Mecanismos de resistencia del Streptococcus pneumoniae

1. Review
Mechanisms of antibiotic resistance and tolerance
in Streptococcus pneumoniae
Emmanuelle Charpentiera
, Elaine Tuomanenb
*
a
Department of Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, New York, NY 10016, USA
b
Department of Infectious Diseases, St Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105, USA
ABSTRACT – Streptococcus pneumoniae is a major pathogen causing potentially life-threatening
community-acquired diseases in both the developed and developing world. Since 1967, there has been a
dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci
worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and
alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent
studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the
control of bacterial cell death. © 2000 Éditions scientifiques et médicales Elsevier SAS
Streptococcus pneumoniae / antibiotic resistance / penicillin-binding proteins / antibiotic tolerance / autolysin /
signal transduction / cell death
1. Introduction
Streptococcus pneumoniae is a Gram-positive patho-
gen and is one of the most common causes of community-
acquired diseases, such as pneumonia, otitis media, sep-
ticemia, bacterial meningitis and others. The morbidity
and mortality of infections caused by S. pneumoniae
remain high despite appropriate antibiotic therapy. Since
1940, penicillin has been the drug of choice for the
treatment of pneumococcal infections. The first clinical
isolate resistant to penicillin was described in 1967, where
it was recovered from a patient in Papua New Guinea [1].
The tremendous increase in antibiotic usage worldwide
has strongly contributed to the emergence of multidrug-
resistant pneumococci. The sentinel event in the epidemi-
ology of antibiotic-resistant pneumococci was the out-
break in 1977 in South Africa of pneumococcal diseases
caused by multidrug-resistant strains [2]. In addition to
being highly resistant to penicillin (a 1 000-fold increase
of the MIC), these strains were found to be resistant to
erythromycin, clindamycin, tetracycline and chloram-
phenicol. In this article, we review the different mecha-
nisms that have been employed by S. pneumoniae to
develop resistance against penicillins, cephalosporins,
fluoroquinolones, macrolides, tetracycline, chlorampheni-
col and trimethoprim-sulfamethoxazole (table I). Recent
discoveries about pneumococcal signal transduction path-
ways involved in bacterial cell death and their role in
antibiotic tolerance will be discussed.
2. Mechanisms of antibiotic resistance
in S. pneumoniae
2.1. β-lactams
The mechanism of action of β-lactams is based on the
binding of the antibiotic to cell wall synthesizing enzymes,
the penicillin-binding proteins (PBPs), thereby interfering
with the biosynthesis and remodeling of the bacterial
peptidoglycan. Binding of β-lactams to PBPs leads to a
covalently deacylated complex removing the PBPs from
the metabolically active pool [3].
The mechanism of penicillin resistance in clinical iso-
lates of S. pneumoniae involves the alteration of PBPs so
as to reduce their affinity for the antibiotic molecule (table
I). Mutations leading to resistance to penicillin are usually
present in the transpeptidase-penicillin-binding domain
[4]. To lead to reduced affinity to penicillin, a PBP has to
acquire multiple mutations so that high-level resistance is
reached by the acquisition of more than one low-affinity
PBP variant. In pneumococcus, five PBPs of high molecu-
lar weight (PBPs 1a, 1b, 2x, 2a and 2b) and one PBP of low
molecular weight have been described [5]. Alterations in
PBP2x and PBP2b confer low-level resistance and are the
prerequisite for high-level resistance mediated by muta-
tions in other PBPs, like PBP1a [4, 6]. Resistance in many
pneumococcal clinical isolates is due to changes in only
these three PBPs. In addition to its central role in confer-
ring high-level resistance, PBP2b seems to be related to
the bacteriolytic activity of penicillins [6]. The observation
that third generation cephalosporins, another group of
β-lactam antibiotics, induce less lysis in pneumococci is
* Correspondence and reprints.
E-mail address: elaine.tuomanen@stjude.org (E. Tuomanen).
Microbes and Infection, 2, 2000, 1855−1864
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
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2000, 1855-0
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2. based on the fact that they do not interact with PBP2b.
Pneumococci more resistant to the extended-spectrum
cephalosporins than to penicillin G have been described;
this pattern of resistance appears to be due to unique
alterations in PBPs such as PBP2x and PBP1a (table I) [7].
In pneumococcus, the genes that encode the altered
PBPs are called mosaic genes. This feature refers to the
existence of long, contiguous nucleotide sequences within
the PBP genes, which appear to be divergent, i.e. non-
pneumococcal origin [8]. Mosaic genes have emerged in
naturally transformable organisms like neisseriae and strep-
tococci most likely due to the ability to exchange genetic
material via homologous recombination of distinct alleles
[5]. The presence of extended DNA sequences in the PBP
genes modifies not only the active site of these proteins but
perhaps also some secondary domains involved in the
recognition of the muropeptide structure that these bacte-
ria use for building their particular clone-specific pepti-
doglycan [4, 9]. The origin of these mosaic blocks seems
to be traceable to other commensal species of strepto-
cocci, since closely related or even identical blocks of
sequences have been identified in resistant strains of Strep-
tococcus sanguis, Streptococcus mitis and Streptococcus
oralis [10–12]. The existence of identical PBP genes in
genetically distinct clones of penicillin-resistant S. pneu-
moniae demonstrates the horizontal spread of resistance
determinants within one species. A model for the origin of
penicillin resistance and the mechanism by which resis-
tance levels increase has been proposed [13, 14]. Acqui-
sition of mosaic genes may occur in a stepwise manner.
Incorporation of one of such altered low-affinity PBP gene
marks the beginning of a resistant clone, which then
expands through cell division until one of this lineage
engages in a second recombinational event that results in
the modification of another of the high-molecular-weight
PBP genes in the recipient pneumococcus. The progeny of
such a cell (which now has an increased MIC to penicillin)
may undergo further recombination events, each of which
increases the resistance level further [14].
Two alternative mechanisms of β-lactam resistance have
recently been described in vitro in pneumococcus. Both
mechanisms would most likely be involved in the biosyn-
thesis of cell wall components acting upstream of the
biosynthetic function of PBPs [4]. The first mechanism
involves a putative glycosyltransferase, CpoA, which
seems to act as the primary determinant. It was found in a
laboratory mutant obtained upon selection with piperacil-
lin, a highly lytic β-lactam that has high affinity to all
pneumococcal PBPs [15]. CpoA could be involved in
teichoic acid biosynthesis by transferring carbohydrates to
the lipid intermediate [4]. The second mechanism refers to
a putative histidine kinase encoded by the gene ciaH and
identified in a laboratory mutant resistant to cefotaxime, a
third generation cephalosporin that does not induce much
lysis [16]. It was proposed that the cia system might be
involved in sensing and counteracting cell wall damage
induced upon β-lactam treatment. No clinical correlate
implicating these alternative pathways of penicillin resis-
tance has been identified yet. No mechanism of penicillin
resistance involving β-lactamase has been reported thus
far in S. pneumoniae.
2.2. Fluoroquinolones
Quinolones such as the new fluoroquinolones, trova-
floxacin and moxifloxacin, appeared as alternative thera-
peutic agents for the treatment of penicillin-resistant pneu-
mococcal infections. Fluoroquinolones principally target
the type II topoisomerase A2B2 complex, also called DNA
gyrase, that catalyzes DNA supercoiling during replica-
tion, and the topoisomerase IV complex C2E2 that is essen-
tial for chromosome segregation [17].
In clinical isolates of pneumococci, fluoroquinolone
resistance is mediated by target modifications that involve
mutations in the gyrase genes, gyrA and gyrB, and in the
topoisomerase IV genes, parC and parE (table I). However,
in vitro studies have indicated that some strains may use
an efflux mechanism resulting in reduced intracellular
accumulation of the antibiotic [18, 19]. The presence of
mutations in gyrA and parC, the order of appearance of the
mutations and the type of fluoroquinolone that induce the
mutations constitute factors in the development of resis-
tance to fluoroquinolones. Ciprofloxacin resistance in
pneumococcus results from initial and necessary parC
mutations leading to low level of resistance, and subse-
quent gyrA mutations lead to higher levels of resistance
[20, 21]. The mutations in parC that have been described
thus far in clinical isolates and laboratory mutants involve
substitutions of Ser-79 to Tyr/Phe or Asp-83 to Gly/Ala, and
the mutations in gyrA include substitutions of Ser-83 to
Tyr/Phe or Glu-88 to Gln/Lys [20–23]. In contrast to cipro-
floxacin resistance, sparfloxacin resistance results initially
from mutations in gyrA and subsequently, additional muta
Table I. Mechanisms of antibiotic resistance in S. pneumoniae.
Antibiotic family Antibiotic agent Target Resistance mechanism
β-lactams penicillin PBPa
altered target
cephalosporin PBP altered target
Fluoroquinolones ciprofloxacin sparfloxacin DNA gyrase and topoisomerase IV altered target, efflux
DNA gyrase and topoisomerase IV altered target, efflux
Macrolides erythromycin 23S ribosomal RNA altered target, efflux
Chloramphenicol chloramphenicol 50S ribosomal subunit antibiotic enzymatic modification
Tetracycline tetracycline 30S ribosomal subunit altered target
Diaminopyrimidine trimethoprim DHFRa
altered target
Sulphonamide sulfamethoxazole DHPSa
altered target
a
PBP, penicillin-binding protein; DHFR, dihydrofolate reductase; DHPS, dihydropteroate reductase.
Review Charpentier and Tuomanen
1856 Microbes and Infection
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3. tions in parC. A mutation in gyrA resulting in substitution
of Ser-83 to Tyr/Phe and mutations in parC leading to
changes of Ser-79 to Tyr and Asp-83 to Asn were detected
in clinical isolates and laboratory mutants resistant to
sparfloxacin [23, 24]. High level of resistance to clina-
floxacin in laboratory mutants of S. pneumoniae requires
stepwise and multiple mutations in gyrA and parC [25]. By
aligning the DNA sequences of gyrA and parC, it is obvi-
ous that the mutation hotspots in gyrA (Ser-83 and Glu-88)
correspond to those in parC (Ser-79 and Asp-83). It was
thus proposed that the interactions of fluoroquinolones
with GyrA would be similar to those with ParC. The gyrB
and parE genes share significant homology. A mutation in
parE leading to a single amino acid substitution of Asp-435
to Asn was described in pneumococcal clinical and labo-
ratory mutants conferring low-level resistance to fluoro-
quinolone, whereas sequential acquisitions of mutations
in parE and gyrA are required to reach higher levels of
resistance [26, 27]. A mutation in gyrB changing Ser-127
to Leu that resulted in novobiocin resistance was reported
in laboratory mutants [22]. No mutation in gyrB conferring
quinolone resistance has yet been reported in pneumo-
coccal clinical isolates.
Antibiotic efflux was recently suggested to be a likely
relevant mechanism in clinical isolates of S. pneumoniae
resistant to fluoroquinolones (table I) [28, 29]. An active
efflux mechanism of fluoroquinolones similar to that con-
ferred by NorA, a membrane-associated active efflux pump
in Staphylococus aureus, was identified in a pneumococ-
cal laboratory mutant [30]. An efflux protein, PmrA, which
confers resistance to norfloxacin was recently character-
ized in vitro in S. pneumoniae [31].
2.3. Macrolide-lincosamide-streptogramins (MLS)
Although MLS antibiotics are chemically distinct, they
competitively interact when binding to the ribosomal 50S
subunit, where only one molecule is able to bind [32].
Two mechanisms of resistance to MLS in clinical iso-
lates of pneumococci have already been reported: modi-
fication of the target that results in co-resistance to MLS
and efflux of the antibiotic that mediates resistance to
14-membered and 15-membered macrolides only result-
ing in a so-called M phenotype (table I) [18, 33].
Co-resistance to MLS involves the gene erm encoding an
S-adenosylmethionine-dependent methylase that methy-
lates an adenine residue in the peptidyl transferase domain
of the 23S rRNA. The rRNA methylation leads most likely
to a conformational change in the ribosome, thus reducing
the affinity of MLS antibiotics for the rRNA [34]. Descrip-
tion of the gene ermAM carried on the conjugative trans-
poson Tn1545 or a transposon similar to Tn917 was
reported in pneumococcal clinical isolates [35]. The M
resistance phenotype is conferred by a mechanism of
efflux of the antibiotic from the cell [36]. The gene mefE
encodes a transmembrane hydrophobic protein that plays
a role of efflux pump by most likely using the proton
motive force. This mechanism appears to be rapidly emerg-
ing as the predominant mechanism of resistance to eryth-
romycin in clinical isolates of pneumococci isolated in
many countries [37].
2.4. Chloramphenicol
Chloramphenicol inhibits bacterial protein synthesis by
targeting the peptidyl transferase during translation [38].
In pneumococci, resistance to chloramphenicol is due
to the production of the chloramphenicol acetyltrans-
ferase enzyme catalyzing the conversion of chlorampheni-
col to derivatives, which are unable to bind the ribosomal
50S subunit and therefore are no longer capable of inac-
tivating the peptidyltransferase (table I) [39]. Pneumococ-
cal clinical isolates harboring the gene cat carried on the
conjugative transposon Tn5253, a composite transposon
consisting of the tetracycline resistance transposon Tn5251
and Tn5252 were identified [40]. Chloramphenicol-
resistant pneumococcal clinical strains containing
sequences homologous or identical to the cat gene
encoded by the plasmid pC194 from S. aureus have also
been reported [41, 42].
2.5. Tetracycline
Tetracyclines cause bacteriostasis by binding to either
the acceptor site (A-site) or the peptidyl-donor site (P-site)
of the 30S subunit of the bacterial ribosome, thus prevent-
ing binding of the aminoacyl-tRNA to the A-site [38].
Ribosomal protection mediated by the genes tet(M) and
tet(O) is the only resistance mechanism that has been
described thus far in pneumococcus (table I) [43, 44].
Pneumococcal resistant strains harboring tet(M) located
on the transposons Tn1545 and Tn5251 were isolated [40,
45]. The precise mechanism by which the proteins Tet(M)
and Tet(O) protect the ribosome from the action of tetra-
cycline is still unclear. It was suggested that Tet(M) would
promote the release of tetracycline from the ribosome in a
mechanism involving GTP as an energy source and that it
could function either as a tetracycline-resistant analog of
this elongation factor(s) or by modifying the target sites on
the ribosome in a catalytic fashion [46, 47]. It was also
considered that Tet(M) might be involved in modifying the
tRNA in such a way that its binding to the ribosome is not
affected by the presence of tetracycline [48].
2.6. Trimethoprim-sulfamethoxazole
The combination of trimethoprim with sulfamethox-
azole (cotrimoxazole) has been used extensively for the
treatment of lower respiratory tract infections in develop-
ing countries because of its attractive cost and effective-
ness [49]. Trimethoprim and sulfamethoxazole interfere
with the biosynthesis of folic acid [50]. Trimethoprim
selectively inhibits bacterial dihydrofolate reductase
(DHFR) thus preventing the reduction of dihydrofolate to
tetrahydrofolate. Sulfamethoxazole competes with para-
aminobenzoate for dihydropteroate synthetase (DHPS),
preventing the production of 7,8-dihydropteroate and thus
stopping DNA synthesis [50].
Trimethoprim resistance in clinical isolates of S. pneu-
moniae results from a single amino acid substitution (Ile-
100 to Leu) in the chromosomal-encoded DHFR (table I).
It was suggested that this amino acid change would prob-
ably disrupt the hydrogen bonding of the DHFR to the
4-amino group of trimethoprim thus altering the DHFR
function [51]. The nature of the mechanisms resulting in
high levels of trimethoprim resistance in pneumococcus
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2000, 1855-0
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4. remains unknown. Resistance to sulfamethoxazole in
pneumococcal clinical isolates is due to altered
chromosomal-encoded DHPS (table I) [49]. Duplication
of either three or six bases resulting in the repetition of one
or two amino acids in the region from Arg-58 to Tyr-63 of
the chromosomal-encoded DHPS was identified in a resis-
tant isolate. In a laboratory mutant, a duplication of amino
acids 66 and 67 in the chromosomal-encoded DHPS was
also described [52]. More recently, a duplication of Ser-
61, a duplication of Arg-58 and Pro-59 and an insertion of
an arginine residue between Gly-60 and Ser-61 in DHPS
were detected in South African clinical strains of S. pneu-
moniae resistant to trimethoprim-sulfamethoxazole [53].
2.7. Glycopeptides
The glycopeptide antibiotics, vancomycin and teico-
planin, exert their antimicrobial action by preventing both
the transglycosylation and transpeptidation reactions that
mediate the formation of mature cell wall [54]. They have
been considered as the drugs of last resort for infections
due to penicillin-resistant pneumococci. No resistance to
glycopeptides in S. pneumoniae has been thus far identi-
fied. Nevertheless, of great concern is the possibility that
the vancomycin-resistance genes found in enterococci
may be transferred to pneumococci. These enterococcal
genes encoding modified cell wall precursors with
decreased affinity for vancomycin could confer high levels
of resistance and are carried by transmissible elements
[55].
3. Epidemiology of antibiotic resistance
in S. pneumoniae
3.1. β-lactams
It was not until the 1960s that reports of strains of
pneumococci with intermediate levels of penicillin resis-
tance (MICs, 0.1–0.6 µg/mL) began to appear. The first
penicillin-resistant clinical isolate of S. pneumoniae (MIC,
0.5 µg/mL) was described in 1967 in Papua New Guinea
[1, 56].
Between 1967 and 1977, sporadic reports of penicillin-
resistant clinical isolates were published from various
parts of the world. The first dramatic report was the out-
break of epidemic pneumococcal disease caused by
multidrug-resistant strains in South Africa in 1977. In
addition to exhibiting greatly increased MICs of penicillin
of 4 to up to 8 µg/mL, these isolates were also resistant to
chloramphenicol or to tetracycline, erythromycin, clinda-
mycin and chloramphenicol [57, 58].
Since then, penicillin-resistant clinical isolates of pneu-
mococci have spread increasingly worldwide [2, 59]. By
the early 1980s, geographic areas where more than 10%
of isolates were found to be penicillin-resistant included
Israel, France, Hungary, Poland, Spain, South Africa, New
Guinea and the United States from New Mexico to Alaska.
During the 1980s in the United States, several large
multicenter studies showed that the prevalence of S. pneu-
moniae with decreased susceptibility to penicillin was
about 4–5% and bacteria with higher level resistance
(≥ 4 µg/mL) were extremely rare [60, 61]. During the same
period in a number of countries including South Korea,
Hungary and Spain, dramatic increases in penicillin resis-
tance were reported. In 1988 and 1989 in Hungary, an
epidemiological survey revealed that 58% of all pneumo-
coccal isolates and 70% of pneumococcal isolates from
children were resistant to penicillin [62]. In most parts of
the world where surveillance for resistant pneumococci
was performed at several time intervals, appearance of
isolates with low to intermediate resistance levels usually
preceded the appearance of more highly resistant bacte-
ria.
During the last decade, the areas with the highest
prevalence of penicillin-resistant pneumococci included
South Africa, Spain, France, eastern Europe, Israel, South
Korea, Japan, New Guinea and the most southerly areas of
South America [63, 64]. In the United States, the figure
changed abruptly with the proportion of penicillin-resistant
strains increasing to about 25% in certain geographic
locations [63, 65–68]. In some countries, like in Iceland,
penicillin- and multiply antibiotic-resistance emerged in
the 1990s, rapidly reaching frequencies close to 20% in
S. pneumoniae isolated from children [69]. Recent sur-
veillance studies in Latin America, eastern Europe and the
United States demonstrated evidence for similar importa-
tion of two distinct multiply antibiotic-resistant clones of
S. pneumoniae [70–72]. Although the mechanisms of resis-
tance are not directly linked, strains resistant to penicillin
are much more likely to be resistant to macrolides, tetra-
cycline, chloramphenicol and trimethoprim-sulfa-
methoxazole [59].
3.2. Fluoroquinolones
A surveillance study performed in Canada in 1988 and
between 1993 and 1998 on 7 551 isolates of S. pneumo-
niae revealed that reduced susceptibility to fluoroquino-
lones increased from 0% in 1993 to 1.7% in 1997 and
1998 and was associated with penicillin resistance [73]. In
Spain, among 2 822 pneumococcal strains isolated from
1991 to 1998, 2% were resistant to ciprofloxacin (MIC
≥ 4 µg/mL) with an increase from 0.9% in 1991–1992 to
3% in 1997–1998. A relation was observed between
ciprofloxacin resistance and penicillin resistance but also
with MLS resistance [74]. Of 1 037 clinical isolates exam-
ined from the United Kingdom, 273 showed reduced
susceptibility to norfloxacin or ciprofloxacin [28]. From a
recent study on 8 419 worldwide clinical isolates of
S. pneumoniae obtained during 1997–1998, 69 isolates
showed reduced susceptibility or resistance to fluoroqui-
nolones [23]. Recently, in Hong Kong, among 181 clinical
isolates of S. pneumoniae, 12.1% were found resistant to
ciprofloxacin (MIC > 2 µg/mL) [75].
3.3. MLS
Macrolide resistance has been frequently observed,
significantly limiting the usefulness of this class of drugs in
the treatment of pneumonia. S. pneumoniae resistant to
erythromycin was first observed in 1967 in Toronto [18].
In 1992 in France 27.5% of the pneumococcal strains
studied were resistant to erythromycin. Between 1991 and
1992 in the United States 3.7 and 2.2% of pneumococcal
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1858 Microbes and Infection
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5. strains isolated in children aged 1–2 years and 3–4 years,
respectively, were resistant to erythromycin [76]. Impor-
tantly, penicillin-resistant strains are frequently cross-
resistant to macrolides [77]. Since the first observation of
M resistance in pneumococci in Houston, Texas, the M
phenotype was shown to be present in as many as 85% of
erythromycin-resistant isolates in the United States [78]
and to be significantly increasing in clinical strains iso-
lated in South Africa [18]. In a recent study performed in
Taiwan, among 200 clinical isolates of S. pneumoniae
obtained from January 1996 to December 1997, a very
high rate of 82% were erythromycin resistant and 90%
clarithromycin resistant [79].
3.4. Tetracycline
Wide use of tetracyclines has resulted in resistance
developing in pneumococcal infections. The first pneu-
mococcal isolate resistant to tetracycline was isolated in
New South Wales in 1963 from a 10-month-old child with
pneumococcal meningitis [80]. Since then, reports on
tetracycline-resistant pneumococcal clinical isolates have
been described in the literature. As an example, among 91
pneumococcal strains isolated in children in Spain, 72.5%
were resistant to tetracycline [81].
3.5. Chloramphenicol
Chloramphenicol resistance in pneumococci was first
reported in 1970 in Poland, but since has not become a
major problem worldwide [18]. Although in Spain 30–50%
of clinical isolates of pneumococci have been reported to
be resistant to chloramphenicol, less than 5% of pneumo-
cocci isolated from other countries showed resistance
[82]. In developing countries, where the antibiotic is still
widely used, chloramphenicol resistance may be more
common.
3.6. Trimethoprim-sulfamethoxazole
The first clinical strain of pneumococcus resistant to
trimethoprim-sulfamethoxazole was first isolated in 1972
from a patient with an acute exacerbation of chronic
bronchitis [83]. The resistance impact in clinical isolates is
high, with the highest rate reported in Spain between 1984
and 1986, where the resistance rate among clinical iso-
lates was 67% [81]. More than 90% of co-trimoxazole-
resistant pneumococcal strains isolated in South Africa are
also resistant to penicillin and chloramphenicol [51]. Such
a high co-resistance to penicillin prevents the use of
co-trimoxazole for the treatment of penicillin-resistant
pneumococcal infections. In a recent study performed in
Taiwan, among 200 clinical isolates of S. pneumoniae
obtained from January 1996 to December 1997, a very
high rate of 87% were trimethoprim-sulfamethoxazole
resistant [79].
4. Mechanisms of antibiotic tolerance
and bacterial cell death
4.1. Autolytic enzymes
Cell wall hydrolases are required to maintain the pep-
tidoglycan during bacterial growth and split the septum
during cell separation. The expression of most hydrolases
is constitutive throughout the cell cycle, but the enzyme is
only active during stationary-phase lysis. To act as auto-
lysins, the hydrolases completely deregulate and entirely
degrade the cell wall [84]. Autolysis due to activation of
autolysins like the major autolysin LytA (an
N-acetylmuramoyl-L-alanine-amidase) is characteristic for
pneumococci.
In current models, the antibacterial effects of β-lactam
antibiotics are initiated by the binding of antibiotic to
PBPs. This binding inhibits specific steps in cell wall
synthesis, leading to the cessation of bacterial growth. The
bacteria then actively cooperate using their own enzy-
matic death machinery to achieve the final killing out-
come. Although fundamental to the action of penicillins,
the mechanism that explains how the inhibition of cell
wall synthesis or the binding of penicillins to PBPs acti-
vates autolysins remains unknown [85]. A secondary pro-
cess arising from the bacteria itself is necessary to trigger
these cell wall hydrolases to lead to cell death.
Antibiotic tolerance, a phenomenon distinct from anti-
biotic resistance, was first described in 1970 in pneumo-
cocci [86]. Antibiotic tolerance is best described by the
fact that antibiotic-binding to the bacterium becomes dis-
connected from the mechanism of killing. Antibiotic-
tolerant pneumococcal strains stop growing in the pres-
ence of conventional concentrations of antibiotics, but do
not go on to rapidly die. In most cases, antibiotic tolerance
goes with reduced lysis of the bacteria. Nevertheless, in
some instances, bacteria do not lyse upon binding to a
bactericidal antibiotic, but still undergo considerable cell
death [87]. Tolerance occurs due to two different settings:
phenotypic tolerance and genotypic tolerance.
4.2. Phenotypic tolerance
In response to deprivation of an essential nutrient, all
bacteria develop resistance to lysis by most β-lactam
antibiotics, a phenomenon termed phenotypic tolerance.
During this specific metabolic process, called the stringent
response, the bacterium shuts down the synthesis of mac-
romolecules such as DNA, phospholipids and cell wall
peptidoglycan [88]. One major characteristic of pheno-
typic tolerance had already been noted in the early 1940s,
where it became evident that non-growing bacteria are not
killed by penicillin. Since β-lactams bind normally to PBPs
of non-growing bacteria, the protection from the bacteri-
cidal antibiotic must arise by the control of activity of
autolytic enzymes, a process that is poorly understood.
This hypothesis is further substantiated by the fact that
autolysin preparations from non-growing strains retain
their hydrolytic activities when transferred to growing
cells. Phenotypic tolerance is not only restricted to depri-
vation of essential nutrients, non-growing or slow-growing
bacteria. It can also be induced by changes of the bacterial
environment, e.g., by lowering the pH of the medium or
by adding proteolytic enzymes or inhibitors of the autolytic
enzymes [89]. Similarly, addition of lipoteichoic acid
(Forssman antigen) to the growth medium of pneumococ-
cal cultures causes resistance to stationary-phase lysis and
penicillin tolerance, suggesting that lipoteichoic acids
might be involved in the in vivo control of autolysin
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6. activity. This assumption is supported by the observation
that lipoteichoic acids appeared to inhibit autolysin activ-
ity in several bacterial species [90–92].
4.3. Genotypic tolerance
In contrast to phenotypic tolerance (a response of all
bacteria to environmental changes), tolerance to antibiot-
ics can result from genetic mutations. Tolerance arises if
either the pneumococcal autolysin, which lyses the cell
wall, is not triggered or the autolysin itself is not active or
present. The most obvious example of tolerance is the
loss-of-function pneumococcal mutant in the autolysin
gene, lytA, which fails to lyse and dies very slowly [86].
However, no clinical isolates have been identified harbor-
ing a loss-of-function mutation of the autolysin gene.
Some studies suggest that 30% of clinical isolates of pneu-
mococci are genetically tolerant to penicillin [93]. There-
fore, clinical tolerance appears to arise by genetic alter-
ation at the level of regulation of autolysin activity [94].
In recent studies, loss-of-function pneumococcal
mutants were identified from a library of penicillin-tolerant
mutants. Analysis of the strains revealed several different
mechanisms interfering with the control of the pneumo-
coccal autolytic machinery: a two-component regulatory
system (VncS-R), ABC transporters (Psa and Pst), a zinc-
metalloprotease (ZmpB) and a heat-shock protein (ClpC)
[95–99].
4.4. Model for the control of bacterial cell death
One of the pneumococcal mutants from the library
failed to die in the presence of β-lactam antibiotics, includ-
ing vancomycin. The affected gene encoded a histidine
kinase, VncS, belonging to a two-component regulatory
system, VncS–VncR (figure 1) [97]. It was suggested that
the two-component system, VncS–VncR, represents an
early element in the autolytic trigger pathway, controlling
the activity of autolysin via levels of phosphorylation of
the response regulator VncR [97]. This implies that VncS–
VncR functions as a relay station reacting to cell density
signals (stationary-phase lysis) or the binding of antibiotics
to PBPs. Although there is still no evident link between cell
wall inhibition or PBPs and this system, a signal peptide
Pep27
has been identified, which might be a quorum-
sensing signal sensed by the two-component system,
VncS–VncR, necessary to trigger autolytic activity (figure
1) [100].
5. Conclusions and perspectives
The incidence of penicillin-resistant pneumococci has
increased dramatically worldwide, especially in the 1990s.
The spread of penicillin resistance appears to be due to a
global dissemination of several clones carrying both altered
PBP genes and genes encoding resistance to other antibi-
otic classes, including macrolides, tetracycline, chloram-
phenicol and trimethoprim-sulfamethoxazole. This situa-
tion is worsened by the recent emergence of high-level
resistance to extended-spectrum third generation cepha-
losporins [101]. The last-resort antibiotic for the treatment
of multidrug-resistant pneumococcal infections has
Figure 1. Model of autolysin triggering. Environmental signals regulate the addition of a phosphoryl group (P) to the sensor kinase
(VncS). This, in turn, controls whether the response regulator (VncR) is on (phosphorylated) or off (dephosphorylated). When VncR is
phosphorylated, genes that are turned on in response to antibiotics or stationary phase (and induce activation of autolysin, killing the
bacteria) are switched off. One of the trigger signals for bacterial lysis seems to be the peptide Pep27
, which acts in a quorum-sensing
manner. It is sensed by the two-component system, VncS–VncR, and determines with that the dephosphorylation of VncR, leading to cell
death. It remains to be established how and where inhibition of cell wall synthesis by antibiotics feeds into the death peptide pathway.
Review Charpentier and Tuomanen
1860 Microbes and Infection
2000, 1855-0

7. become the glycopeptide vancomycin [102]. The rapid
emergence of enterococcal strains harboring the
vancomycin-resistance gene complex in a highly transfer-
able form raises great concern of a likely transfer of van-
comycin resistance to multidrug-resistant pneumococci.
In addition to a more restricted application of antibiotics,
there is an urgent need for new antimicrobial agents that
are able to overcome the developed antibiotic-resistance
mechanisms.
S. pneumoniae is an autolytic pathogen, which regu-
lates its suicidal enzymatic system. The downregulation of
autolysis leads to tolerance and is of clinical significance
as underscored by reports that failure to eradicate tolerant
bacteria might result in prolongation and even failure of
therapy. Whether this has a broader impact on the general
clinical situation still has to be determined, but it seems
that in body sites of poor defense like the cerebrospinal
fluid compartment, antibiotic-tolerant bacteria might be
responsible for relapsing infections and treatment failures
[103, 104]. A signal transduction pathway involved in
controlling pneumococcal killing was recently uncov-
ered. Understanding of the function and regulation of all
bacterial suicidal participants is critical for the develop-
ment of new antibacterial agents which will not fail in
situations of difficult growth conditions.
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