complexed with antibiotics offer a
good opportunity to develop new antibiotic com-
pounds.16–18,106,107) As to macrolides, the 3D structures
of the ligands adjacent to peptidyl-transferase center on
the 50S ribosomal subunit reveal how an inhibitor binds
to the target, thus facilitating computational analysis of
other factors that contribute to binding, such as shape
and electrostatic complementlity.18,106–108) This struc-
ture-based drug-design approach produces promising
potential macrolide derivatives against respiratory tract
pathogens resistant to macrolides and ketolides, pen-
icillin, chloramphenicol, and sulphamethoxazole.106)
V. New Targets
Historically, the main classes of antibiotics inhibit
four classical targets: (i) cell wall biosynthesis, (ii)
protein biosynthesis, (iii) DNA and RNA biosynthesis,
and (iv) folate coenzyme biosynthesis.8) The recent
advance in bacterial genomics has changed the anti-
bacterial therapeutic environment from target-poor to
target-rich. The genomics provide many molecular
targets to be identified and effectively screened:5,96)
peptide deformylase (PDF),109) aminoacyl-tRNA syn-
thetase,110) fatty acid biosynthesis,111) isoprenoid biosyn-
thesis,112) lipid A biosynthesis,113) efflux pump,114)
aromatic amino acid biosynthesis,96) DNA replication
(GyrB subunit inhibitor),115) protein secretion,116) cell
wall biosynthesis (sortase),117) proteasome,118) glyoxy-
late cycle,119) and signal networks (two-component
systems and quorum sensing pathways).120,121)
1. Peptide deformylase (PDF)
Although there have been few novel antibacterial lead
compounds that produce commercially successful anti-
biotics from these new approaches so far, PDF appears
to have gained intense awareness recently as a promis-
ing target.109) Although the bacterial protein-synthesiz-
ing machinery is similar to those of mammalian cells,
there are sufficient differences to allow selective
inhibition of this process in bacteria. One significant
difference is the transformylation and subsequent de-
formylation of initiating methionine in bacteria.122,123)
Unlike cytosolic protein synthesis in mammalian cells,
protein synthesis in bacteria and mitochondria is
initiated with N-formylmethionine,124,125) which is
formed by N-methionyl-tRNA transformylase.126) In
bacteria, but not in mitochondria, the N-formylmethio-
nine of newly synthesized protein is removed sequen-
 Antibiotic Resistance in Bacteria
tially by PDF and a methionine amino peptidase to
produce mature proteins.125,127) This essential role of
bacterial PDF in protein biosynthesis provides a rational
basis for selective inhibition. PDF inhibitor-resistant
mutants appear easily like other antibiotics; the trans-
formylase gene has changed in E. coli, H. influenzae,
and S. aureus.128–130) But resistant mutants defective in
the transformylase gene have a slow growth pheno-
type,109,128) suggesting diminished virulence of the
pathogenic bacteria. Indeed, a PDF inhibitor-resistant
mutant of S. aureus showed weakened virulence in a
murine thigh-abscess model,130) and hence this type of
resistance might not emerge in a clinical situation.109)
2. Non-mevalonate pathway
Although the mevalonate pathway has been known
for several decades as the source of isoprenoids, an
alternative non-mevalonate pathway has recently been
found,112,131) in which the first intermediate 1-deoxy-D-
xylulose 5-phosphate, also an intermediate in thiamine
and pyridoxal biosynthesis, is formed by condensing
glyceraldehyde 3-phosphate and pyruvate.112) In contrast
to animals that use the mevalonate pathway exclusively,
many bacteria, including clinically important pathogens,
utilize the non-mevalonate pathway.112) Since isoprenoid
biosynthesis is essential and humans do not have
enzymes of the non-mevalonate pathway, all of the
seven enzymes in the non-mevalonate pathway are
attractive antibacterial targets for the development of
novel antibiotics. In fact, the 2C-methyl-D-erythritol 4-
phosphate synthase inhibitor fosmidomycin has been
shown to possess antibacterial activity.132) On the other
hand, bacterial bioinformatics suggests that genes
encoding the mevalonate pathway are essential in
S. aureus and other Gram-positive cocci. Hence inhib-
itors of the mevalonate pathway should work against
these Gram-positive pathogens.133,134)
3. Bacterial fatty acid synthesis
Fatty acid biosynthesis is essential for bacterial
growth.5,96) In mammals, fatty acid is synthesized by a
single large polypeptide called fatty acid synthase
(FAS),135) whereas in the bacterial FAS system individ-
ual enzymes perform the individual synthetic steps,136)
suggesting that the differences in the systems are the
basis for selective inhibition by natural products, such as
cerulenin, thiolactomycin, and isoniazid.5) Recently, an
antibacterial antiseptic, triclosan, was shown to inhibit
enoyl-ACP reductase (FabI) in E. coli,137) and isoniazid,
an important antituberculosis drug, was found to be
activated by mycobacterial KatG catalase to acylate
NAD bound in the active site of InhA, the homolog of
FabI of E. coli, and thus to inhibit the enzyme.138) Thus
the fatty acid biosynthetic pathway is validated to
provide good targets. Screening against FabI has
identified two novel antibacterial leads (benzodiazepine
and imidazole compounds) that inhibit S. aureus en-
zyme selectively, but not the human FAS system.111)
1069
Recent structural analysis of bacterial fatty acid bio-
synthetic enzymes complexed with ligand is also
facilitating the lead optimization process.139–141)
4. Bacterial virulence factors
In the past decades, antibiotic discovery programs
have focused on eliminating infection by bactericidal
and/or bacteriostatic agents that target essential proc-
esses for bacterial viability in vitro. However, patho-
genic bacteria elaborate virulence factors, involved in
adhesion, invasion, and host defense system evasion, to
establish an infection142) the expression of which is
carefully regulated in response to environmental
changes.99) Recently developed techniques, such as
IVET, DFI, and STM, have identified genes that are
involved in the infectious process of pathogenic bacteria
in the context of their expression.100–103) According to
‘‘Koch’s molecular postulates,’’ genetic disruption of an
essential virulence factor should reduce bacterial viru-
lence.143) In fact, allelic replacement of individual genes
causes attenuation of virulence in in vivo models,
confirming their product’s role in infection.99) They are
global regulators of virulence factor expression and
sortase in S. aureus,117,144) the two-component system in
S. pneumoniae145) and in S. aureus,146) the quorum
sensing system in P. aeruginosa,147) host cell invasion
in S. enterica,148) S. flexneri,149) and L. monocyto-
genes,150) and the glyoxylate cycle in M. tuberculo-
sis.119) These factors are unique to bacteria, and hence
the virulence factors can be a target for development of
new antibiotics.99)
The current concept of antibiotic discovery is that
broad-spectrum agents are preferred over narrow-spec-
trum ones, implying that targets should be conserved
among pathogenic bacteria.99) In this context, inhibitors
targeting these virulence factor-related targets are
narrow-spectrum. Among them, sortase, which cova-
lently attaches cell-surface proteins to the peptidoglycan
layer in Gram-positive bacteria, is an attractive drug
target, since sortase has a common substrate specificity
(LPXTG)151–153) and should be easily accessible to drugs
due to its cell-surface location. In addition, the 3D
structure of sortase has been determined by NMR
spectroscopy, enabling rational structure-based drug
design.154) Thus there is an increasing possibility of a
single sortase inhibitor with activity against many Gram-
positive pathogens.99)
VI. Conclusion
The development of antibiotics is among the greatest
achievements of medicine in human history, but it is
clear that infectious diseases are still of major medical
and scientific concern.96,98) The ability of bacterial
pathogens to gain resistance to new antibiotics has been
proved since the early introduction of sulfonamides and
penicillins into clinical use.8,35) To keep pace with the
development of antibiotic resistance in bacterial patho-
1070 H. YONEYAMA and R. KATSUMATA
gens, we must continue the development of novel
antibiotics.4,8,155) In addition, it is of great importance to
rethink strategies for preserving and extending the useful
life of antibiotics, such as rotating the use of anti-
biotics156) and combination antibiotic therapy.8,36) The
challenges can be met only by understanding the
fundamental biology of bacterial pathogens and host-
pathogen interactions, and by valuing the antibiotics as
precious, finite resources.36)
Acknowledgments
We are grateful to Professor Yoshiyuki Kamio of
Tohoku University for making it possible for us to write
this article.
References
1) Cohen, M., Changing patterns of infectious disease.
Nature, 406, 762–767 (2000).
2) Fauch, A. S., Infectious diseases: considerations for the
21st century. Clin. Infect. Dis., 32, 675–685 (2001).
3) Nathan, C., Antibiotics at the crossroads. Nature, 431,
899–902 (2004).
4) Leeb, M., A shot in the arm. Nature, 431, 892–893
(2004).
5) Projan, S. J., New (and not so new) antibacterial
targets: from where and when will the novel drugs
come? Curr. Opin. Pharmacol., 2, 513–522 (2002).
6) Walsh, C., ‘‘Antibiotics, Action, Origins, Resistance,’’
ASM Press, Washington, DC, pp. 3–49 (2003).
7) Mascaretti, O. A., ‘‘Bacteria Versus Antibacterial
Agents, an Integrated Approach,’’ ASM Press, Wash-
ington, DC, pp. 97–105 (2003).
8) Walsh, C., Where will new antibiotics come from? Nat.
Rev. Microbiol., 1, 65–70 (2003).
9) Nikaido, H., Prevention of drug access to bacterial
targes: permeability barriers and active efflux. Science,
264, 382–388 (1994).
10) Spratt, B. G., and Cromie, K. D., Penicillin-binding
proteins of gram-negative bacteria. Rev. Infect. Dis.,
10, 699–711 (1988).
11) Williams, D. H., The glycopeptide story: how to kill
the deadly ‘superbugs.’ Nat. Prod. Rep., 13, 469–477
(1996).
12) Ban, N., Nissen, P., Hansen, J., Moore, P. B., and
Steitz, T. A., The complete atomic structure of the
large ribosomal subunit at 2.4 Å resolution. Science,
289, 905–920 (2000).
13) Wimberly, B. T., Brodersen, D. E., Clemons, W. M.,
Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C.,
Hartsch, T., and Ramakrishnan, V., Structure of the
30S ribosomal subunit. Nature, 407, 327–339 (2000).
14) Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J.,
Gluehmann, M., Janell, D., Bashan, A., Bartels, H.,
Agmon, I., Franceschi, F., and Yonath, A., Structure of
functionally activated small ribosomal subunit at 3.3 Å
resolution. Cell, 102, 615–623 (2000).
15) Yusupov, M. M., Yusupova, G. Z., Baucom, A.,
Lieberman, K., Earnest, T. N., Cate, J. H. D., and
Noller, H. F., Crystal structure of the ribosome at 5.5 Å
resolution. Science, 292, 883–896 (2001).
16) Carter, A. P., Clemons, W. M., Brodersen, D. E.,
Morgan-Warren, R. J., Wimberly, B. T., and
Ramakrishnan, V., Functional insights from the struc-
ture of the 30S ribosomal subunit and its interactions
with antibiotics. Nature, 407, 340–348 (2000).
17) Brodersen, D. E., Clemons, W. M., Carter, A. P.,
Morgan-Warren, R. J., Wimberly, B. T., and
Ramakrishnan, V., The structural basis for the action
of the antibiotics tetracycline, pactamycin, and hy-
gromycin B on the 30S ribosomal subunit. Cell, 103,
1143–1154 (2000).
18) Schlunzen, F., Zarivach, R., Harms, J., Bashan, A.,
Tocilj, A., Albrecht, R., Yonath, A., and Franceschi, F.,
Structural basis for the interaction of antibiotics with
the peptidyl transferase centre in eubacteria. Nature,
413, 814–821 (2001).
19) Sherratt, D. J., Bacterial chromosome dynamics.
Science, 301, 780–785 (2003).
20) Maxwell, A., DNA gyrase as a drug target. Trends
Microbiol., 5, 102–109 (1997).
21) Lesher, G. Y., Forelich, E. D., Gruet, M. D., Bailey,
J. H., and Brundage, R. P., 1,8-Naphthyridone deriv-
atives. A new class of chemotherapeutic agents. J.
Med. Pharm. Chem., 5, 1063–1068 (1962).
22) Mascaretti, O. A., ‘‘Bacteria Versus Antibacterial
Agents, an Integrated Approach,’’ ASM Press, Wash-
ington, DC, pp. 289–328 (2003).
23) Pan, X., and Fisher, L. M., Targeting of DNA gyrase in
Streptococcus pneumoniae by sparfloxacin: selective
targeting of gyrase or topoisomerase IV by quinolones.
Antimicrob. Agents Chemother., 41, 471–474 (1997).
24) Ng, E. Y., Trucksis, M., and Hooper, D. C., Quinolone
resistance mutations in topoisomerase IV: relationship
to the flqA locus and genetic evidence that topoisomer-
ase IV is the primary target and DNA gyrase is the
secondary target of fluoroquinolones in Staphylococcus
aureus. Antimicrob. Agents Chemother., 40, 1881–
1888 (1996).
25) Murakami, K. S., and Darst, S. A., Bacterial RNA
polymerases: the whole story. Curr. Opin. Struct. Biol.,
13, 31–39 (2003).
26) Spratt, B. G., Resistance to antibiotics mediated by
target alterations. Science, 264, 388–393 (1994).
27) Campbell, E. A., Korzheva, N., Mustaev, A.,
Murakami, K., Nair, S., Goldfarb, A., and Darst, S.
A., Structural mechanism for rifampicin inhibition of
bacterial RNA polymerase. Cell, 104, 901–912 (2001).
28) Stryer, L., ‘‘Biochemistry’’ 4th ed., W. H. Freeman and
Company, New York, pp. 719–721 (1995).
29) Hancock, R. E. W., and Chapple, D. S., Peptide
antibiotics. Antimicrob. Agents Chemother., 43, 1317–
1323 (1999).
30) Baba, T., and Schneewind, O., Instruments of micro-
bial warfare: bacteriocin synthesis, toxicity and im-
munity. Trends Microbiol., 6, 66–71 (1998).
31) Breukink, E., Wiedemann, I., van Kraaij, C., Kuuipers,
O. P., Sahl, H.-G., and de Kruijff, B., Use of the cell
wall precursor lipid II by a pore-forming peptide
antibiotic. Science, 286, 2361–2364 (1999).
32) Wiedemann, I., Breukink, E., van Kraaij, C., Kuipers,
O. P., Bierbaum, G., de Kruijff, B., and Sahl, H.-G.,
Specific binding of nisin to the peptidoglycan precursor
lipid II combines pore formation and inhibition of cell
 Antibiotic Resistance in Bacteria
wall biosynthesis for potent antibiotic activity. J. Biol.
Chem., 276, 1772–1779 (2001).
33) Hsu, S.-T. D., Breukink, E., Tishenko, E., Lutters, M.
A. G., de Kuijuff, B., Kaptein, R., Bonvin, A. M. J. J.,
and van Nuland, N. A. J., The nisin-lipid II complex
reveals a pyrophosphate cage that provides a blueprint
for novel antibiotics. Nat. Struct. Mol. Biol., 11, 963–
967 (2004).
34) Palumbi, S. R., Humans as the world’s greatest
evolutionary force. Science, 293, 1786–1790 (2001).
35) Davies, J., Inactivation of antibiotics and the dissem-
ination of resistance genes. Science, 264, 375–382
(1994).
36) Walsh, C., Molecular mechanisms that confer anti-
bacterial drug resistance. Nature, 406, 775–781 (2000).
37) Livermore, D., Can better prescribing turn the tide of
resistance? Nat. Rev. Microbiol., 2, 73–78 (2004).
38) Travis, J., Reviving the antibiotic miracle? Science,
264, 360–362 (1994).
39) Waksman, S. A., Reily, H. C., and Schatz, A., Strain
specificity and production of antibiotic substances. V.
Strain resistance of bacteria to antibiotic substances,
especially to streptomycin. Proc. Natl. Acad. Sci. USA,
31, 157–164 (1945).
40) Murray, E., Vancomycin resistant enterococci. Am. J.
Med., 102, 284–293 (1997).
41) Neu, H. C., The crisis in antibiotic resistance. Science,
257, 1064–1073 (1992).
42) Coates, A., Hu, Y., Bax, R., and Page, C., The future
challenges facing the development of new antimicro-
bial drugs. Nat. Rev. Drug Discov., 1, 895–910 (2002).
43) Sievert, D. M., Boulton, M. L., Stoltman, G., Johnson,
D., Stobierske, M. G., Downes, F. P., Somsel, P. A.,
Rudrik, J. T., Brown, W., Hafeez, W., Lundstrom, T.,
Flanagan, E., Johnson, R., and Mitchell, J., Staph-
ylococcus aureus resistant to vancomycin: United
States, 2002. Morbid. Mortal. Wkly Rep., 51, 565–
567 (2002).
44) Falkow, S., and Kennedy, D., Antibiotics, animals, and
people—again! Science, 291, 397 (2001).
45) Witte, W., Medical consequences of antibiotic use in
agriculture. Science, 279, 996–997 (1998).
46) Gustafson, R. H., and Bowen, R. E., Antibiotic use in
animal agriculture. J. Appl. Microbiol., 83, 531–541
(1997).
47) Wehener, H. C., Antibiotics in animal feed and their
role in resistance development. Curr. Opin. Microbiol.,
6, 439–445 (2002).
48) Casewell, M., Friis, C., Marco, E., McMullin, P., and
Phillips, I., The European ban on growth-promoting
antibiotics and emerging consequences for human and
animal health. J. Antimicrob. Chemother., 52, 159–161
(2003).
49) Tsiodras, S., Gold, H. S., Sakoulas, G., Eliopoulos, G.
M., Wennersten, C., Venkataraman, L., Moellering, R.
C., and Ferraro, M. J., Linezolid resistance in a clinical
isolate of Staphylococcus aureus. Lancet, 358, 207–
208 (2001).
50) Gonzales, R. D., Schreckenberger, P. C., Graham, M.
B., Kelkar, S., DenBesten, K., and Quinn, J. P.,
Infections due to vancomycin-resistant Enterococcus
faecium resistant to linezolid. Lancet, 357, 1179
(2001).
1071
51) Nakae, T., Role of membrane permeability in deter-
mining antibiotic resistance in Pseudomonas aerugi-
nosa. Microbiol. Immunol., 39, 221–229 (1995).
52) McMurry, L., Petrucci, R. E., Jr., and Levy, S. B.,
Active efflux of tetracycline encoded by four genet-
ically different tetracycline resistance determinants in
Escherichia coli. Proc. Natl. Acad. Sci. USA, 77, 3974–
3977 (1980).
53) Yoneyama, H., and Nakae, T., Cloning of the protein
D2 gene of Pseudomonas aeruginosa and its functional
expression in the imipenem-resistant host. FEBS Lett.,
283, 177–179 (1991).
54) Poole, K., Krebes, K., McNally, C., and Neshat, S.,
Multiple antibiotic resistance in Pseudomonas aerugi-
nosa: evidence for involvement of an efflux operon. J.
Bacteriol., 175, 7363–7372 (1993).
55) Morshed, S. R. M., Lei, Y., Yoneyama, H., and Nakae,
T., Expression of genes associated with antibiotic
extrusion in Pseudomonas aeruginosa. Biochem. Bio-
phys. Res. Commun., 210, 356–362 (1995).
56) Yoneyama, H., Ocaktan, A., Tsuda, M., and Nakae, T.,
The role of mex-gene products in antibiotic extrusion
in Pseudomonas aeruginosa. Biochem. Biophys. Res.
Commun., 233, 611–618 (1997).
57) Paulsen, I. T., Multidrug efflux pumps and resistance:
regulation and evolution. Curr. Opin. Microbiol., 6,
446–451 (2003).
58) Bolhuis, H., Molenaar, D., Poelarends, G., van Veen,
H. W., Poolman, B., Driessen, A. J. M., and Konings,
W. N., Proton motive force-driven and ATP-dependent
drug extrusion systems in multidrug-resistant Lacto-
coccus lactis. J. Bacteriol., 176, 6957–6964 (1994).
59) Neyfakh, A. A., Mystery of multidrug transporters: the
answer can be simple. Mol. Microbiol., 44, 1123–1130
(2002).
60) Murakami, S., Nakashima, R., Yamashita, E., and
Yamaguchi, A., Crystal structure of bacterial multidrug
efflux transporter AcrB. Nature, 419, 587–593 (2002).
61) Akama, H., Matsuura, T., Kashiwagi, S., Yoneyama,
H., Narita, S., Tsukihara, T., Nakagawa, A., and
Nakae, T., Crystal structure of the membrane fusion
protein, MexA, of the multidrug transporter in Pseu-
domonas aeruginosa. J. Biol. Chem., 279, 25939–
25942 (2004).
62) Higgins, M. K., Bokma, E., Koronakis, E., Hughes, C.,
and Koronakis, V., Structure of the periplasmic
component of a bacterial drug efflux pump. Proc.
Natl. Acad. Sci. USA, 101, 9994–9999 (2004).
63) Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., and
Hughes, C., Crystal structure of the bacterial mem-
brane protein TolC central to multidrug efflux and
protein export. Nature, 405, 914–919 (2000).
64) Akama, H., Kanemaki, M., Yoshimura, M., Tsukihara,
T., Kashiwagi, T., Yoneyama, H., Narita, S.,
Nakagawa, A., and Nakae, T., Crystal structure of the
drug discharge outer membrane protein, OprM, of
Pseudomonas aeruginosa: dual modes of membrane
anchoring and occluded cavity end. J. Biol. Chem.,
279, 52816–52819 (2004).
65) Fernandez-Recio, J., Walas, F., Federici, L., Pratap, J.
V., Bavro, V. N., Miguel, R. N., Mizuguchi, K., and
Luisi, B., A model of a transmembrane drug-efflux
pump from gram-negative bacteria. FEBS Lett., 578,
1072 H. YONEYAMA and R. KATSUMATA
5–9 (2004).
66) Narita, S., Eda, S., Yoshihara, E., and Nakae, T.,
Linkage of the efflux-pump expression level with
substrate extrusion rate in the MexAB-OprM efflux
pump of Pseudomonas aeruginosa. Biochem. Biophys.
Res. Commun., 308, 922–926 (2003).
67) Murakami, S., and Yamaguchi, A., Multidrug-export-
ing secondary transporters. Curr. Opin. Struct. Biol.,
13, 443–452 (2003).
68) Yu, E. W., Aires, J. R., and Nikaido, H., AcrB
multidrug efflux pump of Escherichia coli: composite
substrate-binding cavity of exceptional flexibility gen-
erates its extremely wide substrate specificity. J.
Bacteriol., 185, 5657–5664 (2003).
69) Yu, E. W., McDermott, G., Zgurskaya, H. I., Nikaido,
H., and Koshland, D. E., Jr., Structural basis of
multiple drug-binding capacity of the AcrB multidrug
efflux pump. Science, 300, 976–980 (2003).
70) Fisher, J., Belasco, J. G., Khosla, S., and Knowles, J.
R.,
-lactamase proceeds via an acyl-enzyme inter-
mediate: interaction of the Escherichia coli RTEM
enzyme with cefoxitin. Biochemistry, 19, 2895–2901
(1980).
71) Knox, J. R., Moews, P. C., and Frere, J. M., Molecular
evolution of bacterial beta-lactam resistance. Chem.
Biol., 3, 937–947 (1996).
72) Knox, J. R., Extended-spectrum and inhibitor-resistant
TEM-type beta-lactamases: mutations, specificity, and
three-dimensional structure. Antimicrob. Agents Che-
mother., 39, 2593–2601 (1995).
73) Kotra, L. P., Haddad, J., and Mobashery, S., Amino-
glycosides: perspectives on mechanisms of action and
resistance and strategies to counter resistance. Anti-
microb. Agents Chemother., 44, 3249–3256 (2000).
74) Wright, G. E., Aminoglycoside-modifying enzymes.
Curr. Opin. Microbiol., 2, 499–503 (1999).
75) Rodriguez-Fonseca, C., Amis, R., and Carrett, R. A.,
Fine structure of the peptidyl transferase center on 23S-
like rRNAs deduced from chemical probing of anti-
biotic-ribosome complexes. J. Mol. Biol., 247, 224–
235 (1995).
76) Moazed, D., and Noller, H. F., Chloramphenicol,
erythromycin, carbamycin and vernamycin B protect
overlapping sites in the peptidyl transferase region of
23S ribosomal RNA. Biochemie, 69, 879–884 (1987).
77) Murray, I. A., and Shaw, W. V., O-Acetyltransferases
for chloramphenicol and other natural products. Anti-
microb. Agents Chemother., 41, 1–6 (1997).
78) Abraham, E. P., and Chain, E., An enzyme from
bacteria able to destroy penicillin. Nature, 146, 837
(1940).
79) Bussiere, D., Muchmore, S. W., Dealwis, C. G.,
Schluckebier, G., Nienaber, V. L., Edalji, R. P.,
Walter, K. A., Ladror, U. S., Holzman, T. F., and
Abad-Zapatero, C., Crystal structure of ErmC0 , an
rRNA methyltransferase which mediates antibiotic
resistance in bacteria. Biochemistry, 37, 7103–7112
(1998).
80) Weisblum, B., Erythromycin resistance by ribosome
modification. Antimicrob. Agents Chemother., 39, 577–
585 (1995).
81) Skinner, R., Cundliffe, E., and Schmidt, F. J., Site of
action of a ribosomal RNA methylase responsible for
resistance to erythromycin and other antibiotics. J.
Biol. Chem., 258, 12702–12705 (1983).
82) Cetinkaya, Y., Falk, P., and Mayhall, C. G., Vanco-
mycin-resistant enterococci. Clin. Microbiol. Rev., 13,
686–707 (2000).
83) Arias, C. A., Couvalin, P., and Reynolds, P. E., vanC
cluster of vancomycin-resistant Enterococcus gallina-
rum BM4174. Antimicrob. Agents Chemother., 44,
1660–1666 (2000).
84) Bugg, T. D., Wright, G. D., Dutka-Malen, S., Arthur,
M., Couvalin, P., and Walsh, C. T., Molecular basis
for vancomycin resistance in Enterococcus faecium
BM4147: biosynthesis of a depsipeptide peptidoglycan
precursor by vancomycin resistance proteins VanH and
VanA. Biochemistry, 30, 10408–10415 (1991).
85) Park, I. S., Lin, C. H., and Walsh, C. T., Bacterial
resistance to vancomycin: overproduction, purification,
and characterization of VanC2 from Enterococcus
casseliflavus as a D-Ala-D-Ser ligase. Proc. Natl.
Acad. Sci. USA, 94, 10040–10044 (1997).
86) Nagai, K., Davies, T. A., Jacobs, M. R., and
Appelbaum, P. C., Effects of amino acid alterations
in penicillin-binding proteins (PBPs) 1a, 2b, and 2x
on PBP affinities of penicillin, ampicillin, amoxicillin,
cefditoren, cefuroxime, cefprozil, and cefaclor in 18
clinical isolates of penicillin-susceptible, -intermediate,
and -resistant pneumococci. Antimicrob. Agents Che-
mother., 46, 1273–1280 (2002).
87) Hakenbeck, R., Grebe, T., Zahner, D., and Stock, J. B.,
Beta-lactam resistance in Streptococcus pneumoniae:
penicillin-binding proteins and non-penicillin-binding
proteins. Mol. Microbiol., 33, 673–678 (1999).
88) Zighelboim, S., and Tomasz, A., Penicillin-binding
proteins of multiply antibiotic-resistant South African
strains of Streptococcus pneumoniae. Antimicrob.
Agents Chemother., 17, 434–442 (1980).
89) Hakenbeck, R., Ellerbrock, H., Briese, T., Handwerger,
S., and Tomasz, A., Penicillin-binding proteins of
penicillin-susceptible and -resistant pneumococci: im-
munological relatedness of altered proteins and
changes in peptides carrying the beta-lactam binding
site. Antimicrob. Agents Chemother., 30, 553–558
(1986).
90) Hakenbeck, R., Mosaic genes and their role in
penicillin-resistant Streptococcus pneumoniae. Elec-
trophoresis, 19, 597–601 (1998).
91) Dowson, C. G., Coffey, T. J., Kell, C., and Whiley, R.
A., Evolution of penicillin resistance in Streptococcus
pneumoniae: the role of Streptococcus mitis in the
formation of a low affinity PBP2B in S. pneumoniae.
Mol. Microbiol., 9, 635–643 (1993).
92) Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa,
H., Kobayashi, I., Cui, L., Oguchi, A., Aoki, K., Nagai,
Y., Lian, J., Ito, T., Kanamori, M., Matsumura, H.,
Maruyama, A., Murakami, H., Hosoyama, A.,
Mizutani-Ui, Y., Takahashi, N. K., Sawano, T., Inoue,
R., Kaito, C., Sekimizu, K., Hirakawa, H., Kuhara, S.,
Goto, S., Yabuzaki, J., Kanehisa, M., Yamashita, A.,
Oshima, K., Furuya, K., Yoshino, C., Shiba, T.,
Hattori, M., Ogasawara, N., Hayashi, H., and
Hiramatsu, K., Whole genome sequencing of methi-
cillin-resistant Staphylococcus aureus. Lancet, 357,
1225–1240 (2001).
 Antibiotic Resistance in Bacteria
93) Ryffel, C., Tesch, W., Birch-Machin, I., Reynolds, P.
E., Barberis-Maino, L., Kayser, F. H., and Berger-
Bachi, B., Sequence comparison of mecA genes
isolated from methicillin-resistant Staphylococcus aur-
eus and Staphylococcus epidermidis. Gene, 94, 137–
138 (1990).
94) Ubukata, K., Nonoguchi, R., Song, M. D., Matsuhashi,
M., and Konno, M., Homology of mecA gene in
methicillin-resistant Staphylococcus haemolyticus and
Staphylococcus simulans to that of Staphylococcus
aureus. Antimicrob. Agents Chemother., 34, 170–172
(1990).
95) Hall, B. G., Predicting the evolution of antibiotic
resistance genes. Nat. Rev. Microbiol., 2, 430–435
(2004).
96) McDevitt, D., and Rosenberg, M., Exploiting genomics
to discover new antibiotics. Trends Microbiol., 9, 611–
617 (2001).
97) Rosamond, J., and Allsop, A., Harnessing the power of
the genome in the sesarch for new antibiotics. Science,
287, 1973–1976 (2000).
98) Allsop, A., New antibiotic discovery, novel screens,
novel targets and impact of microbial genomics. Curr.
Opin. Microbiol., 1, 530–534 (1998).
99) Alksne, L. E., and Projan, S. J., Bacterial virulence as a
target for antimicrobial chemotherapy. Curr. Opin.
Biotechnol., 11, 625–636 (2000).
100) Mahan, M. J., Slauch, J. M., and Mekalanos, J. J.,
Selection of bacterial virulence genes that are specif-
ically induced in host tissues. Science, 259, 686–688
(1993).
101) Chiang, S. L., Mekalanos, J. J., and Holden, D. W., In
vivo genetic analysis of bacterial virulence. Annu. Rev.
Microbiol., 53, 129–154 (1999).
102) Valdivia, R. H., and Falkow, S., Fluorescence-based
isolation of bacterial genes expressed within host cells.
Science, 277, 2007–2011 (1997).
103) Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D.,
Dalton, E., and Holden, D. W., Simultaneous identi-
fication of bacterial virulence genes by negative
selection. Science, 269, 400–403 (1995).
104) Rodriguez, E., and McDaniel, R., Combinatorial bio-
synthesis of antimicrobials and other natural products.
Curr. Opin. Microbiol., 4, 526–534 (2001).
105) McDaniel, R., Thamchaipenet, A., Gustafsson, C., Fu,
H., Betlach, M., and Ashley, G., Multiple genetic
modifications of the erythromycin polyketide synthase
to produce a library of novel ‘‘unnatural’’ natural
products. Proc. Natl. Acad. Sci. USA, 96, 1846–1851
(1999).
106) Sutcliffe, J. A., The search for new antibiotics targeting
the 50S ribozyme. ASM News, 70, 513–519 (2004).
107) Hansen, J. L., Ippolito, J. A., Ban, N., Nissen, P.,
Moore, P. B., and Steitz, T. A., The structure of four
macrolide antibiotics bound to the large ribosomal
subunit. Mol. Cell, 10, 117–128 (2002).
108) Knowles, D. J. C., Foloppe, N. F., Matassova, N. B.,
and Murchie, A. I. H., The bacterial ribosome, a
promising focus for structure-based drug design. Curr.
Opin. Pharmacol., 2, 501–506 (2002).
109) Yuan, Z., Trias, J., and White, R. J., Deformylase as a
novel antibacterial target. Drug Discovery Today, 6,
954–961 (2001).
1073
110) Tao, J., and Schimmel, P., Inhibitors of amino acyl-
tRNA synthetases as novel anti-infectives. Expert.
Opin. Invest. Drugs, 9, 1767–1775 (2000).
111) Payne, D. J., Warren, P. V., Holmes, D. J., and
Lonsdale, J. Y., Bacterial fatty acid biosynthesis: a
genomics-driven target for antibacterial drug discov-
ery. Drug Discovery Today, 6, 537–544 (2001).
112) Rohdich, F., Kis, K., Bacher, A., and Eisenreich, W.,
The non-mevalonate pathway of isoprenoids: genes,
enzymes and intermediates. Curr. Opin. Chem. Biol., 5,
535–540 (2001).
113) Clements, J. M., Coignard, F., Johnson, I., Chandler,
S., Palan, S., Waller, A., Wijkmans, J., and Hunter, M.
G., Antibacterial activities and characterization of
novel inhibitors of LpxC. Antimicrob. Agents Chemo-
ther., 46, 1793–1799 (2002).
114) Renau, T. E., Leger, R., Yen, R., She, M. W., Flamme,
E. M., Sangalang, J., Gannon, C. L., Chamberland, S.,
Lomovskaya, O., and Lee, V. J., Peptidomimetics of
efflux pump inhibitors potentiate the activity of levo-
floxacin in Pseudomonas aeruginosa. Bioorg. Med.
Chem. Lett., 12, 763–766 (2002).
115) Flatman, R. H., Howells, A. J., Heide, L., Fiedler,
H.-P., and Maxwell, A., Simocyclinone D8, an inhib-
itor of DNA gyrase with a novel mode of action.
Antimicrob. Agents Chemother., 49, 1093–1100
(2005).
116) Alksne, L. E., Burgio, P., Hu, W., Feld, B., Singh, M.
P., Tuckman, M., Petersen, P. J., Labthavikul, P.,
McGlynn, M., Barbieri, L., McDonald, L., Bradford,
P., Dushin, R. G., Rothstein, D., and Projan, S. J.,
Identification and analysis of bacterial secretion inhib-
itors utilizing a SecA-LacZ reporter fusion system.
Antimicrob. Agents Chemother., 44, 1418–1427
(2000).
117) Mazmanian, S. K., Liu, G., Jensen, E. R., Lenoy, E.,
and Schneewind, O., Staphylococcus aureus sortase
mutants defective in the display of surface proteins and
in the pathogenesis of animal infections. Proc. Natl.
Acad. Sci. USA, 97, 5510–5515 (2000).
118) Darwin, K. H., Ehrt, S., Gutierrez-Ramos, J.-C.,
Weich, N., and Nathan, C. F., The proteasome of
Mycobacterium tuberculosis is required for resistance
to nitric oxide. Science, 302, 1963–1966 (2003).
119) McKinney, T. K., Sharma, V. K., Craig, W. A., and
Archer, G. L., Persistence of Mycobacterium tuber-
culosis in macrophages and mice requires the glyox-
ylate shunt enzyme isocitrate lyase. Nature, 406, 735–
738 (2000).
120) Barett, J. F., and Hoch, J. A., Two-component signal
transduction as a target for microbial anti-infective
therapy. Antimicrob. Agents Chemother., 42, 1529–
1536 (1998).
121) Mayville, P., Ji, G., Beavis, R., Yang, H., Goger, M.,
Novick, R. P., and Muir, T. W., Structure-activity
analysis of synthetic autoinducing thiolactone peptides
from Stphylococcus aureus responsible for virulence.
Proc. Natl. Acad. Sci. USA, 96, 1218–1223 (1999).
122) Mazel, D., Pochet, S., and Marliere, P., Genetic
characterization of polypeptide deformylase, a distinc-
tive enzyme of eubacterial translation. EMBO J., 13,
914–923 (1994).
123) Mazel, D., Coic, E., Blanchard, S., Saurin, W., and
1074 H. YONEYAMA and R. KATSUMATA
Marliere, P., A survey of polypeptide deformylase
function throughout the eubacterial lineage. J. Mol.
Biol., 266, 939–949 (1997).
124) Adams, J. M., and Capecchi, M. R., N-formylmethio-
nyl-sRNA as the initiator of protein synthesis. Proc.
Natl. Acad. Sci. USA, 55, 147–155 (1966).
125) Lucchini, G., and Bianchetti, R., Initiation of protein
synthesis in isolated mitochondria and chloroplasts.
Biochim. Biophys. Acta, 608, 54–61 (1980).
126) Guillon, J. M., Mechulam, Y., Schmitter, J. M.,
Blanquet, S., and Fayat, G., Disruption of the gene
for Met-tRNA(fMet) formyltransferase severely im-
pairs growth of Escherichia coli. J. Bacteriol., 174,
4294–4301 (1992).
127) Meinnel, T., Mechulam, Y., and Blanquet, S., Methio-
nine as translation start signal: a review of the enzymes
of the pathway in Escherichia coli. Biochimie, 75,
1061–1075 (1993).
128) Apfel, C. M., Locher, H., Evers, S., Takacs, B.,
Hubschwerlen, C., Pirson, W., Page, M. G. P., and
Keck, W., Peptide deformylase as an antibacterial drug
target: target validation and resistance development.
Antimicrob. Agents Chemother., 45, 1058–1064
(2001).
129) Clements, J. M., Beckett, R. P., Brown, A., Catlin, G.,
Lobell, M., Palan, S., Thomas, W., Whittaker, M.,
Wood, S., Salama, S., Baker, P. J., Rodgers, H. F.,
Barynin, V., Rice, D. W., and Hunter, M. G., Antibiotic
activity and characterization of BB-3497, a novel
peptide deformylase inhibitor. Antimicrob. Agents
Chemother., 45, 563–570 (2001).
130) Margolis, P. S., Hackbarth, C. J., Young, D. C., Chen,
W. W. D., Yuan, Z., White, R., and Trias, J., Peptide
deformylase in Staphylococcus aureus: resistance to
inhibition is mediated by mutations in the formyl-
transferase gene. Antimicrob. Agents Chemother., 44,
1825–1831 (2000).
131) Rohmer, M., Knani, M., Simonin, P., Sutter, B., and
Sahm, H., Isoprenoid biosynthesis in bacteria: a novel
pathway for the early steps leading to isopentenyl
diphosphate. Biochem. J., 295, 517–524 (1993).
132) Kazuyama, T., Shimizu, T., Takahashi, S., and Seto,
H., Fosmidomycin, a specific inhibitor of 1-deoxy-D-
xylulose 5-phosphate reductoisomerase in the non-
mevalonate pathway for terpenoid biosynthesis. Tetra-
hedron Lett., 39, 7913–7916 (1998).
133) Hedl, M., Sutherlin, A., Wilding, E. I., Mazaulla, M.,
McDevitt, D., Lane, P., Burgner, J. W., 2nd,
Lehnbeuter, K. R., Stauffacher, C. V., Gwynn, M. N.,
and Rodwell, V. W., Enteroccus faecalis acetoacetyl-
coenzyme A thiolase/3-hydroxy-3-methylglutaryl-
coenzyme A reductase, a dual-function protein of
isopentenyl diphosphate biosynthesis. J. Bacteriol.,
184, 2116–2122 (2002).
134) Wildling, E. I., Brown, J. R., Bryant, A. P., Chalker, A.
F., Holmes, D. J., Ingraham, K. A., Iordanescu, S., So,
S., Rosenberg, M., and Gwynn, M. N., Identification,
evolution, and essentiality of the mevalonate pathway
for isopentenyl diphosphate biosynthesis in gram-
positive cocci. J. Bacteriol., 182, 4319–4327 (2000).
135) Chirala, S. S., Huang, W. Y., Jayakumar, A., Sakai, K.,
and Wakil, S. J., Animal fatty acid synthetase: func-
tional mapping and cloning and expression of the
domain I constituent activities. Proc. Natl. Acad. Sci.
USA, 94, 5588–5593 (1997).
136) Heath, R. J., White, S. W., and Rock, C. O., Lipid
biosynthesis as a target for antibacterial agents. Prog.
Lipid Res., 40, 467–497 (2001).
137) McMurry, L. M., Oethinger, M., and Levy, S. B.,
Triclosan targets lipid synthesis. Nature, 394, 531–532
(1998).
138) Rozwarski, D. A., Grant, G. A., Barton, D. H., Jacobs,
W. R., and Sacchettini, J. C., Modification of the
NADH of the isoniazid target (InhA) from Mycobac-
terium tuberculosis. Science, 279, 98–102 (1998).
139) Qiu, X., Janson, C. A., Court, R. I., Smyth, M. G.,
Payne, D. J., and Abdel-Meguid, S. S., Molecular basis
of triclosan activity involves a flipping loop in the
active site. Protein Sci., 8, 2529–2532 (2001).
140) Moche, M., Schneider, G., Edwards, P., Dehesh, K.,
and Lindqvist, Y., Structure of the complex between
the antibiotic cerulenin and its target, beta-ketoacyl
carrier protein synthase. J. Biol. Chem., 274, 6031–
6034 (1999).
141) Qiu, X., Janson, C. A., Smith, W. W., Head, M.,
Lonsdale, J., and Konstantinidis, A. K., Refined
structures of beta-ketoacyl carrier protein synthase
III. J. Mol. Biol., 307, 341–356 (2001).
142) Strauss, E. J., and Falkow, S., Microbial pathogenesis:
genomics and beyond. Science, 276, 707–712 (1997).
143) Falkow, S., Molecular Koch’s postulates applied to
microbial pathogenicity. Rev. Infect. Dis., 10, S274–
276 (1988).
144) Cheung, A. L., Eberhardt, K. J., Chung, E., Yeaman,
M. R., Sullam, P. M., Ramos, M., and Bayer, A. S.,
Diminished virulence of a sar
/agr
mutant of Staph-
ylococcus aureus in the rabbit model of endocarditis.
J. Clin. Invest., 94, 1815–1822 (1994).
145) Throup, J. P., Koretke, K. K., Bryant, A. P., Ingraham,
K. A., Chalker, A. F., Ge, Y., Marta, A., Wallis, N. G.,
Brown, J. R., Holmes, D. J., Rosenberg, M., and
Burnham, M. K., A genomic analysis of two-compo-
nent signal transduction in Streptococcus pnermoniae.
Mol. Microbiol., 35, 566–576 (2000).
146) Throup, J. P., Zappacosta, F., Lunsford, R. D., Annan,
R. S., Carr, S. A., Lonsdale, J. T., Bryant, A. P.,
McDevitt, D., Rosenberg, M., and Burnham, M. K.,
The srhSR gene pair from Staphylococcus aureus:
genomic and proteomic approaches to the identification
and characterization of gene function. Biochemistry,
40, 10392–10401 (2001).
147) Tang, H. B., DiMango, E., Bryan, R., Gambello, M.,
Iglewski, B. H., Goldberg, J. B., and Prince, A.,
Contribution of specific Pseudomonas aeruginosa
virulence factors to pathogenesis of pneumonia in a
neonatal mouse model of infection. Infect. Immun., 64,
37–43 (1996).
148) Uchiya, K., Barbieri, M. A., Funato, K., Shah, A. H.,
Stahl, P. D., and Groisman, E. A., A Salmonella
virulence protein that inhibits cellular trafficking.
EMBO J., 18, 3924–3933 (1999).
149) Menard, R., Sansonetti, P. J., and Parsot, C., Nonpolar
mutagenesis of the ipa genes defines IpaB, IpaC, and
IpaD as effectors of Shigella flexneri entry into
epithelial cells. J. Bacteriol., 175, 5899–5906 (1993).
150) Gaillot, O., Pellegrini, E., Bregenholt, S., and Berche,
 Antibiotic Resistance in Bacteria
P., The ClpP serine protease is essential for the
intracellular parasitism and virulence of Listeria
monocytogenes. Mol. Microbiol., 35, 1286–1294
(2000).
151) Fischetti, V. A., Pancholi, V., and Schneewind, O.,
Conservation of a hexapeptide sequence in the anchor
region of surface proteins from gram-positive cocci.
Mol. Microbiol., 4, 1603–1605 (1990).
152) Schneewind, O., Mihaylove-Petkov, D., and Model, P.,
Cell wall sorting signals in surface proteins of gram-
positive bacteria. EMBO J., 12, 4803–4811 (1993).
153) Mazmanian, S. K., Liu, G., Ton-That, H., and
Schneewind, O., Staphylococcus aureus sortase, an
enzyme that anchors surface proteins to the cell wall.
Science, 285, 760–763 (1999).
1075
154) Ilangovan, U., Ton-That, H., Isahara, J., Schneewind,
O., and Clubb, R. T., Structure of sortase, the trans-
peptidase that anchors proteins to the cell wall of
Staphylococcus aureus. Proc. Natl. Acad. Sci. USA, 98,
6056–6061 (2001).
155) Couvalin, P., and Davies, J., Antimicrobials: time to
act! Editorial overview. Curr. Opin. Microbiol., 6,
425–426 (2003).
156) Schlaes, D. M., Gerding, D. N., John, J. F., Jr., Craig,
W. A., Bornstein, D. L., Duncan, R. A., Eckman, M.
R., Farrer, W. E., Greene, W. H., Lorian, V., Levy, S.,
McGowan, J. E. Jr., Paul, S. M., Ruskin, J., Tenover, F.
C., and Watanakunakorn, C., Guidelines for the
prevention of antimicrobial resistance in hospitals.
Clin. Infect. Dis., 25, 583–599 (1997).